Mol Gen Genet (1992) 231:332-336

MGG

© Springer-Verlag 1992

Short communication

Cloning and nucleotide sequence of the Escherichia coil K-12 ppsA gene, encoding PEP synthase M. Niersbach 1, F. Kreuzaler 1, R.H. Geerse 2, P.W. Postma

2,

and H.J. Hirseh 1

1 Institut ffir BiologieI, Rheinisch-WestffilischeTechnischeHochschule,WorringerWeg,W-5100 Aachen, FRG 2 E.C. Slater Institutefor BiochemicalResearch, Universityof Amsterdam,Plantage Muidergracht 12, NL-1018 TV Amsterdam, The Netherlands Received July 12, 1991

Summary. We have cloned and sequenced the Escherichia coli K-12 ppsA gene. The ppsA gene codes for PEP synthase, which converts pyruvate into phosphoenolpyruvate (PEP), an essential step in gluconeogenesis when pyruvate or lactate are used as a carbon source. The open reading frame consists of 792 amino acids and shows homology with other phosphohistidinecontaining enzymes that catalyze the conversion between pyruvate and PEP. These enzymes include pyruvate, orthophosphate dikinases from plants and Bacteroides symbiosus and Enzyme I of the bacterial PEP:carbohydrate phosphotransferase system. Key words" PEP synthase -ppsA gene - Escherichia coli K-12

In enteric bacteria such as Escherichia coli and Salmonella typhimurium the starting compound for gluconeogenesis, phosphoenolpyruvate (PEP), can be generated by two different reactions. First, oxaloacetate can be converted to PEP by PEP carboxykinase. However, if lactate or pyruvate is the carbon source, the only way to produce PEP is via the enzyme PEP synthase (ATP:pyruvate, water phosphotransferase, EC 2.7.9.2) which catalyzes the following reaction: pyruvate + ATP ~ P E P + AMP+Pi. PEP synthase was first described in E. coli by Cooper and Kornberg (1965). These authors purified the enzyme and showed also that the enzyme was phosphorylated during the reaction (Cooper and Kornberg 1967). It was subsequently shown by Narindrasorasak and Bridger (1977) that a histidyl residue was the site of phosphorylation. The essential role of PEP synthase in growth on

Offprint requests to ."H.J. Hirsch

C3-compounds was demonstrated by the isolation of E. coli ppsA mutants, which were unable to grow on pyruvate or lactate although growth on C4-carboxylic acids was unimpaired (Brice and Kornberg 1967). The ppsA gene is localized at 37 rain on the E. coli genetic map. In S. typhimurium a second locus affecting PEP synthase activity, ppsB, was found at 3 min on the map (Calvo et al. 1971; Geerse et al. 1986). ppsB mutants displayed the same phenotype as the E. coli ppsA mutants, i.e. no growth on pyruvate and lactate. It was shown later that this locus, renamed fruR (Postma and Lengeler 1985), encodes the FruR protein, which has a dual function. FruR is a repressor of thefruFKA operon which codes for the enzymes of the fructose phosphotransferase system (Geerse et al. 1989a; Jahreis etal. 1991). In addition, FruR is an activator ofppsA transcription (Geerse et al. 1989b). Interestingly, enzymes of the phosphotransferase system (PTS) are, with one exception, phosphohistidine proteins like PEP synthase (Postma and Lengeler 1985). In a number of microorganisms as well as in plants, synthesis of PEP from pyruvate is catalyzed by pyruvate, orthophosphate dikinase (ATP:pyruvate, orthophosphate phosphotransferase, EC 2.7.9.1; PPDK) (Reeves et al. 1968; Evans and Wood 1968). The plant PPDK catalyzes a reaction analogous to PEP synthase, i.e. the conversion of pyruvate, ATP and Pi into PEP, AMP and PPI- In bacteria like Bacteroides syrnbiosus, PPDK functions as a pyruvate kinase, i.e. it catalyzes the conversion of PEP and ADP into pyruvate and ATP. It has been shown that in PPDK a phosphohistidine is an intermediate in the reaction. A similar mechanism has been proposed for both plant and bacterial dikinases (Wang et al. 1988). Genes encoding PPDK from maize (Hudspeth et al. 1986), Flaveria trinervia (Rosche and Westhoff 1990) and B. symbiosus (Pocalyko et al. 1990) have been cloned and sequenced. It seemed of interest to compare the amino acid sequence of PEP synthase

333 with that of PPDK in view of the common mechanism that has been proposed for the two types of enzymes. Cloning of the E. coli K-12 ppsA gene has been reported (Geerse et al. 1989b). In this paper, we report the nucleotide sequence of the E. colippsA gene, determination of its N-terminal amino acid sequence by protein sequencing and the comparison of the deduced amino acid sequence with that of other phosphohistidine-containing enzymes, i.e. PPDK from plants and B. symbiosus and Enzyme I of the phosphotransferase system PTS.

