Accepted Article
1 2
Mannitol-1-phosphate dehydrogenases/phosphatases:
A family of novel bifunctional enzymes for bacterial adaptation to osmotic stress
1
3 4
Miriam Sand1, Marta Rodrigues2, José M. González3, Valérie de Crécy-Lagard4,
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Helena Santos2, Volker Müller1 and Beate Averhoff1*
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1
8
Johann Wolfgang Goethe University Frankfurt am Main, Max-von-Laue-Str. 9, 60438
9
Frankfurt, Germany
Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences,
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2
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Universidade Nova de Lisboa, Av. da República-EAN, 2780-157 Oeiras, Portugal
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3
13
Spain
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4
15
32611, USA
Cell Physiology and NMR Lab, Instituto de Tecnologia Química e Biológica,
Department of Microbiology, University of La Laguna, ES-38206 La Laguna, Tenerife,
Department of Microbiology and Cell Science, University of Florida, Gainesville, FL
16 17
Running title: A bifunctional Mtl-1-P dehydrogenases/phosphatase
18 19
*Corresponding author: Beate Averhoff, Molecular Microbiology & Bioenergetics,
20
Institute of Molecular Biosciences, Johann Wolfgang Goethe University Frankfurt/Main,
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Max-von-Laue-Str. 9, 60438 Frankfurt, Germany. Phone: +49-69-79829509, Fax: +49-69-
22
79829306. E-mail: [email protected]
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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1462-2920.12503 This article is protected by copyright. All rights reserved.
2
Accepted Article
1 2
Summary
3 4
The nutritionally versatile soil bacterium Acinetobacter baylyi ADP1 copes with salt
5
stress by the accumulation of compatible solutes, a strategy that is widespread in
6
nature. This bacterium synthesizes the sugar alcohol mannitol de novo in response to
7
osmotic stress. In a previous study, we identified MtlD, a mannitol-1-phosphate
8
dehydrogenase, which is essential for mannitol biosynthesis and which catalyzes the
9
first step in mannitol biosynthesis, the reduction of fructose-6-phosphate (F-6-P) to
10
the intermediate mannitol-1-phosphate (Mtl-1-P). Until now, the identity of the
11
second enzyme, the phosphatase that catalyzes the dephosphorylation of Mtl-1-P to
12
mannitol, was elusive. Here we show that MtlD has a unique sequence among known
13
mannitol-1-phosphate dehydrogenases with a haloacid dehalogenases (HAD)-like
14
phosphatase domain at the N-terminus. This domain is indeed shown to have a
15
phosphatase activity. Phosphatase activity is strictly Mg2+ dependent. NMR analysis
16
revealed that purified MtlD catalyzes not only reduction of F-6-P but also
17
dephosphorylation of Mtl-1-P. MtlD of A. baylyi is the first bifunctional enzyme of
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mannitol biosynthesis that combines Mtl-1-P dehydrogenase and phosphatase
19
activities in a single polypeptide chain. Bioinformatic analysis revealed that the
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bifunctional enzyme is wide-spread among Acinetobacter strains but only rarely
21
present in other phylogenetic tribes.
22 23 24 25
This article is protected by copyright. All rights reserved.
3
Accepted Article
1 2
Introduction
3 4
Water is the most important compound on earth and its availability is the prerequisite of
5
life. As cellular membranes are permeable to water, an increase of the external osmolality
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or desiccation will lead to a loss of water, followed by shrinkage and finally cell death if no
7
countermeasures are taken (Wood et al., 2001). Living cells respond to a decrease in water
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activities in two ways: halophilic aerobic archaea (halobacteria) accumulate potassium
9
chloride up to molar concentrations in the cells. This requires an adapted cellular
10
machinery that has to work at molar KCl concentrations and restricts growth to high salt
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concentrations (Galinski and Trüper, 1994; Roeßler and Müller, 2001). More flexible and
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thus widespread in archaea, bacteria and eukaryotes is the accumulation of small organic
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molecules that are compatible with the cellular metabolism and thus, they are termed
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„compatible solutes“, which can be divided in different classes such as polyols, sugars,
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amino acids and derivatives thereof (da Costa et al., 1998).
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The metabolically versatile and widespread aerobic soil bacterium Acinetobacter
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baylyi responds to increasing salinities with the uptake of glycine betaine or its precursor
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choline from the medium (Sand et al., 2011; Sand et al., 2013b). If glycine betaine is not
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available it synthesizes glutamate and mannitol de novo (Sand et al., 2013a). The finding
20
of the polyol mannitol as compatible solute was rather surprising, since it had hitherto only
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been found in eukaryotes such as plants, algae and fungi, and in one bacterial species,
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Pseudomonas putida, where it is produced together with N-acetylglutaminylglutamine
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amide (Kets et al., 1996).
24
Mannitol is the most abundant sugar alcohol in nature. It serves as carbon and
25
energy source or radical scavenger and in plants and fungi it is well known as compatible
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4 solute (Jennings, 1984; Stoop et al., 1996). In lactic acid bacteria reduction of fructose to
2
mannitol is used to reoxidize NADH produced during glycolysis (Wisselink et al., 2002).
3
However, some homofermentative lactic acid bacteria synthesize mannitol from the
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glycolytic intermediate, F-6-P, to yield Mtl-1-P that is subsequently dephosphorylated to
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mannitol (Neves et al., 2000). This pathway has been used to engineer mannitol producing
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strains by altering the pattern of endproducts (Gaspar et al., 2011). Biotechnological
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production of mannitol is of high interest since mannitol is widely used in the food,
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pharmaceutical, medical and chemical industries.
Accepted Article
1
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So far there are three known routes for the biosynthesis of mannitol in nature. One
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is the direct reduction of fructose to mannitol by a mannitol-2-dehydrogenase. This
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enzyme is found in heterofermentative lactic acid bacteria (Wisselink et al., 2002). In a
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second route mannose-6-phosphate is converted by a mannose-6-phosphate reductase and a
13
phosphatase to mannitol (Rumpho et al., 1983; Loescher et al., 1992). The third
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mechanism involves the reduction of F-6-P to mannitol via the intermediate Mtl-1-P. This
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pathway requires two distinct enzymes, a Mtl-1-P dehydrogenase catalyzing the first step
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and a Mtl-1-P phosphatase catalyzing the second step. This mannitol production pathway
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is found in homofermentative lactic acid bacteria or algae for instances (Neves et al., 2000;
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Wisselink et al., 2002; Iwamoto et al., 2003).
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The biosynthetic route for the production of mannitol as compatible solute in A.
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baylyi or P. putida was unknown until recently. NMR analysis using A. baylyi revealed
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Mtl-1-P as intermediate. A rather unusual NADPH-dependent, salt-induced and salt-
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dependent Mtl-1-P dehydrogenase MtlD (ACIAD1672) catalyzing the reaction F-6-P +
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NADPH Mtl-1-P + NADP+ was discovered. Mtl-1-P was further dephosphorylated
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leading to mannitol (Sand et al., 2013a), but the phosphatase catalyzing the second reaction
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in this two-step process of mannitol biosynthesis remained to be identified. We report here
This article is protected by copyright. All rights reserved.
5 that MtlD of A. baylyi is a unique bifunctional enzyme mediating both, dehydrogenase and
2
phosphatase activity thus converting F-6-P to mannitol. These data provide first insights
3
into a novel class of Mtl-1-P dehydrogenases/phosphatases which open a new avenue for
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the biotechnological production of mannitol.
Accepted Article
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5 6
Results
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MtlD of A. baylyi contains a phosphatase domain
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10
A close inspection of the amino acid sequence of MtlD of A. baylyi revealed that the C-
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terminus is similar to dehydrogenase domains found in other dehydrogenases with a
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glycine-rich conserved domain (Rossmann-fold) starting at position 247 for cosubstrate
13
binding (GIHGFGAIGGG). Interestingly, the N-terminal domain of MtlD is similar to
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members
15
phosphoesterases, ATPases, phosphatases, phosphonatases, dehalogenases and sugar
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phosphomutases (Burroughs et al., 2006). The first 240 amino acids of MtlD share 43% of
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the residues with a DL-glycerol-3-phosphatase (At5g57440) and 44% with another
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phosphatase (At2g38740) of the HAD superfamily, present in Arabidopsis thaliana (Fig.
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1). Sequence alignment of the phosphatase domain with the mannitol-1-phosphate
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phosphatase (EsM1Pase) of Ectocarpus siliculosus, also a HAD-like phosphatase, showed
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a similarity of 35%. All members of the HAD-family have a high sequence divergence but
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share the α/β core domain catalytic scaffold that catalyzes the transfer of the phosphoryl
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group. This core domain consists of four loops that contain highly conserved sequence
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motifs (motif 1, DXD; motif 2, T/S; motif 3, K/R; motif 4, E/DD, GDXXXD or
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GDXXXXD) (Caparros-Martin et al., 2013) (Fig. 1). Besides that core domain the
26
members of the HAD-family have a second, smaller cap domain which is used to divide
of
the
HAD
(haloacid
dehalogenase)
superfamily,
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that
includes
6 the members into three subfamilies (Selengut, 2001). This second domain serves for
2
substrate recognition. Members of subfamily I have a small α-helical bundle cap domain
3
located between motif I and motif II, members of subfamily II have a mixed α/β domain
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that is located between motif II and motif III and members of subfamily III have no cap
5
domain (Selengut, 2001; Lu et al., 2005). MtlD of A. baylyi has a large domain consisting
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of α- helices between motif I and motif II which might serve as the cap and therefore we
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conclude that MtlD belongs to subfamily I. The presence of this HAD-like domain
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prompted us to look for a potential phosphatase activity of MtlD.
