Appl Biochem Biotechnol (2014) 173:1849–1857 DOI 10.1007/s12010-014-0971-6

Expression, Purification, and Characterization of NADP+-Dependent Malic Enzyme from the Oleaginous Fungus Mortierella Alpina Jiayu Yang & Xinjie Hu & Huaiyuan Zhang & Haiqin Chen & M’balu. R. Kargbo & Jianxin Zhao & Yuanda Song & Yong Q. Chen & Hao Zhang & Wei Chen

Received: 26 January 2014 / Accepted: 16 May 2014 / Published online: 27 May 2014 # Springer Science+Business Media New York 2014

Abstract Malic enzymes are a class of oxidative decarboxylases that catalyze the oxidative decarboxylation of malate to pyruvate and carbon dioxide, with concomitant reduction of NAD(P)+ to NAD(P)H. The NADP+-dependent malic enzyme in oleaginous fungi plays a key role in fatty acid biosynthesis. In this study, the malic enzyme-encoding complementary DNA (cDNA) (malE1) from the oleaginous fungus Mortierella alpina was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant protein (MaME) was purified using Ni-NTA affinity chromatography. The purified enzyme used NADP+ as the cofactor. The Km values for + L-malate and NADP were 2.19±0.01 and 0.38±0.02 mM, respectively, while the Vmax values were 147±2 and 302±14 U/mg, respectively, at the optimal condition of pH 7.5 and 33 °C. MaME is active in the presence of Mn2+, Mg2+, Co2+, Ni2+, and low concentrations of Zn2+ rather than Ca2+, Cu2+, or high concentrations of Zn2+. Oxaloacetic acid and glyoxylate inhibited the MaME activity by competing with malate, and their Ki values were 0.08 and 0.6 mM, respectively. Keywords Malic enzyme . Mortierella alpina . Characterization

Jiayu Yang and Xinjie Hu contributed equally to this work.

J. Yang : X. Hu : H. Zhang : H. Chen : M. R. Kargbo : J. Zhao : Y. Song (*) : Y. Q. Chen : H. Zhang : W. Chen (*) State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122 Jiangsu Province, People’s Republic of China e-mail: [email protected] e-mail: [email protected] X. Hu : H. Chen : J. Zhao : Y. Q. Chen : H. Zhang : W. Chen Synergistic Innovation Center for Food Safety and Nutrition, Wuxi 214122 Jiangsu Province, People’s Republic of China X. Hu College of Food Science, Sichuan Agricultural University, Yaan 625014 Sichuan Province, People’s Republic of China

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Introduction Arachidonic acid (ARA, 20:4, n-6) plays an important role as a structural component of membrane phospholipids especially in the brain and the retina [1]. ARA has been widely used in medicine, cosmetics, food industry, and other fields [2]. Its traditional sources are egg yolks and animal livers, which are unacceptable for vegetarians. Alternatively, it can be produced by oleaginous fungi, for example, Mortierella alpina, which has been used industrially for ARA production for over 10 years. Under the condition of nitrogen starvation, this microorganism can accumulate lipids up to a level of 50 % (w/w, cell dry weight), 40 % of which is ARA. The biochemistry of lipid accumulation in M. alpina has been extensively studied [3–8], and malic enzyme (ME) has been found to be a major provider of nicotinamide adenine dinucleotide phosphate (NADPH) for lipid biosynthesis [9]. ME activity is highly consistent with the process and extent of lipid accumulation in oleaginous fungi M. alpina and Mucor circinelloides which is another commercially important oleaginous fungus. Inhibition of ME activity with sesamol leads to the diminution of lipid accumulation in M. circinelloides [10]. ME is hypothesized to control the extent of lipid accumulation in oleaginous fungi, and no other NADPH-generating enzyme shows an equal capacity of providing reducing power for lipid biosynthesis [9]. However, ME can simultaneously fulfill other roles and thus may exist in multiple isoforms encoded by different genes. At least seven isoforms (A–G) of ME have been identified in M. alpina. However, only isoform E, which is converted from isoform D, has been demonstrated to be associated with lipid accumulation [11]. A similar phenomenon was also observed in M. circinelloides, which possesses at least six ME isoforms (isoforms I–VI), and only isoform IV (converted from isoform III) is associated with lipid accumulation [12]. This finding has been confirmed by the overexpression of isoform III or D, respectively, in M. circinelloides which resulted in 2.5-fold increases of lipid accumulation [13]. However, a later study suggested that the 2.5-fold increase in lipid accumulation is due to the integrity of the leucine biosynthetic pathway comparing with leucine auxotrophic strains; this finding could indicate that ME is not the only bottleneck in lipid accumulation [14]. A recent study, however, showed that when the ME gene from M. circinelloides was cloned and overexpressed in the oleaginous yeast Rhodotorula glutinis, it led to a 2-fold increase in lipid accumulation [15]. In another oleaginous microorganism Rhodosporidium toruloides, it was found that the transcription of ME is downregulated, but the protein level of ME is significantly increased during the lipid accumulation phase. This finding suggests that the regulation of ME activity is complicated [16]. The biochemical mechanism of lipid accumulation and its relationship with the regulation of ME activity are far from clear. In this study, the complementary DNA (cDNA) of malE1 from M. alpina encoding the ME isoform D/E was cloned into the vector pET-28a (+) and expressed in Escherichia coli BL21 (DE3). The recombinant protein (MaME) was purified and characterized.

