Biochemical and kinetic characterization of the eukaryotic phosphotransacetylase class iia enzyme from phytophthora ramorum

Abstract

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 (PtaI) or presence (PtaII) of an N-terminal regulatory domain. PtaI has been fairly well studied in bacteria and one genus of archaea; however, only the Escherichia coli and Salmonella enterica PtaII 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 PrPtaII1 and PrPtaII2, both belong to class II. PrPtaII1 displayed positive cooperativity for both acetyl phosphate and CoA and is allosterically regulated. We compared the effects of different metabolites on PrPtaII1 and the S. enterica PtaII 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 PtaII 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.