Accepted Article
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Identification and biochemical characterization of the phosphatase activity of MtlD
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To determine a potential phosphatase activity of MtlD we performed an enzyme assay with
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pNPP (p-nitrophenylphosphate) as artificial substrate. These analyses revealed that purified
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MtlD indeed dephosphorylates pNPP to pNP. Since phosphatases often require Mg2+ for
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full activity (Zhang et al., 2004), Mg2+ dependence was analyzed. As can be seen from Fig.
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2 phosphatase activity of MtlD was strongly dependent on the Mg2+ concentration and
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maximal activity (103 mU/mg) was reached with 5 mM MgSO4. Activity was also
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stimulated by other cations such as ZnCl2 (30%), CaCl2 (14%) or CuSO4 (15%) but Mg2+
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stimulated the enzyme most effectively (MgCl2 100%; MgSO4 91%). Furthermore, there
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was no phosphatase activity in the presence of the phosphatase inhibitor NaF (1 mM). The
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pH optimum of the phosphatase reaction with pNPP as substrate was 5.0 but the enzyme
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still retains an activity of 60% at pH 6.0 and 47% at pH 4.5 (Fig. S1).
23 24
MtlD is a bifunctional enzyme and exhibits Mtl-1-P phosphatase activity
25
This article is protected by copyright. All rights reserved.
7 31
To address the question whether MtlD is able to produce mannitol from F-6-P
2
was used as analytical technique to monitor online substrate consumption and product
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formation. The pools of the phosphorylated metabolites, Mtl-1-P, Pi, and NADP+
4
produced, as well as F-6-P and NADPH consumption were monitored. The enzyme was
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incubated in the absence of Mg2+ to prevent possible dephosphorylation of Mtl-1-P and the
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reaction was started by addition of F-6-P at time zero (Fig. 3).
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acquired sequentially for up to 50 minutes. From the array of spectra shown in Fig. 3 it is
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apparent that the resonances due to F-6-P and NADPH decreased with concomitant
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increase of the resonances corresponding to Mtl-1-P, Pi and NADP+. Control assays
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without enzyme showed no release of Pi (Fig. S2). This confirms that MtlD catalyzes the
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reduction of F-6-P to Mtl-1-P. Addition of 5 mM MgCl2 at time 21.3 min (indicated by an
12
arrow) restored phosphatase activity of MtlD and the peak due to Mtl-1-P decreased
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immediately while Pi increased in the same proportion. The presence of mannitol as end-
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product of the reaction was confirmed by 1H NMR (Fig. S3). These experiments clearly
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prove that MtlD is a bifunctional enzyme possessing Mtl-1-P dehydrogenase activity and
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Mg2+-dependent Mtl-1-P phosphatase activity.
Accepted Article
1
31
P-NMR
P-NMR spectra were
17 18
MtlD is highly specific for Mtl-1-P
19 20
To confirm that pure commercial Mtl-1-P is used as substrate by MtlD we analyzed the
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dephosphorylation of Mtl-1-P in a similar assay monitored by 31P-NMR. In the presence of
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MgCl2 (5 mM), Mtl-1-P was completely dephosphorylated by MtlD within 1.2 minutes of
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reaction (Fig. 4A). In contrast, when the assay was repeated without addition of MgCl2, the
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reaction proceeded at a much slower rate (Fig. 4B). The 1H-NMR spectra acquired at the
25
end of the assay revealed that in both cases Mtl-1-P was fully dephosphorylated to
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8 mannitol. These results provide definite evidence for the presence of an Mg2+-dependent
2
phosphatase activity in MtlD. This phosphatase activity was highly specific for Mtl-1-P
3
and the activity determined was 83 U/mg. Substrates such as F-6-P, ribose-5-phosphate,
4
glucose-6-phosphate and glucose-1-phosphate were not dephosphorylated.
Accepted Article
1
5
The dehydrogenase activity of MtlD was previously found to increase with
6
increasing NaCl concentrations with highest activities at 500 mM NaCl (Sand et al.,
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2013a). However still 40% of the maximal activity was observed with 300 mM NaCl. In
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contrast, phosphatase activity (with pNPP as substrate) decreased with elevated NaCl
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concentrations. However, in the presence of 300 mM NaCl still 20% of the maximal
10
activity were detected.
11 12
Distribution and phylogenetic analysis of bifunctional MtlD homologs
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An analysis of around 8000 genomes retrieved from GenBank revealed that the
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bifunctional MtlD enzyme is encoded in all 199 Acinetobacter strains except for six
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Acinetobacter radioresistens strains. No other MtlD-like dehydrogenase was found in the
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genomes of Acinetobacter. Genes encoding the bifunctional enzyme were also detected in
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P. putida and a few other pseudomonads, and also in a few strains that belong to the
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Clostridiales
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Alicycliphilus and Methylobacillus as well as in Arcobacter, an ε-proteobacterium (Fig. 5).
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In every case, the central region of the bifunctional enzyme contains the GXGXXG motif
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that is typically found in the Rossmann fold and is responsible for binding to the
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dinucleotide phosphate. Most of the strains that have the bifunctional MtlD encoded, have
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no other MtlD-like domains encoded in their genomes. Phylogenetic analyses group all
(genera
Clostridium
and
Syntrophobotulus),
the
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β-proteobacteria
9 Acinetobacter MtlDs in one cluster distant from homologs in other γ-proteobacteria and
2
homologs in other phyla.
Accepted Article
1
3 4
Discussion
5 6
Although mannitol is a major compatible solute in the soil bacterium A. baylyi, its
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biosynthesis route remained elusive. Previous analysis revealed a pathway starting from
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F-6-P via Mtl-1-P to mannitol but the phosphatase was not identified. The data presented
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here provide clear evidence that MtlD catalyzes both steps of the pathway, the reduction of
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F-6-P to Mtl-1-P and the subsequent dephosphorylation of Mtl-1-P to mannitol.
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In general, Mtl-1-P dehydrogenases belong to the heterogenous group of long-chain
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secondary alcohol dehydrogenases comprising 66 recognized members. The peptides of
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this mainly prokaryotic protein family are typically between 350 and 560 amino acids long.
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Mtl-1-P dehydrogenases (MtlDs) exhibit the shortest primary sequence within this protein
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family (Kavanagh et al., 2003; Klimacek and Nidetzky, 2002). MtlD of A. baylyi
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comprises 713 amino acids and thereby significantly extends the characteristic length of
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secondary alcohol dehydrogenases. Moreover, only the conserved C-terminal domain, the
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dehydrogenase part of MtlD, is similar to known MtlDs.
19
Sequence analysis revealed that A. baylyi MtlD contains indeed a phosphatase
20
domain at the N-terminus which could not be detected in an alignment with other known
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MtlDs. This domain is conserved in members of the HAD superfamily which is a large
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group of phosphatases with diverse substrate specifity (Burroughs et al., 2006; Caparros-
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Martin et al., 2013). The sequence divergence of phosphatases in general is high but they
24
share an active site formed by four loops that accomodate the four consensus motifs
25
(motifs 1 - 4) (Morais et al., 2000; Lu et al., 2005). They were also detected at the N-
This article is protected by copyright. All rights reserved.
10 terminus of MtlD from A. baylyi. Loop 1 has an aspartate which acts as nucleophile and
2
mediates phosphoryl group transfer in HAD-like phosphatases. Loop 2 assists in substrate
3
binding and contributes threonine or serine and loop 3 has amino acids with positively
4
charged groups such as arginine or lysine and helps in orientation of the nucleophile or
5
electrophile (Caparros-Martin et al., 2013). Loop 4, together with loop 1, is essential for
6
cofactor binding and within the HAD superfamily of phosphatases there is a group of
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magnesium dependent acid phosphatases which comprise a conserved aspartate box
8
(DXDX) near the N-terminus acting as the nucleophile residue (Collet et al., 1998)). In
9
these phosphatases, substrate binding is followed by closure of the active site. Interaction
10
of Mg2+ with the negatively charged phosphate is followed by a nucleophilic attack by the
11
first conserved aspartate (Caparros-Martin et al., 2013). Mg2+ which is essential for the
12
reaction, is bound to aspartate residues which are orientated in loop 4 that is also conserved
13
in MtlD of A. baylyi (Fig. 1).
Accepted Article
1
14
The phosphatase activity of the bifunctional MtlD is highly specific for Mtl-1-P.
15
This is consistent with the findings for other mannitol-1-phosphatases such as the
16
phosphatase from the red algae Caloglossa continua or from the brown algae
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Spatoglossum pacificum and Dictyota dichotomoa that are also specific for Mtl-1-P (Ikawa
18
et al., 1972; Iwamoto et al., 2001; Kavanagh et al., 2003). Groisillier et al. (2014) also
19
described an HAD-like mannitol-1-phosphatase from Ectocarpus siliculosus that is also
20
highly specific for Mtl-1-P and they indicated that these enzymes constitute a new family
21
of phosphatases exhibiting a limited substrate specificity within the HAD superfamily that
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usually show a broad substrate spectrum (Groisillier et al., 2014).
