Biochemical and Kinetic Characterization of the Eukaryotic Phosphotransacetylase Class IIa Enzyme from Phytophthora ramorum.

Phosphotransacetylase (Pta), a key enzyme in bacterial metabolism, catalyzes the reversible transfer of an acetyl group from acetyl phosphate to coenzyme A (CoA) to produce acetyl-CoA and Pi. Two classes of Pta have been identified based on the absence (Pta(I)) or presence (Pta(II)) of an N-terminal regulatory domain. Pta(I) has been fairly well studied in bacteria and one genus of archaea; however, only the Escherichia coli and Salmonella enterica Pta(II) enzymes have been biochemically characterized, and they are allosterically regulated. Here, we describe the first biochemical and kinetic characterization of a eukaryotic Pta from the oomycete Phytophthora ramorum. The two Ptas from P. ramorum, designated PrPta(II)1 and PrPta(II)2, both belong to class II. PrPta(II)1 displayed positive cooperativity for both acetyl phosphate and CoA and is allosterically regulated. We compared the effects of different metabolites on PrPta(II)1 and the S. enterica Pta(II) and found that, although the N-terminal regulatory domains share only 19% identity, both enzymes are inhibited by ATP, NADP, NADH, phosphoenolpyruvate (PEP), and pyruvate in the acetyl-CoA/Pi-forming direction but are differentially regulated by AMP. Phylogenetic analysis of bacterial, archaeal, and eukaryotic sequences identified four subtypes of Pta(II) based on the presence or absence of the P-loop and DRTGG subdomains within the N-terminal regulatory domain. Although the E. coli, S. enterica, and P. ramorum enzymes all belong to the IIa subclass, our kinetic analysis has indicated that enzymes within a subclass can still display differences in their allosteric regulation.

Identification of cysteine and arginine residues essential for the phosphotransacetylase from Methanosarcina thermophila.

Phosphotransacetylase catalyzes the following reaction: CoASH + CH3CO2PO3(2-) <==> CH3COSCoA + HPO4(2-) (where CoA is coenzyme A). Based on biochemical characterization of the enzyme from the obligate anaerobe Clostridium kluyveri, a ternary mechanism was proposed in which an unspecified cysteine abstracts a proton from CoASH forming a nucleophilic thiolate anion which attacks acetyl phosphate (J. Henkin and R. H. Abeles, Biochemistry 15:3472-3479, 1976). Heterologous production in Escherichia coli of the phosphotransacetylase from Methanosarcina thermophila, an obligately anaerobic methanoarchaeon, allowed site-specific replacements to identify essential residues. All four cysteines present in the sequence were individually replaced with alanine, and the kinetic constants of the altered enzymes were determined. The results indicated that only C159 is essential for activity; however, replacement with serine resulted in a fully active enzyme. Activity of the unaltered phosphotransacetylase was sensitive to N-ethylmaleimide. Inhibition kinetics of altered enzymes indicated that this sensitivity resulted from modification of C312, which is at the active site but itself is nonessential for catalysis. Five arginines were individually replaced with glutamine. Kinetic analysis of the altered enzymes identified R310 as essential for activity. Of the four nonessential for activity, R87 and R133 appear to be involved in binding CoA.