Results and discussion

N-terminal amino acid sequence of PEP synthase To establish the N-terminal amino acid sequence of PEP synthase, the enzyme was overproduced in E. coli containing the ppsA gene cloned in pUCI8 (p29, see below). Preparation of the crude extract, protamine precipitation and dialysis were according to Cooper and Kornberg (1969). Ammonium sulphate precipitation and DEAE-cellulose chromatography were according to Berman and Cohn (1970). PEP synthase was further purified by isoelectric focussing with Serva ampholines (pH 3-7) in 5% acrylamide rod gels. The active band (pH 4.8) was cut out and further purified by SDS gel electrophoresis. One prominent band with an apparent molecular weight of approximately 80000 was obtained. Proteins were transferred to Immobilon-P membrane (PVDV, Millipore). PEP synthase was cut out and the N-terminal amino acid sequence was determined by Dr. U. Jahnke, Kernforschungsanlage, Jfilich on an Applied Biosystems 477A protein sequencer. The resulting sequence was SNNGS-PLVL-YNQLGMN-V (residues denoted by dashes could not be identified unequivocally).

Nucleotide sequence of the ppsA gene Plasmid p29 contained a 6.1 kb insert that complemented E. coli ppsA strains (Fig. 1). The positions of the restriction sites corresponded (with one exception) to those found at 37 min on the physical map of the E. coli chromosome (Kohara et al. 1987), the site of the ppsA locus. Deletion of a 2.8 kb BamHI-XbaI fragment from p29 resulted in p29-2 (Fig. i). Plasmid p29-2 could still complement a ppsA mutant at 30°C but complementation at 37° C was strongly reduced. When a further 49 bp were deleted (digestion of p29 with EcoRV and XbaI, yielding p29-1), complementation for growth of a ppsA strain on D,L-lactate was strongly reduced. The nucleotide sequence of the chromosomal insert of p29-2, containing the ppsA gene and flanking regions, was determined (Fig. 2). The amino acid sequence deduced from the single open reading frame found is also shown in Fig. 2. The N-terminal amino acid sequence as determined by protein sequencing corresponded to that of the translated nucleotide sequence between positions 658 and 717. A methionine codon precedes the first serine codon. Seven nucleotides upstream, a poten-

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Fig. 1. Restriction map of the insert in p29 and location of the ppsA genes. DNA was isolated from E. coli DH1 (Maniatis et al. 1982), partially digested with Sau3AI, cloned into the cosmid pHC79 BamHI site, and packaged in vitro according to Hohn (1979). Clones complementing HG4 (ppsA pek-i tyrA his pyrD lac edd-i thi rpsL; Goldieand Sanwa11980) were partiallydigested with Sau3AI and fragments(> 1 kb) were subclonedin the BamHI site of pUCI 8. Plasmidp29 contained a 6.1 kb insert that complemented the ppsA mutation of E. coli 1120 (J)psA,Hansen and Juni 1979). Plasmidp29-2 was derived from p29 by deletion of a 2.8 kb BamHI-XbaI fragment. B4 and B7 are derivatives of p29-2, constructed with the Erase-a-Base procedure (Promega). Restriction enzymes between brackets are positioned in the multiple cloning site. Restriction enzymes marked with an asterisk can be found on the physicalmap (Kohara et al. 1987).

tial consensus AGGA sequence for the ribosomal binding site can be found. The ppsA gene encodes a protein of 792 amino acids with a calculated molecular weight of 87430, close to that determined from SDS polyacrylamide gel electrophoresis (Geerse etal. 1989b), but larger than the 77000 as determined with the purified protein (Narindrasorasak and Bridger 1977). No typical promoter consensus sequence could be detected. Interestingly, the region of approximately 650 bp preceding the start codon at position 655, is required for PEP synthase synthesis. Removal of 49 bp from p292 at the 5' end resulted in a drastic reduction of growth of HG4/p29-1 on lactate. Using the Erase-a-Base procedure (Promega), several C-terminal deletions were constructed. Deletion B4, which eliminates 33 nucleotides at the 3' end of the ppsA gene (Fig. 1) could still complement ppsA strains but a further deletion, eliminating the sequence distal from position 2851 (B7, Fig. 1) resulted in complete absence of PEP synthase activity. At the 3' end of the ppsA gene, a hairpin loop can be constructed, beginning at position 3041 and ending at 3065, with a stem of 8 bp and a loop of 9 bp.