23
Mannitol is produced in response to osmotic stress and we have previously reported
24
that the Mtl-1-P dehydrogenase activity of MtlD increased with increasing salt
25
concentrations, with maximal activities at 500 mM NaCl (Sand et al., 2013a). In contrast,
This article is protected by copyright. All rights reserved.
11 the phosphatase activity decreased with increasing NaCl concentrations. This is consistent
2
with previous reports on the activity of other Mtl-1-P phosphatases such as the Mtl-1-P
3
phosphatase of E. siliculosus which decreased in the presence of 400 mM NaCl by 70% or
4
the Mtl-1-P phosphatase of C. continua which decreased at high salinity by 60% whereas
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the dehydrogenase activity was stimulated (Iwamoto et al., 2001; Groisillier et al., 2014).
6
However, MtlD of A. baylyi exhibits both, dehydrogenase and phosphatase activities, in the
7
presence of moderate salt concentrations (300 mM NaCl) and even at lower salt
8
concentrations (< 300 mM NaCl) and in the absence of NaCl phosphatase activity was still
9
detected.
Accepted Article
1
10
It is interesting to note that bifunctional Mtl-1-P dehydrogenases/phosphatases are
11
widespread in members of the genus Acinetobacter but are mainly restricted to this genus.
12
A bioinformatics approach to identify candidate genes involved in carbon storage and
13
metabolism in the algae Micromonas spp. led to the detection of a potential Mtl-1-P
14
dehydrogenase gene encoding a HAD domain (Michel et al., 2010), although no
15
experimental evidence is available so far. Here, we provide clear experimental evidence
16
that MtlD of A. baylyi is a bifunctional enzyme mediating dehydrogenase and phosphatase
17
activity. Phylogenetic analyses however show that the resulting peptide is novel exclusive
18
of Acinetobacter and distant from orthologs in other taxa even within the same γ-
19
proteobacteria class. It is interesting to note that modules of MltD are more conserved
20
between proteins comprising of both modules than with others that contain just one, either
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the phophatase or dehydrogenase module. Due to this finding it is tempting to speculate
22
that the two modules in the bifunctional phophatases/dehydrogenases have undergone
23
coevolution for an optimal cofunctioning of these different enzymatic activities.“
24
The fusion of two activites in one protein has taken place also in many other cases.
25
For instance, many pathways for di-myo-inositol-phosphate synthesis include bifunctional
This article is protected by copyright. All rights reserved.
12 enzymes while others include monofunctional proteins (Brito et al., 2011). Likewise, genes
2
involved in the biosynthetic pathways of amino acids such as tryptophan (HisB) and
3
histidine (AKAI) biosynthesis and others have been fused (Brilli and Fani, 2004; Fondi et
4
al., 2007).
Accepted Article
1
5
Gene duplication and fusion are two main mechanisms of the evolution of
6
metabolic pathways. Fusions serve for the physical association of different catalytic
7
domains or for generation of regulatory networks (Jensen, 1976). They often occur within
8
genes that code for proteins or enzymes that catalayze sequential steps within the same
9
metabolic pathway (Yanai et al., 2002). An advantage could be to simplify the regulation
10
of metabolic pathways and an enhanced catalytic activity due to channeling of
11
intermediates, such as Mtl-1-P, that are unstable or toxic for the cell (Fondi et al., 2009;
12
Nakano et al., 2013). The toxicity of sugar-phosphates in bacterial cells is well known. For
13
instances, an E. coli fructose-1-phosphate (F-1-P) kinase mutant which was unable to use
14
F-1-P or fructose as substrates was impaired in growth in the presence of these substrates
15
due to an accumulation of toxic F-1-P (Ferenci and Kornberg, 1973). Furthermore, an
16
MtlD-mutant of Salmonella typhimurium was also impaired in growth and lysed after the
17
addition of mannitol as nutrient. It is proposed that this effect was due to the accumulation
18
of Mtl-1-P which was toxic (Berkowitz, 1971).
19
The synthesis of compatible solutes such as mannitol occurs as response to high
20
salinity in the environment of a bacterial cell and has to be quite fast to prevent cell death.
21
As the cytoplasm of a cell is extremely complex and crowded this might be a barrier for
22
free diffusion and movement of proteins (Zimmerman and Trach, 1991; Dauty and
23
Verkman, 2004). Enzymes that are separated in the cytoplasm and have to interact are
24
therefore rate-limited in a fast interaction. The fusion of two polypeptides might support
25
substrate channeling and bypass this problem preventing the cell of wasting energy (Fani et
This article is protected by copyright. All rights reserved.
13 al., 2007). Combining the Mtl-1-P dehydrogenase and Mtl-1-P phosphatase domains in one
2
polypeptide might therefore lead to a more efficient reaction in the synthesis of mannitol in
3
response to osmotic stress.
Accepted Article
1
4
MtlD of A. baylyi represents a novel enzyme with a regulatory network that is
5
likewise expected to be previously unrecognized in the pathways leading to the synthesis
6
of mannitol. This could have implications in the design of chemicals that target
7
Acinetobacter as a pathogen or in biotechnological applications for the synthesis of
8
mannitol.
9
10
Experimental procedures
11 12
Enzyme purification and enzyme assays
13
MtlD (ACIAD1672) from A. baylyi ADP1 was heterologously expressed in E. coli grown
14
in LB medium and purified as described in Sand et al. (Sand et al., 2013a) except that for
15
the phosphatase assay with Mtl-1-P and other sugar substrates MOPS instead of sodium
16
phosphate buffer was used. A coupled enzymatic assay was used to determine phosphatase
17
activity of MtlD with pNPP as artificial substrate. A typical assay was carried out in a total
18
volume of 2 ml containing Na-acetate buffer (50 mM Na-acetate, 10 mM MgSO4), pH 5, at
19
37°C and 5 mM pNPP (p-nitrophenylphosphate). The amount of pNP released was
20
quantified by determining the absorbance at 405 nm.
21
Determination of the optimal pH was performed with pNPP in Na-acetate-MES-
22
HEPES buffer (each 50 mM). The determination of the activity in the presence of different
23
cations (5 mM) was performed in Na-acetate buffer (50 mM, pH 5.0) with pNPP as
24
substrate.
This article is protected by copyright. All rights reserved.
14 The phosphatase assays with Mtl-1-P and other sugar phosphates (each 0.5 mM) as
2
substrates were carried out by determining the amount of Pi released according to
3
Heinonen and Lahti (Heinonen and Lahti, 1981).
Accepted Article
1
4
Analysis of the substrate specificity was performed in 50 mM MOPS buffer (pH
5
7.0, 10 mM MgSO4) at 30°C. The enzyme was preincubated in the buffer for 10 min at
6
30°C before the reaction was started by addition of the substrates.
7 8
NMR analysis
9
31
P-NMR spectra were recorded at 202.45 MHz on a Bruker AVANCEII spectrometer
10
using a 5-mm selective probe head at 30ºC. Spectra were acquired with a 60º pulse and a
11
repetition delay of 1.2 seconds; each spectrum represents an average of 30 or 60 scans,
12
corresponding to 0.6 and 1.2 min acquisition, respectively. Proton broadband decoupling
13
was applied during the acquisition time only to avoid heating. 1H-NMR spectra were
14
acquired with the same spectrometer operating at 500.13 MHz using a 5-mm inverse
15
detection probe head. Water presaturation was applied.
16
Reactions were done in a total volume of 0.5 ml and assays with F-6-P as substrate
17
were prepared as follows: in a 5 mm NMR tube, 50 mM PIPES buffer (pH 6.5), 10 % (v/v)
18
D2O, 0.3 M NaCl, 10 mM NADPH, and 50 μg of MtlD, were combined and pre-incubated
19
at 30ºC before starting the reaction with the addition of 10 mM F-6-P. The reaction was
20
monitored online by acquiring successive
21
Mg2+, the sequence of spectra was interrupted, 5 mM MgCl2 was added to the same tube
22
and acquisition resumed under the same conditions until complete exustion of F-6-P. When
23
Mtl-1-P was studied as substrate the reaction mixture contained 50 mM PIPES buffer (pH
24
6.5), 10 % (v/v) D2O, 0.3 M NaCl, and 3.6 mM Mtl-1-P were combined in the presence or
25
absence of 5 mM MgCl2. The reaction was started by addition of 25 μg of MtlD. 3-
31
P-NMR spectra. To determine the effect of
This article is protected by copyright. All rights reserved.
15 (Trimethylsilyl)-1-propanesulfonic acid and 85% phosphoric acid contained in a cappillary
2
tube, both designated at 0 ppm were used as chemical shift references for 1H- and
3
NMR, respectively.
Accepted Article
1
31
P-
4 5
Phylogenetic analysis
6
The peptide files from available genomes in GenBank were downloaded and searched for
7
specific protein families using Pfam v25.0 run with HMMER3 (Eddy, 2008). A hit was
8
considered valid if its score was equal or bigger than the "gathering score" for the model.