Materials and Methods Microorganisms, Plasmid, and Growth Conditions E. coli BL21 (DE3) was purchased from Novagen (Shanghai, China). The vector pET-28a (+) was purchased from Invitrogen (Shanghai, China). The T vector was purchased from Takara (Dalian, China). The malE1 from M. alpina ATCC32222 was sub-cloned to T vector in our laboratory. E. coli was cultivated on Luria-Bertani (LB) medium at 37 °C and shaken at 200 rpm.

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Construction of Expression Plasmid pET28a-malE1 The coding region of malE1 encoding ME isoform E was amplified from cDNA (clone constructed previously in our laboratory) by PCR with a forward primer (malE1-F 5′-CTAG CTAGCACTGTCAGCGAAAACACCACTC-3′) and a reverse primer (malE1-R 5′-CCCA AGCTTTTAGAGGTGAGGGGCAAAG-3′) including restrictive endonuclease sites NheI and HindIII, respectively. The PCR fragments were ligated into vector pET-28a (+). The resulting plasmids were confirmed by DNA sequencing (Sangon Inc, Shanghai, China). The expression plasmid pET28a-malE1 was transformed into E. coli BL21 through heat shock [17], and transformants were selected using an LB agar plate containing 50 μg/ml of kanamycin sulfate. Expression and Purification of Recombinant MaME Protein Transformants of E. coli BL21 (DE3) with plasmid pET28a-malE1 were inoculated in 50 ml of LB medium (with 50 μg/ml kanamycin sulfate) and cultivated at 37 °C. When the optical density (at 600 nm) reached 0.6, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the culture to reach a final concentration of 0.1 mM. The culture was kept at 20 °C with shaking for 12 h for the expression of the desired MaME. The induced cell pellets were harvested by centrifugation (4,800g for 8 min at 4 °C) and washed and resuspended in buffer A [containing 100 mM KH2PO4-KOH at pH 7.5, 20 % (w/v) glycerol, 1 mM benzamidine·HCl, and 1 mM DTT]. The proteins of the culture were extracted by ultrasonication on ice. The crude cell-free extracts were collected by centrifugation (16,100g for 20 min at 4 °C), and the supernatant was further purified by affinity chromatography with a Ni-NTA column. The column was washed with 30-ml washing buffer (containing 80 mM imidazole, 20-mM phosphate-buffered saline (PBS), and 500 mM NaCl), and the recombinant protein was eluted with eluting buffer (containing 200 mM imidazole, 20 mM PBS, and 500 mM NaCl). The whole purification process was carried out at 4 °C. The eluted protein was dialyzed in buffer A overnight at 4 °C. The purified MaME protein was stored at −80 °C in storage buffer with 50 % (v/v) glycerol for further studies. Protein Determination and MaME Activity Staining Protein concentration was measured by the method of Bradford with bovine serum albumin (BSA) as the standard. Protein samples were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), which was performed in 12 % (v/v) acrylamide gels according to the established procedure [17], and visualized with Coomassie Brilliant Blue R 250. The molecular weight of the recombinant MaME was estimated using broad-range molecular mass standards as marker (purchased from Fermentas). Native PAGE was performed using 10 % (v/v) acrylamide gels without adding SDS. Electrophoresis was run at a constant 60 V on ice. Gels were assayed for MaME activity by incubating in a pH 7.5 solution containing 100 mM KH2PO4/KOH, 20 mM L-malate, 0.26 mM NADP+, 70 μg phenazine methosulfate (PMS)/ml, 100 μM DTT, 3 mM MgCl2, and 0.5 mg/ml nitroblue tetrazolium (NBT) chloride at 30 °C. MaME Activity Assays ME specific activity was determined from the rate of NADPH formation at 340 nm [18] with a UV spectrophotometer at 30 °C. The standard reaction mixture contained 80 mM KH2PO4/