Comparison with other phosphohistidine-containingenzymes The translated ppsA sequence was compared with all proteins present in the PIR and Swiss-Prot databases. The following enzymes showed homologies with PEP synthase: PPDK from Flavaria trinervia, maize and B. symbiosus and Enzyme I of the phosphoenolpyruvate: carbohydrate phosphotransferase system. Similarity between the dikinases and Enzyme I was noted previously by Pocalyko et al. (1990). These enzymes have in common that each reacts with PEP and is phosphorylated on a histidine residue. Fig. 3 shows the alignment of regions of these five proteins that are highly conserved.

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335 Acknowledgements. We thank Dr. A.H. Goldie, Dr. E. Juni and

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Dr. J. Collins for their generous gifts of strains and plasmids, Dr. U. Jahnke for amino acid sequencing and P. Subai for excellent technical assistance. During the preparation of this manuscript, the nucleotide sequence of ppsA from E. coli was submitted to GenBank (Accession number M69116) by DL Holzschu and coworkers. The sequences differ in 20 places (mainly outside the coding region) but analysis of our gels failed to provide any explanation for the discrepancies.

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Fig. 3. Comparison of stretches of the deduced amino acid sequence of PEP synthase with corresponding regions of pyruvate, phosphate dikinases and Enzyme I. Amino acid residues have been shaded if they occur at least twice in equivalent positions. PPDKM, PPDK from maize; PPDKF, PPDK from Flaveria trinervia; PPDKB, PPDK from B. symbiosus; PPSA, E. co6 PEP synthase; PTSI, E. cog Enzyme I

The percentage identity outside these regions is lower and the overall percentage identity between P E P synthase a n d P P D K f r o m B. symbiosus, P P D K f r o m Flavaria trinervia and E n z y m e I is 21%, 19% and 19%, respectively. As can be seen, the region a r o u n d the p h o s p h o r y lated histidine (His-455 in the B. symbiosus P P D K , equivalent to His-421 in P E P synthase) is strongly conserved in all five enzymes. It has still be shown that His-421 is indeed the site o f p h o s p h o r y l a t i o n in P E P synthase, however. As noted by P o c a l y k o et al. (1990), a threonine residue two residues u p s t r e a m o f the histidine is conserved in the dikinases. In the plant P P D K this threonine residue can be p h o s p h o r y l a t e d and serves as a site o f regulation. N o i n f o r m a t i o n exists a b o u t the possible role o f the threonine residue in the bacterial P P D K . Interestingly, this residue is also conserved in b o t h P E P synthase and E n z y m e I. It has been suggested that P P D K can be divided into d o m a i n s which e n c o m p a s s an A T P binding site (N-terminal), the histidine residue which is p h o s p h o r y l a t e d and a P E P / p y r u v a t e binding site (C-terminal), respectively. E n z y m e I which reacts only with P E P but n o t with A T P should lack the A T P - b i n d i n g domain. Indeed, alignment suggested that the first d o m a i n is lacking in E n z y m e I (Fig. 3 A ; P o c a l y k o et al. 1990). P E P synthase, on the other hand, catalyzes a reaction similar to P P D K and is expected to contain all three domains. A l i g n m e n t shows that there exist four segments in P E P synthase (Fig. 3 B - E ) that show m u c h a higher degree o f identity with P P D K and E n z y m e I than the regions in between.

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Rosche E, Westhoff P (1990) Primary structure of pyruvate, orthophosphate dikinase in the dicotyledonous C4 plant Flavaria trinervia. FEBS Lett 273:116-121 Wang HC, Ciskanik L, Dunaway-Mariano D, von der Saal W, Villafranca JJ (1988) Investigations of the partial reactions catalyzed by pyruvate phosphate dikinase. Biochemistry 27:625 633

C o m m u n i c a t e d b y J.W. Lengeler