9
Bifunctional MltD homologs were retrieved when the same peptide contained the domains
10
PF13419 (haloacid dehalogenase-like hydrolase that corresponds to the phosphatase
11
domain) and PF08125 (mannitol dehydrogenase, C-terminal). Analysis was carried out by
12
customized Python scripts.
13
The alignments to generate the maximum likelihood trees were generated using
14
MUSCLE (Edgar, 2004) and trimmed using the trimAl software (Capella-Gutierrez et al.,
15
2009) to eliminate highly diverged regions. The maximum-likelihood tree was inferred
16
with RAxML (Stamatakis, 2006) using the LG model with gamma distribution of rates and
17
invariant site categories (implemented as “PROTGAMMAILG”). To obtain the statistical
18
confidence of branching order, 100 pseudoreplicates were generated. The results with
19
maximum-likelihood were confirmed by Bayesian inference using MrBayes v. 3.2.2
20
(Ronquist and Huelsenbeck, 2003). Two independent 1,000,000 generations were run with
21
the WAG model and trees were sampled every 500 generations. The average standard
22
deviation of split frequencies was below 0.007 at the end, which indicates that the MCMC
23
chains converged. The Bayesian tree of the bifunctional MltD amino acid sequences
24
constructed yielded a branching order similar to the maximum likelihood tree.
25
This article is protected by copyright. All rights reserved.
16 Acknowledgments
Accepted Article
1 2 3
This work was funded by a grant from the Deutsche Forschungsgemeinschaft. JMG was
4
funded
5
MarineGems (CTM2010-20361) from the Spanish Ministry of Science and Innovation.
6
The NMR spectrometers are part of The National NMR Facility, supported by Fundação
7
para a Ciência e a Tecnologia (RECI/BBB-BQB/0230/2012).
by
the
CONSOLIDER-INGENIO2010
Program
(CSD2008-00077)
and
8 9
References
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17 Edgar, R.C. (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32: 1792-1797. Fani, R., Brilli, M., Fondi, M., and Lio, P. (2007) The role of gene fusions in the evolution of metabolic pathways: the histidine biosynthesis case. BMC Evol Biol 7 Suppl 2: S4. Ferenci, T., and Kornberg, H.L. (1973) The utilization of fructose by Escherichia coli. Properties of a mutant defective in fructose 1-phosphate kinase activity. Biochem J 132: 341-347. Fondi, M., Brilli, M., and Fani, R. (2007) On the origin and evolution of biosynthetic pathways: integrating microarray data with structure and organization of the common pathway genes. BMC Bioinformatics 8 Suppl 1: S12. Fondi, M., Emiliani, G., and Fani, R. (2009) Origin and evolution of operons and metabolic pathways. Res Microbiol 160: 502-512. Galinski, E.A., and Trüper, H.G. (1994) Microbial behaviour in salt-stressed ecosystems. FEMS Microbiol Rev 15: 95-108. Gaspar, P., Neves, A.R., Gasson, M.J., Shearman, C.A., and Santos, H. (2011) High yields of 2,3-butanediol and mannitol in Lactococcus lactis through engineering of NAD+ cofactor recycling. Appl Environ Microbiol 77: 6826-6835. Groisillier, A., Shao, Z., Michel, G., Goulitquer, S., Bonin, P., Krahulec, S. et al. (2014) Mannitol metabolism in brown algae involves a new phosphatase family. J Exp Bot 65: 559-570. Heinonen, J.K., and Lahti, R.J. (1981) A new and convenient colorimetric determination of inorganic orthophosphate and its application to the assay of inorganic pyrophosphatase. Anal Biochem 113: 313-317. Ikawa, T., Watanabe, T., and Nisizawa, K. (1972) Enzymes involved in the last steps of the biosynthesis of mannitol in brown algae. Plant and Cell Physiol 13: 1017-1029. Iwamoto, K., Kawanobe, H., Shiraiwa, Y., and Ikawa, T. (2001) Purification and characterization of mannitol-l-phosphatase in the red alga Caloglossa continua (Ceramiales, Rhodophyta). Mar Biotechnol 3: 493-500. Iwamoto, K., Kawanobe, H., Ikawa, T., and Shiraiwa, Y. (2003) Characterization of saltregulated mannitol-1-phosphate dehydrogenase in the red alga Caloglossa continua. Plant Physiol 133: 893-900. Jennings, D.H. (1984) Polyol metabolism in fungi. Adv Microb Physiol 25: 149-193. Jensen, R.A. (1976) Enzyme recruitment in evolution of new function. Annu Rev Microbiol 30: 409-425. Kavanagh, K.L., Klimacek, M., Nidetzky, B., and Wilson, D.K. (2003) Crystal structure of Pseudomonas fluorescens mannitol 2-dehydrogenase: evidence for a very divergent long-chain dehydrogenase family. Chem Biol Interact 143-144: 551-558. Kets, E.P., Galinski, E.A., de Wit, M., de Bont, J.A., and Heipieper, H.J. (1996) Mannitol, a novel bacterial compatible solute in Pseudomonas putida S12. J Bacteriol 178: 6665-6670. Klimacek, M., and Nidetzky, B. (2002) A catalytic consensus motif for D-mannitol 2dehydrogenase, a member of a polyol-specific long-chain dehydrogenase family, revealed by kinetic characterization of site-directed mutants of the enzyme from Pseudomonas fluorescens. Biochem J 367: 13-18. Loescher, W.H., Tyson, R.H., Everard, J.D., Redgwell, R.J., and Bieleski, R.L. (1992) Mannitol synthesis in higher plants: evidence for the role and characterization of a NADPH-dependent mannose 6-phosphate reductase. Plant Physiol 98: 1396-1402. Lu, Z., Dunaway-Mariano, D., and Allen, K.N. (2005) HAD superfamily phosphotransferase substrate diversification: structure and function analysis of HAD subclass IIB sugar phosphatase BT4131. Biochemistry 44: 8684-8696.
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18 Michel, G., Tonon, T., Scornet, D., Cock, J.M., Kloareg, B. (2010) Central and storage carbon metabolism of the brown alga Ectocarpus siliculosus: insights into the origin and evolution of storage carbohydrates in Eukaryotes. New Phytol 188: 67-81. Morais, M.C., Zhang, W., Baker, A.S., Zhang, G., Dunaway-Mariano, D., and Allen, K.N. (2000) The crystal structure of Bacillus cereus phosphonoacetaldehyde hydrolase: insight into catalysis of phosphorus bond cleavage and catalytic diversification within the HAD enzyme superfamily. Biochemistry 39: 10385-10396. Nakano, T., Ohki, I., Yokota, A., and Ashida, H. (2013) MtnBD is a multifunctional fusion enzyme in the methionine salvage pathway of Tetrahymena thermophila. PloS one 8: e67385. Neves, A.R., Ramos, A., Shearman, C., Gasson, M.J., Almeida, J.S., and Santos, H. (2000) Metabolic characterization of Lactococcus lactis deficient in lactate dehydrogenase using in vivo 13C-NMR. Eur J Biochem 267: 3859-3868. Roeßler, M., and Müller, V. (2001) Osmoadaptation in bacteria and archaea: common principles and differences. Environ Microbiol 3: 743-754. Ronquist, F., and Huelsenbeck, J.P. (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572-1574. Rumpho, M.E., Edwards, G.E., and Loescher, W.H. (1983) A pathway for photosynthetic carbon flow to mannitol in celery leaves: activity and localization of key enzymes. Plant Physiol 73: 869-873. Sand, M., Mingote, A.I., Santos, H., Müller, V., and Averhoff, B. (2013a) Mannitol, a compatible solute synthesized by Acinetobacter baylyi in a two-step mechanism including a salt-induced and salt-dependent mannitol-1-phosphate dehydrogenase. Environ Microbiol 15: 2187-2197. Sand, M., Stahl, J., Waclawska, I., Ziegler, C., and Averhoff, B. (2013b) Identification of an osmo-dependent and an osmo-independent choline transporter in Acinetobacter baylyi: implications in osmostress protection and metabolic adaptation. Environ Microbiol doi: 10.1111/1462-2920.12188 Sand, M., de Berardinis, V., Mingote, A., Santos, H., Göttig, S., Müller, V., and Averhoff, B. (2011) Salt adaptation in Acinetobacter baylyi: identification and characterization of a secondary glycine betaine transporter. Arch Microbiol 193: 723-730. Selengut, J.D. (2001) MDP-1 is a new and distinct member of the haloacid dehalogenase family of aspartate-dependent phosphohydrolases. Biochemistry 40: 12704-12711. Stamatakis, A. (2006) RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22: 2688-2690. Stoop, M.H., Williamson, J.D., and Pharr, D.M. (1996) Mannitol metabolism in plants: a method for coping with stress. Trends Plant Sci 1: 139-144. Wisselink, H.W., Weusthuis, R.A., Eggink, G., Hugenholtz, J., and Grobben, G.J. (2002) Mannitol production by lactic acid bacteria: a review. Int Dairy J 12: 151-161. Wood, J.M., Bremer, E., Csonka, L.N., Krämer, R., Poolman, B., van der Heide, T., and Smith, L.T. (2001) Osmosensing and osmoregulatory compatible solute accumulation by bacteria. Comp Biochem Physiol A Mol Integr Physiol 130: 437-460. Yanai, I., Wolf, Y.I., and Koonin, E.V. (2002) Evolution of gene fusions: horizontal transfer versus independent events. Genome Biol 3: research0024. Zhang, G., Morais, M.C., Dai, J., Zhang, W., Dunaway-Mariano, D., and Allen, K.N. (2004) Investigation of metal ion binding in phosphonoacetaldehyde hydrolase identifies sequence markers for metal-activated enzymes of the HAD enzyme superfamily. Biochemistry 43: 4990-4997.