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KOH as a buffer (pH 7.5), 30 mM L-malate, 3 mM MgCl2, and 0.6 mM NADP+ in a final volume of 1 ml. The reaction was initiated by the addition of L-malate. One unit of activity was defined as the amount of enzyme required for the production of 1 μmol of NADPH per minute. All of the parameters were calculated by triplicate determinations. The effects of seven divalent metal ions on MaME activity were tested. MaME activity was measured in the presence of 0.5 and 5 mM of each divalent metal ion (Mn2+, Mg2+, Co2+, Ni2+, Zn2+, Ca2+, and Cu2+) or without adding any metal ions. The results were represented as percentages over control value, which were obtained by the standard ME assay system. The effect of adding 0.1 mM EDTA into the enzyme assay system was also tested. MaME activity was also measured in the presence of several metabolic intermediates: oxaloacetic acid (OAA), glyoxylate, ketoglutarate, succinate, pyruvate, D-glucose-6photosphate (D-G6P), fructose-6-photosphate (F6P), L-aspartate, L-glutamate, citrate, isocitrate, CoA, acetyl-CoA, propionyl-CoA, AMP, ADP, and ATP, while L-malate concentration was maintained at its Km value (2 mM). The results were presented as percentages of the activity value in the absence of any metabolite. For the metal ions and metabolite regulatory property assay, 100 mM Tris-HCl (pH 7.5) was used instead of a phosphate buffer.

Results Expression of Recombinant MaME The malE1 fragment amplified from malE1 sub-clone, which was constructed in our laboratory, was cloned into pET-28a (+) vector. Then, MaME was expressed as a His-tag fusion protein in E. coli BL21 (DE3) after being induced with 0.1 mM IPTG for 12 h at 20 °C. As shown in Fig. 1 (lane 2), the fusion protein with 606 amino acids had an approximately 64-kDa molecular weight, which was consistent with its theoretical value of 63.51 kDa. The purified fusion protein (lane 3) showed a single band with molecular weight around 64 kDa on SDS-

Fig. 1 SDS-PAGE of the crude extracts and purified MaME. Lane 1 crude extracts of E. coli BL21 (DE3)/pET28a-malE1 before being induced by IPTG, lane 2 crude extracts of E. coli BL21 (DE3)/pET28a-malE1 after being induced by IPTG, lane 3 the purified MaME through Ni-NTA affinity chromatography

kDa 66.2 45.0 35.0 25.0 18.4 6.4

M

1

2

3

64 kDa

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PAGE (Fig. 1). The Native PAGE also revealed a single band and showed high enzyme activity. The purity of the purified recombinant protein was estimated to be more than 95 %. Kinetic Analysis of MaME The optimal pH for the activity of recombinant MaME was about 7.5 in the presence of Mg2+ (Fig. 2a). The maximum activity was observed at 33 °C, and the enzyme was completely inactivated above 45 °C (Fig. 2b). The optimal activity of MaME was 147±2 U/mg at pH 7.5 and 33 °C. MaME activity assays suggested that only NADP+ could be used as the cofactor. In the kinetic assay system, the concentrations of L-malate and NADP+ varied in the ranges of 0.05– 25 and 0.01–0.6 mM, respectively. The Michaelis constants (Km values) and Vmax values of MaME for L-malate and NADP+ were estimated according to the Lineweaver-Burk plot (Table 1). The Km values of MaME (determined at pH 7.5) for L-malate and NADP+ were 2.19±0.01 and 0.38±0.02 mM, respectively. The Vmax values for L-malate and NADP+ were 147±2 and 302±14 U/mg, respectively. The Effect of Divalent Metal Ions on MaME Activity Divalent metal ions are essential for ME activity as activators. Effects of some divalent metal ions at both low concentration (0.5 mM) and high concentration (5 mM), as well as 0.1 mM EDTA, were tested on the activity of MaME (Fig. 3), using 3 mM Mg2+ as control. The activity of MaME was reduced to 11 % of the control without divalent metal ions, and it is completely abolished in the presence of 0.1 mM EDTA. Usually, Mg2+ serves as the preferred divalent metal; however, the most stimulating effect was obtained by adding Mn2+ to the assay system. The enzyme activity attained its plateau at 0.2 mM, and this activity was 1.4 times over control (not shown). MaME also showed a considerable activity with Co2+ and Ni2+ although it is lower than that of Mg2+. For Mg2+, Co2+, and Ni2+, the reaction prefers a high concentration (5 mM) to a low concentration (0.5 mM). Other divalent metal ions such as Ca2+ and Cu2+ exhibited a much less stimulating effect on MaME. The minimal activity was obtained in the presence of 5 mM Cu2+, which is less than 1 % that of the control. To clarify the inhibition mechanism of Ca2+ and Cu2+, the MaME activity was assayed in various concentrations of Cu2 or Ca2+ in the presence of 1 mM Mg2+. Figure 4a shows that the MaME activity decreased significantly while increasing concentrations of Cu2+ in the presence of 1 mM Mg2+. MaME activity diminished to near zero with the

