Cloning, sequence analysis, and hyperexpression of the genes encoding phosphotransacetylase and acetate kinase from Methanosarcina thermophila

Abstract

The genes for the acetate-activating enzymes, acetate kinase and phosphotransacetylase (ack and pta), from Methanosarcina thermophila TM-1 were cloned and sequenced. Both genes are present in only one copy per genome, with the pta gene adjacent to and upstream of the ack gene. Consensus archaeal promoter sequences are found upstream of the pta coding region. The pta and ack genes encode predicted polypeptides with molecular masses of 35,198 and 44,482 Da, respectively. A hydropathy plot of the deduced phosphotransacetylase sequence indicates that it is a hydrophobic polypeptide; however, no membrane-spanning domains are evident. Comparison of the amino acid sequences deduced from the M. thermophila and Escherichia coli ack genes indicate similar subunit molecular weights and 44% identity (60% similarity). The comparison also revealed the presence of several conserved arginine, cysteine, and glutamic acid residues. Arginine, cysteine, and glutamic acid residues have previously been implicated at or near the active site of the E. coli acetate kinase. The pta and ack genes were hyperexpressed in E. coli, and the overproduced enzymes were purified to homogeneity with specific activities higher than those of the enzymes previously purified from M. thermophila. The overproduced phosphotransacetylase and acetate kinase migrated at molecular masses of 37,000 and 42,000 Da, respectively. The activity of the acetate kinase is optimal at 65°C and is protected from thermal inactivation by ATP. Diethylpyrocarbonate and phenylglyoxal inhibited acetate kinase activity in a manner consistent with the presence of histidine and arginine residues at or near the active site; however, the thiol-directed reagents 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide were ineffective.The metabolism of acetate, whether through the production or consumption of the principal intermediate acetyl coenzyme A (acetyl-CoA), is of major importance in the physiology of both procaryotes (Bacteria and Archaea domains) and eucaryotes (Eucarya domain). Acetate is an end product in the energy-yielding metabolism of most facultatively and strictly anaerobic microbes. Phosphotransacetylase (EC 2.3.1.8) and acetate kinase (EC 2.7.2.1) are synthesized by these anaerobes to catalyze the conversion of acetyl-CoA and ADP to acetate and ATP,

New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes for Methanosarcina species

“…The observation that many Methanosarcina genes utilize start codons other than ATG raises potential problems in com-paring the results obtained using translational reporter genes fusions. For example, mcrB uses a GTG start site, while frhA (encoding a hydrogenase subunit) and pta (encoding phosphotransacetylase) use TTG start sites (Bokranz and Klein 1987, Latimer and Ferry 1993, Vaupel and Thauer 1998. At least one gene, the repA gene of the pC2A plasmid, is predicted to utilize a CTG translation start (Metcalf et al 1997).…”

Structural and Kinetic Analyses of Arginine Residues in the Active Site of the Acetate Kinase from Methanosarcina thermophila

“…Access to the crystal structure (7) and production of the M. thermophila acetate kinase in E. coli (17) have allowed experimental approaches not previously employed to investigate the catalytic mechanism of this enzyme. The structure of the homodimeric acetate kinase co-crystallized with ATP (the ATP-AK structure) reveals ADP in a cleft with contacts that are conserved in the nucleotide binding sites of other ASKHA family members, which identifies the active site of the M. thermophila acetate kinase.…”

Crystallization of acetate kinase from Methanosarcina thermophila and prediction of its fold

“…Defining the relationship of monomers within the asymmetric unit: The content of the asymmetric unit of the acetate kinase crystals can be estimated from the unit cell dimensions of the crystals and the molecular weight of the protein calculated from its DNA sequence (44 kDa; Latimer & Ferry, 1993), assuming typical protein density in the crystals (K,, = 2.5 A3/Da) (Matthews, 1968).…”

“…Crystallization: Homogeneous acetate kinase was isolated as described previously (Aceti & Ferry, 1988;Latimer & Ferry, 1993). In a typical crystallization experiment, a 10 pL hanging drop containing 4.9 p g purified acetate kinase in a solution of 0.75 mM ATP, 0.75 mM MgC12, 315 mM (NH4)>SO4, 0.8 mM dithiothreitol, 25 mM NaHEPES (pH 7.5) is equilibrated against a reservoir of 1.7 M (NH4)2S04 at room temperature.…”

“…The cloning and expression of acetate kinase from Methanosarcina thermophila (Aceti & Ferry, 1988; Latimer & Ferry, 1993), an acetate-utilizing methane-producing anaerobe from the Archaea domain, has inaugurated a new era in the study of acetate kinase and afforded an opportunity to reexamine mechanistic issues that were incompletely resolved in studies on acetate kinase from E. coli. The amino acid sequences of acetate kinase from the two organisms are 4 4 % identical, so the two enzymes are likely to use the same catalytic mechanism.…”