Accepted Article
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19 Zimmerman, S.B., and Trach, S.O. (1991) Estimation of macromolecule concentrations and excluded volume effects for the cytoplasm of Escherichia coli. J Mol Biol 222: 599-620.
Accepted Article
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Figure legends
7 8
Fig. 1. Conserved domains of the mannitol-1-phosphate dehydrogenase MtlD of A. baylyi.
9
MtlD has a HAD-like domain at the N-terminus and a mannitol-1-phosphate
10
dehydrogenase-domain (MtlD) and the Rossmann-fold at the C-terminus (A). The amino
11
acid sequence of the phosphatase domain of MtlD of A. baylyi, the two phosphatases of A.
12
thaliana At5g57440 and At2g38740 and the mannitol-1-phosphate phosphatase of
13
Ectocarpus siliculosus EsM1Pase were aligned with the program ClustalW. Residues that
14
are identical or similar in At5g57440, At2g38740 and EsM1Pase are highlighted with
15
asterisks or dots, respectively, and shared motifs are highlighted in grey (B).
16 17
Fig. 2. Mg2+ dependence of MtlD activity. The assay was performed in 50 mM Na-acetate
18
buffer, pH 5.0, with 20 µg enzyme which was preincubated for 10 minutes at 37°C in
19
buffer with the MgSO4 concentrations as indicated. The reaction was started by addition of
20
pNPP to a final concentration of 5 mM. At different time points, 200 µl samples were
21
taken and the reaction was stopped with 800 µl 2 M NaOH. The release of pNP was
22
measured at 405 nm. The figure shows one of three replicates.
23
31
24
Fig. 3. Sequence of
P-NMR spectra monitoring the reaction products of MtID activity.
25
The reaction mixture contained 50 mM PIPES, pH 6.5, 0.3 M NaCl, 10 mM F-6-P, 10 mM
26
NADPH and 50 μg MtID. The reaction was performed at 30ºC. F-6-P was added at time
27
point zero and each spectrum was acquired for 1.2 min, starting at the times indicated. At
28
time 21.3 min, 5 mM MgCl2 (indicated by the arrow) was added. The shift in the
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20 resonances is due to pH changes due to insufficient buffer capacity. ●, Pi; ■, Mtl-1-P; ▲,
2
NADP+; △, F6P; ☐, NADPH.
Accepted Article
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3 4
Fig. 4. Dephosphorylation of Mtl-1-P by MtlD followed by 31P-NMR. Sequences of 31P-
5
NMR acquired consecutively after addition of MtID (25 μg) in the presence of 5 mM
6
MgCl2 (A) or in the absence of MgCl2 (B). Reaction mixture contained 50 mM PIPES, pH
7
6.5, 0.3 M NaCl, 25 μg MtID, and 3.6 mM Mtl-1-P. MtlD was added at time point zero.
8
Each spectrum was acquired for 0.6 min, starting at the time points indicated. ●, Pi; ■, Mtl-
9
1-P.
10 11
Fig. 5. Maximum-likelihood phylogenetic tree of representative bifunctional MltD amino
12
acid sequences. The tree was constructed using RAxML v. 7.8.6. Acinetobacter peptides
13
cluster away from the rest of bifunctional MltD homologs. This topology was found with
14
both Bayesian inference and maximum likelihood methods. Numbers at nodes are
15
bootstrap values in 100 replicates. The scale bar indicates substitutions per site.
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Accepted Article
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emi_12503_f1
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This article is protected by copyright. All rights reserved.
1 2
Mannitol-1-phosphate dehydrogenases/phosphatases:
A family of novel bifunctional enzymes for bacterial adaptation to osmotic stress
1
3 4
Miriam Sand1, Marta Rodrigues2, José M. González3, Valérie de Crécy-Lagard4,
5
Helena Santos2, Volker Müller1 and Beate Averhoff1*
6 7
1
8
Johann Wolfgang Goethe University Frankfurt am Main, Max-von-Laue-Str. 9, 60438
9
Frankfurt, Germany
Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences,
10
2
11
Universidade Nova de Lisboa, Av. da República-EAN, 2780-157 Oeiras, Portugal
12
3
13
Spain
14
4
15
32611, USA
Cell Physiology and NMR Lab, Instituto de Tecnologia Química e Biológica,
Department of Microbiology, University of La Laguna, ES-38206 La Laguna, Tenerife,
Department of Microbiology and Cell Science, University of Florida, Gainesville, FL
16 17
Running title: A bifunctional Mtl-1-P dehydrogenases/phosphatase
18 19
*Corresponding author: Beate Averhoff, Molecular Microbiology & Bioenergetics,
20
Institute of Molecular Biosciences, Johann Wolfgang Goethe University Frankfurt/Main,
21
Max-von-Laue-Str. 9, 60438 Frankfurt, Germany. Phone: +49-69-79829509, Fax: +49-69-
22
79829306. E-mail: [email protected]
23 24
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1462-2920.12503 This article is protected by copyright. All rights reserved.
2
Accepted Article
1 2
Summary
3 4
The nutritionally versatile soil bacterium Acinetobacter baylyi ADP1 copes with salt
5
stress by the accumulation of compatible solutes, a strategy that is widespread in
6
nature. This bacterium synthesizes the sugar alcohol mannitol de novo in response to
7
osmotic stress. In a previous study, we identified MtlD, a mannitol-1-phosphate
8
dehydrogenase, which is essential for mannitol biosynthesis and which catalyzes the
9
first step in mannitol biosynthesis, the reduction of fructose-6-phosphate (F-6-P) to
10
the intermediate mannitol-1-phosphate (Mtl-1-P). Until now, the identity of the
11
second enzyme, the phosphatase that catalyzes the dephosphorylation of Mtl-1-P to
12
mannitol, was elusive. Here we show that MtlD has a unique sequence among known
13
mannitol-1-phosphate dehydrogenases with a haloacid dehalogenases (HAD)-like
14
phosphatase domain at the N-terminus. This domain is indeed shown to have a
15
phosphatase activity. Phosphatase activity is strictly Mg2+ dependent. NMR analysis
16
revealed that purified MtlD catalyzes not only reduction of F-6-P but also
17
dephosphorylation of Mtl-1-P. MtlD of A. baylyi is the first bifunctional enzyme of
18
mannitol biosynthesis that combines Mtl-1-P dehydrogenase and phosphatase
19
activities in a single polypeptide chain. Bioinformatic analysis revealed that the
20
bifunctional enzyme is wide-spread among Acinetobacter strains but only rarely
21
present in other phylogenetic tribes.
22 23 24 25
This article is protected by copyright. All rights reserved.
3
Accepted Article
1 2
Introduction
3 4
Water is the most important compound on earth and its availability is the prerequisite of
5
life. As cellular membranes are permeable to water, an increase of the external osmolality
6
or desiccation will lead to a loss of water, followed by shrinkage and finally cell death if no
7
countermeasures are taken (Wood et al., 2001). Living cells respond to a decrease in water
8
activities in two ways: halophilic aerobic archaea (halobacteria) accumulate potassium
9
chloride up to molar concentrations in the cells. This requires an adapted cellular
10
machinery that has to work at molar KCl concentrations and restricts growth to high salt
11
concentrations (Galinski and Trüper, 1994; Roeßler and Müller, 2001). More flexible and
12
thus widespread in archaea, bacteria and eukaryotes is the accumulation of small organic
13
molecules that are compatible with the cellular metabolism and thus, they are termed
14
„compatible solutes“, which can be divided in different classes such as polyols, sugars,
15
amino acids and derivatives thereof (da Costa et al., 1998).
16
The metabolically versatile and widespread aerobic soil bacterium Acinetobacter
17
baylyi responds to increasing salinities with the uptake of glycine betaine or its precursor
18
choline from the medium (Sand et al., 2011; Sand et al., 2013b). If glycine betaine is not
19
available it synthesizes glutamate and mannitol de novo (Sand et al., 2013a). The finding
20
of the polyol mannitol as compatible solute was rather surprising, since it had hitherto only
21
been found in eukaryotes such as plants, algae and fungi, and in one bacterial species,
22
Pseudomonas putida, where it is produced together with N-acetylglutaminylglutamine
23
amide (Kets et al., 1996).
24
Mannitol is the most abundant sugar alcohol in nature. It serves as carbon and
25
energy source or radical scavenger and in plants and fungi it is well known as compatible
This article is protected by copyright. All rights reserved.
4 solute (Jennings, 1984; Stoop et al., 1996). In lactic acid bacteria reduction of fructose to
2
mannitol is used to reoxidize NADH produced during glycolysis (Wisselink et al., 2002).