Fig. 2 The effect of temperature (a) and pH value (b) on MaME activity

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Table 1 Km and Vmax values of MaME Km (mM)

Vmax (U/mg)

L-Malate

2.19±0.01

147±2

+b

0.38±0.02

Substrate (mM) a

NADP

302±14

a

+

The Km and Vmax values for L-malate were determined at 0.6 mM NADP

b

The Km and Vmax values for NADP+ were determined at 30 mM L-malate

addition of 5 mM Cu2+. Figure 4b shows that Ca2+ could also inhibit MaME activity in the presence of Mg2+, but the activity decreased slowly while increasing concentrations of Ca2+. With the addition of 5 mM Ca2+, the MaME activity retained more than 50 % that of the control. Interestingly, compared with the assay without the addition of metals, MaME was slightly activated at low concentrations of Zn2+ but was inhibited at high concentrations. Furthermore, the MaME activity decreased from 58 to 10 % that of the control when the concentration of Zn2+ increased from 0.2 to 10 mM (not shown). The Effect of Metabolites on MaME Activity Several compounds were tested as possible effectors of MaME activity while the L-malate concentration was maintained at its Km value (2 mM). The results (Fig. 5) suggest that MaME activity was significantly inhibited by OAA and glyoxylate but not influenced by other metabolites (α-ketoglutarate, D-G6P, F6P, pyruvate, citrate, isocitrate, succinate, L-aspartate, and L-glutamate at 2 mM and CoA, acetyl-CoA, propionyl-CoA, AMP, ADP, or ATP at 10 and 100 μM). In the presence of 2 mM OAA and glyoxylate, MaME activity was reduced to 23

0.5mM

120

5mM

80

60

40

˅

ME activity (% of control)

100

Zn2+

Ca2+

Cu2+

Ni2+

Co2+

Mg2+

Mn2+

EDTA

Control

0

None

20

Divalent metal ion

Fig. 3 Effect of divalent metal ions on MaME activity. The control was the standard malic enzyme assay system with 3 mM Mg2+. The results are represented as percentages of the control value (153±1 U/mg)

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Fig. 4 Effect of various concentrations of Cu2+ (a) and Ca2+ (b) on MaME activity in the presence of 1 mM Mg2+. The results are represented as percentages of the control value (114±1 U/mg). The control was assayed in the presence of 1 mM Mg2+ without any other ions

and 60 % of the control, respectively. Both OAA and glyoxylate showed competitive inhibition with L-malate, and the Ki values were 0.08 and 0.6 mM, respectively.

Discussion MEs are common in nature and are a class of oxidative decarboxylases that catalyze the oxidative decarboxylation of L-malate to pyruvate and carbon dioxide through the concomitant reduction of NAD(P)+ to NAD(P)H. ME plays an important role in the fatty acid biosynthesis and desaturation in M. alpina, but its biochemical characteristics are still not clear. The recombinant enzyme MaME (expressed in E. coli) uses NADP+ as a cofactor. In this study, the Km value for NADP+ was 0.38±0.02 mM, which indicates that it is possible to use NADP+ at low concentrations in lipid synthesis. Mg2+ and Mn2+ are commonly used as cofactors for MEs in many other microorganisms [19–23]. Mg2+ activated MaME significantly, and Mn2+ had a much greater activation effect. Both Mn2+ and Mg2+ are octahedrally coordinated by the enzyme with six oxygen atoms, of which three are from amino acid

