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 (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.

FIG 1

Phylogeny of the Pta^I and Pta^II family. The phylogenetic tree was constructed based on the sequences of the Pta catalytic domains. Pta^I sequences are shown in black, Pta^IIa sequences are shown in blue, Pta^IIb sequences are shown in purple, Pta^IIc sequences are shown in green, and Pta^IId sequences are shown in pink. The scale bar indicates the expected number of amino acid replacements per site. Only bootstrap values of 75% or higher are shown.

FIG 2

Subdomain structures of the Pta^I and Pta^II classes. Pta^I enzymes have only a catalytic domain; Pta^II enzymes have a catalytic domain and an N-terminal domain. Four subclasses of Pta^II enzymes have been identified based on the presence or absence of the P-loop and DRTGG subdomains, as shown. The domains are not drawn to scale.

FIG 3

Regulation of PrPta^II1 by allosteric effectors. PrPta^II1 activity in the acetyl-CoA-forming direction was monitored in the absence and presence of allosteric effector molecules. All data were normalized to the control, which represents the enzymatic activity observed in the absence of an effector molecule. (A) The results are displayed as percent activity in the presence of 100 μM and 750 μM NAD⁺, NADH, NADP, and NADPH. (B) The results are displayed as percent activity in the presence of 100 μM and 750 μM ATP, ADP, and AMP and 100 μM and 1,000 μM PEP and pyruvate. The data are expressed as means ± SD.

FIG 4

Effects of NADP on CoA and acetyl phosphate utilization by PrPta^II1. (A) Enzymatic activity was measured in the acetyl-CoA-forming direction in the presence of 5 mM acetyl phosphate with various concentrations of NADP using the thioester-forming assay. ●, 0 μM NADP; ■, 67.1 μM NADP; ◆, 134.2 μM NADP. (B) Enzymatic activity in the acetyl-CoA-forming direction was measured in the presence of 4 mM CoA with various concentrations of NADP using the thioester-forming assay. ●, 0 μM NADP; ■; 67.1 μM NADP; ◆, 134.2 μM NADP.

FIG 5

AMP and pyruvate activate and inhibit SePta^II in the acetyl-CoA-forming direction. SePta^II activity in the acetyl-CoA-forming direction was determined in the presence of increasing concentrations of AMP (■) and pyruvate (●). All data were normalized to the control, which represents the enzymatic activity observed in the absence of an effector molecule. The data are expressed as means ± SD.

FIG 6

Alteration of the Gly³⁰⁰ residue in PrPta^II1 changes the effect of pyruvate. Enzyme activity was determined in the presence of increasing amounts of pyruvate in the acetyl-CoA-forming direction for PrPta1 (●) and PrPta1^G300D (■). The data are expressed as means ± SD.