3
However, some homofermentative lactic acid bacteria synthesize mannitol from the
4
glycolytic intermediate, F-6-P, to yield Mtl-1-P that is subsequently dephosphorylated to
5
mannitol (Neves et al., 2000). This pathway has been used to engineer mannitol producing
6
strains by altering the pattern of endproducts (Gaspar et al., 2011). Biotechnological
7
production of mannitol is of high interest since mannitol is widely used in the food,
8
pharmaceutical, medical and chemical industries.
Accepted Article
1
9
So far there are three known routes for the biosynthesis of mannitol in nature. One
10
is the direct reduction of fructose to mannitol by a mannitol-2-dehydrogenase. This
11
enzyme is found in heterofermentative lactic acid bacteria (Wisselink et al., 2002). In a
12
second route mannose-6-phosphate is converted by a mannose-6-phosphate reductase and a
13
phosphatase to mannitol (Rumpho et al., 1983; Loescher et al., 1992). The third
14
mechanism involves the reduction of F-6-P to mannitol via the intermediate Mtl-1-P. This
15
pathway requires two distinct enzymes, a Mtl-1-P dehydrogenase catalyzing the first step
16
and a Mtl-1-P phosphatase catalyzing the second step. This mannitol production pathway
17
is found in homofermentative lactic acid bacteria or algae for instances (Neves et al., 2000;
18
Wisselink et al., 2002; Iwamoto et al., 2003).
19
The biosynthetic route for the production of mannitol as compatible solute in A.
20
baylyi or P. putida was unknown until recently. NMR analysis using A. baylyi revealed
21
Mtl-1-P as intermediate. A rather unusual NADPH-dependent, salt-induced and salt-
22
dependent Mtl-1-P dehydrogenase MtlD (ACIAD1672) catalyzing the reaction F-6-P +
23
NADPH Mtl-1-P + NADP+ was discovered. Mtl-1-P was further dephosphorylated
24
leading to mannitol (Sand et al., 2013a), but the phosphatase catalyzing the second reaction
25
in this two-step process of mannitol biosynthesis remained to be identified. We report here
This article is protected by copyright. All rights reserved.
5 that MtlD of A. baylyi is a unique bifunctional enzyme mediating both, dehydrogenase and
2
phosphatase activity thus converting F-6-P to mannitol. These data provide first insights
3
into a novel class of Mtl-1-P dehydrogenases/phosphatases which open a new avenue for
4
the biotechnological production of mannitol.
Accepted Article
1
5 6
Results
7 8
MtlD of A. baylyi contains a phosphatase domain
9
10
A close inspection of the amino acid sequence of MtlD of A. baylyi revealed that the C-
11
terminus is similar to dehydrogenase domains found in other dehydrogenases with a
12
glycine-rich conserved domain (Rossmann-fold) starting at position 247 for cosubstrate
13
binding (GIHGFGAIGGG). Interestingly, the N-terminal domain of MtlD is similar to
14
members
15
phosphoesterases, ATPases, phosphatases, phosphonatases, dehalogenases and sugar
16
phosphomutases (Burroughs et al., 2006). The first 240 amino acids of MtlD share 43% of
17
the residues with a DL-glycerol-3-phosphatase (At5g57440) and 44% with another
18
phosphatase (At2g38740) of the HAD superfamily, present in Arabidopsis thaliana (Fig.
19
1). Sequence alignment of the phosphatase domain with the mannitol-1-phosphate
20
phosphatase (EsM1Pase) of Ectocarpus siliculosus, also a HAD-like phosphatase, showed
21
a similarity of 35%. All members of the HAD-family have a high sequence divergence but
22
share the α/β core domain catalytic scaffold that catalyzes the transfer of the phosphoryl
23
group. This core domain consists of four loops that contain highly conserved sequence
24
motifs (motif 1, DXD; motif 2, T/S; motif 3, K/R; motif 4, E/DD, GDXXXD or
25
GDXXXXD) (Caparros-Martin et al., 2013) (Fig. 1). Besides that core domain the
26
members of the HAD-family have a second, smaller cap domain which is used to divide
of
the
HAD
(haloacid
dehalogenase)
superfamily,
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that
includes
6 the members into three subfamilies (Selengut, 2001). This second domain serves for
2
substrate recognition. Members of subfamily I have a small α-helical bundle cap domain
3
located between motif I and motif II, members of subfamily II have a mixed α/β domain
4
that is located between motif II and motif III and members of subfamily III have no cap
5
domain (Selengut, 2001; Lu et al., 2005). MtlD of A. baylyi has a large domain consisting
6
of α- helices between motif I and motif II which might serve as the cap and therefore we
7
conclude that MtlD belongs to subfamily I. The presence of this HAD-like domain
8
prompted us to look for a potential phosphatase activity of MtlD.
Accepted Article
1
9
10
Identification and biochemical characterization of the phosphatase activity of MtlD
11 12
To determine a potential phosphatase activity of MtlD we performed an enzyme assay with
13
pNPP (p-nitrophenylphosphate) as artificial substrate. These analyses revealed that purified
14
MtlD indeed dephosphorylates pNPP to pNP. Since phosphatases often require Mg2+ for
15
full activity (Zhang et al., 2004), Mg2+ dependence was analyzed. As can be seen from Fig.
16
2 phosphatase activity of MtlD was strongly dependent on the Mg2+ concentration and
17
maximal activity (103 mU/mg) was reached with 5 mM MgSO4. Activity was also
18
stimulated by other cations such as ZnCl2 (30%), CaCl2 (14%) or CuSO4 (15%) but Mg2+
19
stimulated the enzyme most effectively (MgCl2 100%; MgSO4 91%). Furthermore, there
20
was no phosphatase activity in the presence of the phosphatase inhibitor NaF (1 mM). The
21
pH optimum of the phosphatase reaction with pNPP as substrate was 5.0 but the enzyme
22
still retains an activity of 60% at pH 6.0 and 47% at pH 4.5 (Fig. S1).
23 24
MtlD is a bifunctional enzyme and exhibits Mtl-1-P phosphatase activity
25
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7 31
To address the question whether MtlD is able to produce mannitol from F-6-P
2
was used as analytical technique to monitor online substrate consumption and product
3
formation. The pools of the phosphorylated metabolites, Mtl-1-P, Pi, and NADP+
4
produced, as well as F-6-P and NADPH consumption were monitored. The enzyme was
5
incubated in the absence of Mg2+ to prevent possible dephosphorylation of Mtl-1-P and the
6
reaction was started by addition of F-6-P at time zero (Fig. 3).
7
acquired sequentially for up to 50 minutes. From the array of spectra shown in Fig. 3 it is
8
apparent that the resonances due to F-6-P and NADPH decreased with concomitant
9
increase of the resonances corresponding to Mtl-1-P, Pi and NADP+. Control assays
10
without enzyme showed no release of Pi (Fig. S2). This confirms that MtlD catalyzes the
11
reduction of F-6-P to Mtl-1-P. Addition of 5 mM MgCl2 at time 21.3 min (indicated by an
12
arrow) restored phosphatase activity of MtlD and the peak due to Mtl-1-P decreased
13
immediately while Pi increased in the same proportion. The presence of mannitol as end-
14
product of the reaction was confirmed by 1H NMR (Fig. S3). These experiments clearly
15
prove that MtlD is a bifunctional enzyme possessing Mtl-1-P dehydrogenase activity and
16
Mg2+-dependent Mtl-1-P phosphatase activity.
Accepted Article
1
31
P-NMR
P-NMR spectra were
17 18
MtlD is highly specific for Mtl-1-P
19 20
To confirm that pure commercial Mtl-1-P is used as substrate by MtlD we analyzed the
21
dephosphorylation of Mtl-1-P in a similar assay monitored by 31P-NMR. In the presence of
22
MgCl2 (5 mM), Mtl-1-P was completely dephosphorylated by MtlD within 1.2 minutes of
23
reaction (Fig. 4A). In contrast, when the assay was repeated without addition of MgCl2, the
24
reaction proceeded at a much slower rate (Fig. 4B). The 1H-NMR spectra acquired at the
25
end of the assay revealed that in both cases Mtl-1-P was fully dephosphorylated to
This article is protected by copyright. All rights reserved.
8 mannitol. These results provide definite evidence for the presence of an Mg2+-dependent
2
phosphatase activity in MtlD. This phosphatase activity was highly specific for Mtl-1-P
3
and the activity determined was 83 U/mg. Substrates such as F-6-P, ribose-5-phosphate,
4
glucose-6-phosphate and glucose-1-phosphate were not dephosphorylated.
Accepted Article
1
5
The dehydrogenase activity of MtlD was previously found to increase with
6
increasing NaCl concentrations with highest activities at 500 mM NaCl (Sand et al.,
7
2013a). However still 40% of the maximal activity was observed with 300 mM NaCl. In
8
contrast, phosphatase activity (with pNPP as substrate) decreased with elevated NaCl
9
concentrations. However, in the presence of 300 mM NaCl still 20% of the maximal
10
activity were detected.
11 12
Distribution and phylogenetic analysis of bifunctional MtlD homologs
13 14
An analysis of around 8000 genomes retrieved from GenBank revealed that the
15
bifunctional MtlD enzyme is encoded in all 199 Acinetobacter strains except for six
16
Acinetobacter radioresistens strains. No other MtlD-like dehydrogenase was found in the
17
genomes of Acinetobacter. Genes encoding the bifunctional enzyme were also detected in
18
P. putida and a few other pseudomonads, and also in a few strains that belong to the
19
Clostridiales
20
Alicycliphilus and Methylobacillus as well as in Arcobacter, an ε-proteobacterium (Fig. 5).