Fig. 5 Regulatory properties of recombinant MaME. MaME activity was measured at pH 7.5 in the presence of 10 and 100 μM of each effector indicated on the y axes. The L-malate concentration was kept at approximately the Km value (2 mM). The results are presented as percentages of the control value (118±1 U/mg), which was assayed in the presence of 2 mM L-malate without adding any metabolites

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residues, two are from carboxylate groups in the substrate, and one is from a water molecule in the active center [24]. The similar activity obtained with Ni2+ and Co2+ indicates that these ions might have the same mechanism in binding to MaME. The MaME activity was inhibited significantly by Cu2+ or Ca2+ in the presence of Mg2+ in a dose-dependent manner, suggesting that Ca2+ and Cu2+ compete with Mg2+ for the binding site. The MaME activity detected in the presence of Cu2+ was much lower than that with Ca2+ at the same concentration, indicating that Cu2+ has a stronger competitive ability than Ca2+. In Aspergillus niger [25], adding Cu2+ at the beginning of fermentation is an effective strategy to reduce total lipid and increase citric acid accumulation by abolishing ME activity. The inhibition of ME activity by Zn2+ is different from Cu2+ and Ca2+, as it only occurs at high Zn2+ concentrations (compared to the assay without the addition of metals, Cu2+ and Ca2+, whether at high or low concentration, inhibited ME activity). The same phenomenon had been reported with ME from pigeon liver [26] and Rhizobium meliloti [27]. The coordination number between essential ions and enzyme is six, but in Cu2+ and Zn2+, it is just four. In the metalloenzymes of Cu2+ and Zn2+, metal coordination geometry is usually tetrahedral or distorted tetrahedral, which leads to deactivation of the ME. The catalysis by NAD(P)+-MEs generally proceeds in two steps. The first (step a) involves the dehydrogenation of L-malate to OAA, with a product of NADPH, and then, step b involves the decarboxylation of OAA to pyruvate [28, 29]. The theory of L-malate transformation to pyruvate provides an explanation for the inhibition of OAA on MaME. OAA can feedback inhibit the reaction of step a. As a result, the output of NADPH for lipid is reduced. Furthermore, OAA has a similar structure to L-malate that may accommodate the active site and thus compete with L-malate, inhibiting the enzyme activity. This mechanism for the inhibition of OAA may also apply with glyoxylate. This two-carbon carboxylic acid, which is an analogue to L-malate and OAA, contains a keto group which is considered crucial for effective inhibition of ME. The extent of the inhibition depends on the size of the analogues [28]. Glyoxylate is obviously smaller than L-malate, and this may be the reason why glyoxylate presents less inhibition than OAA. α-Ketoglutarate is also an analogue to L-malate and has a keto group in position 2. However, α-ketoglutarate showed little inhibitive effect on MaME. The extra carbon in α-ketoglutarate may make it unable to bind the active site. As a product of the MaME catalyzing reaction, pyruvate may be expected to have a feedback inhibition effect on the enzyme activity. However, in this study, the velocity loss in step b caused by adding excess pyruvate had little influence on the complete reaction catalyzed by MaME. Therefore, it is beneficial for lipid accumulation. The result is similar with other NAD(P)+-dependent MEs [30, 31]. Acetyl-CoA also has a significant inhibitive effect on NADP+-ME activity in Rhodopseudomonas palustris [30] and R. meliloti [27] but not on NADP+-ME. In this study, neither 10 nor 100 μM of acetyl-CoA had any effect on MaME activity. Succinate is an activator of human NAD(P)+-ME activity [32], and its stimulation is implemented by entering the allosteric site. However, succinate does not show any stimulation on MaME activity. Aspartate, D-G6P, and glutamate show significant stimulation on NAD(P)+ME of E. coli DH5α [31], but they have no effect on MaME activity. In conclusion, we expressed the MaME gene from the oleaginous fungus M. alpina ATCC32222 in E. coli, purified the recombinant protein, and characterized the biochemical property of MaME. Our results provided useful information for further exploring the regulation of ME in M. alpina. Acknowledgments This work was supported by the National Natural Science Foundation of China (Nos. 31271812, 21276108, and 31171636), the National High Technology Research and Development Program of China (2012AA022105C and 2011AA100905), the National Science Fund for Distinguished Young Scholars

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(31125021), the National Basic Research Program 973 of China (2012CB720802), and the Fundamental Research Funds for the Central Universities (No. JUSRP51320B).

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