21
In every case, the central region of the bifunctional enzyme contains the GXGXXG motif
22
that is typically found in the Rossmann fold and is responsible for binding to the
23
dinucleotide phosphate. Most of the strains that have the bifunctional MtlD encoded, have
24
no other MtlD-like domains encoded in their genomes. Phylogenetic analyses group all
(genera
Clostridium
and
Syntrophobotulus),
the
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β-proteobacteria
9 Acinetobacter MtlDs in one cluster distant from homologs in other γ-proteobacteria and
2
homologs in other phyla.
Accepted Article
1
3 4
Discussion
5 6
Although mannitol is a major compatible solute in the soil bacterium A. baylyi, its
7
biosynthesis route remained elusive. Previous analysis revealed a pathway starting from
8
F-6-P via Mtl-1-P to mannitol but the phosphatase was not identified. The data presented
9
here provide clear evidence that MtlD catalyzes both steps of the pathway, the reduction of
10
F-6-P to Mtl-1-P and the subsequent dephosphorylation of Mtl-1-P to mannitol.
11
In general, Mtl-1-P dehydrogenases belong to the heterogenous group of long-chain
12
secondary alcohol dehydrogenases comprising 66 recognized members. The peptides of
13
this mainly prokaryotic protein family are typically between 350 and 560 amino acids long.
14
Mtl-1-P dehydrogenases (MtlDs) exhibit the shortest primary sequence within this protein
15
family (Kavanagh et al., 2003; Klimacek and Nidetzky, 2002). MtlD of A. baylyi
16
comprises 713 amino acids and thereby significantly extends the characteristic length of
17
secondary alcohol dehydrogenases. Moreover, only the conserved C-terminal domain, the
18
dehydrogenase part of MtlD, is similar to known MtlDs.
19
Sequence analysis revealed that A. baylyi MtlD contains indeed a phosphatase
20
domain at the N-terminus which could not be detected in an alignment with other known
21
MtlDs. This domain is conserved in members of the HAD superfamily which is a large
22
group of phosphatases with diverse substrate specifity (Burroughs et al., 2006; Caparros-
23
Martin et al., 2013). The sequence divergence of phosphatases in general is high but they
24
share an active site formed by four loops that accomodate the four consensus motifs
25
(motifs 1 - 4) (Morais et al., 2000; Lu et al., 2005). They were also detected at the N-
This article is protected by copyright. All rights reserved.
10 terminus of MtlD from A. baylyi. Loop 1 has an aspartate which acts as nucleophile and
2
mediates phosphoryl group transfer in HAD-like phosphatases. Loop 2 assists in substrate
3
binding and contributes threonine or serine and loop 3 has amino acids with positively
4
charged groups such as arginine or lysine and helps in orientation of the nucleophile or
5
electrophile (Caparros-Martin et al., 2013). Loop 4, together with loop 1, is essential for
6
cofactor binding and within the HAD superfamily of phosphatases there is a group of
7
magnesium dependent acid phosphatases which comprise a conserved aspartate box
8
(DXDX) near the N-terminus acting as the nucleophile residue (Collet et al., 1998)). In
9
these phosphatases, substrate binding is followed by closure of the active site. Interaction
10
of Mg2+ with the negatively charged phosphate is followed by a nucleophilic attack by the
11
first conserved aspartate (Caparros-Martin et al., 2013). Mg2+ which is essential for the
12
reaction, is bound to aspartate residues which are orientated in loop 4 that is also conserved
13
in MtlD of A. baylyi (Fig. 1).
Accepted Article
1
14
The phosphatase activity of the bifunctional MtlD is highly specific for Mtl-1-P.
15
This is consistent with the findings for other mannitol-1-phosphatases such as the
16
phosphatase from the red algae Caloglossa continua or from the brown algae
17
Spatoglossum pacificum and Dictyota dichotomoa that are also specific for Mtl-1-P (Ikawa
18
et al., 1972; Iwamoto et al., 2001; Kavanagh et al., 2003). Groisillier et al. (2014) also
19
described an HAD-like mannitol-1-phosphatase from Ectocarpus siliculosus that is also
20
highly specific for Mtl-1-P and they indicated that these enzymes constitute a new family
21
of phosphatases exhibiting a limited substrate specificity within the HAD superfamily that
22
usually show a broad substrate spectrum (Groisillier et al., 2014).
23
Mannitol is produced in response to osmotic stress and we have previously reported
24
that the Mtl-1-P dehydrogenase activity of MtlD increased with increasing salt
25
concentrations, with maximal activities at 500 mM NaCl (Sand et al., 2013a). In contrast,
This article is protected by copyright. All rights reserved.
11 the phosphatase activity decreased with increasing NaCl concentrations. This is consistent
2
with previous reports on the activity of other Mtl-1-P phosphatases such as the Mtl-1-P
3
phosphatase of E. siliculosus which decreased in the presence of 400 mM NaCl by 70% or
4
the Mtl-1-P phosphatase of C. continua which decreased at high salinity by 60% whereas
5
the dehydrogenase activity was stimulated (Iwamoto et al., 2001; Groisillier et al., 2014).
6
However, MtlD of A. baylyi exhibits both, dehydrogenase and phosphatase activities, in the
7
presence of moderate salt concentrations (300 mM NaCl) and even at lower salt
8
concentrations (< 300 mM NaCl) and in the absence of NaCl phosphatase activity was still
9
detected.
Accepted Article
1
10
It is interesting to note that bifunctional Mtl-1-P dehydrogenases/phosphatases are
11
widespread in members of the genus Acinetobacter but are mainly restricted to this genus.
12
A bioinformatics approach to identify candidate genes involved in carbon storage and
13
metabolism in the algae Micromonas spp. led to the detection of a potential Mtl-1-P
14
dehydrogenase gene encoding a HAD domain (Michel et al., 2010), although no
15
experimental evidence is available so far. Here, we provide clear experimental evidence
16
that MtlD of A. baylyi is a bifunctional enzyme mediating dehydrogenase and phosphatase
17
activity. Phylogenetic analyses however show that the resulting peptide is novel exclusive
18
of Acinetobacter and distant from orthologs in other taxa even within the same γ-
19
proteobacteria class. It is interesting to note that modules of MltD are more conserved
20
between proteins comprising of both modules than with others that contain just one, either
21
the phophatase or dehydrogenase module. Due to this finding it is tempting to speculate
22
that the two modules in the bifunctional phophatases/dehydrogenases have undergone
23
coevolution for an optimal cofunctioning of these different enzymatic activities.“
24
The fusion of two activites in one protein has taken place also in many other cases.
25
For instance, many pathways for di-myo-inositol-phosphate synthesis include bifunctional
This article is protected by copyright. All rights reserved.
12 enzymes while others include monofunctional proteins (Brito et al., 2011). Likewise, genes
2
involved in the biosynthetic pathways of amino acids such as tryptophan (HisB) and
3
histidine (AKAI) biosynthesis and others have been fused (Brilli and Fani, 2004; Fondi et
4
al., 2007).
Accepted Article
1
5
Gene duplication and fusion are two main mechanisms of the evolution of
6
metabolic pathways. Fusions serve for the physical association of different catalytic
7
domains or for generation of regulatory networks (Jensen, 1976). They often occur within
8
genes that code for proteins or enzymes that catalayze sequential steps within the same
9
metabolic pathway (Yanai et al., 2002). An advantage could be to simplify the regulation
10
of metabolic pathways and an enhanced catalytic activity due to channeling of
11
intermediates, such as Mtl-1-P, that are unstable or toxic for the cell (Fondi et al., 2009;
12
Nakano et al., 2013). The toxicity of sugar-phosphates in bacterial cells is well known. For
13
instances, an E. coli fructose-1-phosphate (F-1-P) kinase mutant which was unable to use
14
F-1-P or fructose as substrates was impaired in growth in the presence of these substrates
15
due to an accumulation of toxic F-1-P (Ferenci and Kornberg, 1973). Furthermore, an
16
MtlD-mutant of Salmonella typhimurium was also impaired in growth and lysed after the
17
addition of mannitol as nutrient. It is proposed that this effect was due to the accumulation
18
of Mtl-1-P which was toxic (Berkowitz, 1971).
19
The synthesis of compatible solutes such as mannitol occurs as response to high
20
salinity in the environment of a bacterial cell and has to be quite fast to prevent cell death.
21
As the cytoplasm of a cell is extremely complex and crowded this might be a barrier for
22
free diffusion and movement of proteins (Zimmerman and Trach, 1991; Dauty and
23
Verkman, 2004). Enzymes that are separated in the cytoplasm and have to interact are
24
therefore rate-limited in a fast interaction. The fusion of two polypeptides might support
25
substrate channeling and bypass this problem preventing the cell of wasting energy (Fani et
This article is protected by copyright. All rights reserved.
13 al., 2007). Combining the Mtl-1-P dehydrogenase and Mtl-1-P phosphatase domains in one
2
polypeptide might therefore lead to a more efficient reaction in the synthesis of mannitol in
3
response to osmotic stress.
Accepted Article
1
4
MtlD of A. baylyi represents a novel enzyme with a regulatory network that is
5
likewise expected to be previously unrecognized in the pathways leading to the synthesis
6
of mannitol. This could have implications in the design of chemicals that target
7
Acinetobacter as a pathogen or in biotechnological applications for the synthesis of
8
mannitol.
9
10
Experimental procedures
11 12
Enzyme purification and enzyme assays
13
MtlD (ACIAD1672) from A. baylyi ADP1 was heterologously expressed in E. coli grown
14
in LB medium and purified as described in Sand et al. (Sand et al., 2013a) except that for
15
the phosphatase assay with Mtl-1-P and other sugar substrates MOPS instead of sodium
16
phosphate buffer was used. A coupled enzymatic assay was used to determine phosphatase
17
activity of MtlD with pNPP as artificial substrate. A typical assay was carried out in a total
18
volume of 2 ml containing Na-acetate buffer (50 mM Na-acetate, 10 mM MgSO4), pH 5, at
19
37°C and 5 mM pNPP (p-nitrophenylphosphate). The amount of pNP released was
20
quantified by determining the absorbance at 405 nm.
21
Determination of the optimal pH was performed with pNPP in Na-acetate-MES-
22
HEPES buffer (each 50 mM). The determination of the activity in the presence of different
23
cations (5 mM) was performed in Na-acetate buffer (50 mM, pH 5.0) with pNPP as
24
substrate.
This article is protected by copyright. All rights reserved.
14 The phosphatase assays with Mtl-1-P and other sugar phosphates (each 0.5 mM) as
2
substrates were carried out by determining the amount of Pi released according to
3
Heinonen and Lahti (Heinonen and Lahti, 1981).
Accepted Article
1
4
Analysis of the substrate specificity was performed in 50 mM MOPS buffer (pH
5
7.0, 10 mM MgSO4) at 30°C. The enzyme was preincubated in the buffer for 10 min at
6
30°C before the reaction was started by addition of the substrates.
7 8
NMR analysis
9
31
P-NMR spectra were recorded at 202.45 MHz on a Bruker AVANCEII spectrometer
10
using a 5-mm selective probe head at 30ºC. Spectra were acquired with a 60º pulse and a
11
repetition delay of 1.2 seconds; each spectrum represents an average of 30 or 60 scans,
12
corresponding to 0.6 and 1.2 min acquisition, respectively. Proton broadband decoupling
13
was applied during the acquisition time only to avoid heating. 1H-NMR spectra were
14
acquired with the same spectrometer operating at 500.13 MHz using a 5-mm inverse
15
detection probe head. Water presaturation was applied.
16
Reactions were done in a total volume of 0.5 ml and assays with F-6-P as substrate
17
were prepared as follows: in a 5 mm NMR tube, 50 mM PIPES buffer (pH 6.5), 10 % (v/v)
18
D2O, 0.3 M NaCl, 10 mM NADPH, and 50 μg of MtlD, were combined and pre-incubated
19
at 30ºC before starting the reaction with the addition of 10 mM F-6-P. The reaction was
20
monitored online by acquiring successive
21
Mg2+, the sequence of spectra was interrupted, 5 mM MgCl2 was added to the same tube
22
and acquisition resumed under the same conditions until complete exustion of F-6-P. When
23
Mtl-1-P was studied as substrate the reaction mixture contained 50 mM PIPES buffer (pH
24
6.5), 10 % (v/v) D2O, 0.3 M NaCl, and 3.6 mM Mtl-1-P were combined in the presence or
25
absence of 5 mM MgCl2. The reaction was started by addition of 25 μg of MtlD. 3-
31
P-NMR spectra. To determine the effect of
This article is protected by copyright. All rights reserved.
15 (Trimethylsilyl)-1-propanesulfonic acid and 85% phosphoric acid contained in a cappillary
2
tube, both designated at 0 ppm were used as chemical shift references for 1H- and
3
NMR, respectively.
Accepted Article
1
31
P-
4 5
Phylogenetic analysis
6
The peptide files from available genomes in GenBank were downloaded and searched for
7
specific protein families using Pfam v25.0 run with HMMER3 (Eddy, 2008). A hit was
8
considered valid if its score was equal or bigger than the "gathering score" for the model.
9
Bifunctional MltD homologs were retrieved when the same peptide contained the domains
10
PF13419 (haloacid dehalogenase-like hydrolase that corresponds to the phosphatase
11
domain) and PF08125 (mannitol dehydrogenase, C-terminal). Analysis was carried out by
12
customized Python scripts.
13
The alignments to generate the maximum likelihood trees were generated using
14
MUSCLE (Edgar, 2004) and trimmed using the trimAl software (Capella-Gutierrez et al.,
15
2009) to eliminate highly diverged regions. The maximum-likelihood tree was inferred
16
with RAxML (Stamatakis, 2006) using the LG model with gamma distribution of rates and
17
invariant site categories (implemented as “PROTGAMMAILG”). To obtain the statistical
18
confidence of branching order, 100 pseudoreplicates were generated. The results with
19
maximum-likelihood were confirmed by Bayesian inference using MrBayes v. 3.2.2
20
(Ronquist and Huelsenbeck, 2003). Two independent 1,000,000 generations were run with
21
the WAG model and trees were sampled every 500 generations. The average standard
22
deviation of split frequencies was below 0.007 at the end, which indicates that the MCMC
23
chains converged. The Bayesian tree of the bifunctional MltD amino acid sequences
24
constructed yielded a branching order similar to the maximum likelihood tree.
25
This article is protected by copyright. All rights reserved.
16 Acknowledgments
Accepted Article
1 2 3
This work was funded by a grant from the Deutsche Forschungsgemeinschaft. JMG was
4
funded
5
MarineGems (CTM2010-20361) from the Spanish Ministry of Science and Innovation.
6
The NMR spectrometers are part of The National NMR Facility, supported by Fundação
7
para a Ciência e a Tecnologia (RECI/BBB-BQB/0230/2012).
by
the
CONSOLIDER-INGENIO2010
Program
(CSD2008-00077)
and
8 9
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Accepted Article
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19 Zimmerman, S.B., and Trach, S.O. (1991) Estimation of macromolecule concentrations and excluded volume effects for the cytoplasm of Escherichia coli. J Mol Biol 222: 599-620.
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Figure legends
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Fig. 1. Conserved domains of the mannitol-1-phosphate dehydrogenase MtlD of A. baylyi.
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MtlD has a HAD-like domain at the N-terminus and a mannitol-1-phosphate
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dehydrogenase-domain (MtlD) and the Rossmann-fold at the C-terminus (A). The amino
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acid sequence of the phosphatase domain of MtlD of A. baylyi, the two phosphatases of A.
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thaliana At5g57440 and At2g38740 and the mannitol-1-phosphate phosphatase of
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Ectocarpus siliculosus EsM1Pase were aligned with the program ClustalW. Residues that
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are identical or similar in At5g57440, At2g38740 and EsM1Pase are highlighted with
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asterisks or dots, respectively, and shared motifs are highlighted in grey (B).
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Fig. 2. Mg2+ dependence of MtlD activity. The assay was performed in 50 mM Na-acetate
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buffer, pH 5.0, with 20 µg enzyme which was preincubated for 10 minutes at 37°C in
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buffer with the MgSO4 concentrations as indicated. The reaction was started by addition of
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pNPP to a final concentration of 5 mM. At different time points, 200 µl samples were
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taken and the reaction was stopped with 800 µl 2 M NaOH. The release of pNP was
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measured at 405 nm. The figure shows one of three replicates.
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Fig. 3. Sequence of
P-NMR spectra monitoring the reaction products of MtID activity.
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The reaction mixture contained 50 mM PIPES, pH 6.5, 0.3 M NaCl, 10 mM F-6-P, 10 mM
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NADPH and 50 μg MtID. The reaction was performed at 30ºC. F-6-P was added at time
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point zero and each spectrum was acquired for 1.2 min, starting at the times indicated. At
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time 21.3 min, 5 mM MgCl2 (indicated by the arrow) was added. The shift in the
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20 resonances is due to pH changes due to insufficient buffer capacity. ●, Pi; ■, Mtl-1-P; ▲,
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NADP+; △, F6P; ☐, NADPH.
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Fig. 4. Dephosphorylation of Mtl-1-P by MtlD followed by 31P-NMR. Sequences of 31P-
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NMR acquired consecutively after addition of MtID (25 μg) in the presence of 5 mM
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MgCl2 (A) or in the absence of MgCl2 (B). Reaction mixture contained 50 mM PIPES, pH
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6.5, 0.3 M NaCl, 25 μg MtID, and 3.6 mM Mtl-1-P. MtlD was added at time point zero.
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Each spectrum was acquired for 0.6 min, starting at the time points indicated. ●, Pi; ■, Mtl-
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1-P.
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Fig. 5. Maximum-likelihood phylogenetic tree of representative bifunctional MltD amino
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acid sequences. The tree was constructed using RAxML v. 7.8.6. Acinetobacter peptides
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cluster away from the rest of bifunctional MltD homologs. This topology was found with
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both Bayesian inference and maximum likelihood methods. Numbers at nodes are
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bootstrap values in 100 replicates. The scale bar indicates substitutions per site.
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