Characterization and Kinetics of Phosphopantothenoylcysteine Synthetase from Enterococcus faecalis

Phosphopantetheine ((R)-3-hydroxy-4-(3-(2-mercaptoethylamino)-3-oxopropylamino)-2,2-dimethyl-4-oxobutyl dihydrogen phosphate) is an fundamental feature in many biological acyl transfer reactions (1–3). The molecule is found imbedded within coenzyme A (CoA), as well as on a post-translationally modified, conserved serine of acyl carrier protein (ACP) (1). Both CoA and ACP play essential roles in activating various acyl groups for enzyme catalyzed transfer in several reactions associated with intermediary metabolism and cell membrane assembly in living organisms (4). The critical nature of phosphopantetheine-containing molecules to the integrity and viability of cells makes the biosynthetic pathway leading to the production of these compounds an intriguing target for antimicrobial development (5, 6). While it has been known for decades that CoA is biosynthesized from nucleotide triphosphate (NTP), d-pantothenate, and l-cysteine, the genes involved have only been identified in recent years (7–9). In eukaryotic systems that have been characterized, the phosphopantetheine portion of CoA is synthesized from pantothenate via three chemical reactions catalyzed by three polypeptides (Scheme 1) (10). First, pantothenate is phosphorylated by pantothenate kinase (PanK; EC 2.7.1.33; coaA), using ATP as the phosphate donor, to yield phosphopantothenate (PPA). This is followed by amide bond formation between the carboxyl group of PPA and the amino group of l-cysteine catalyzed by an ATP-dependent phosphopantothenoylcysteine synthetase (PPCS; EC 6.3.2.5; coaB). Subsequent decarboxylation of the carboxylic acid moiety on the ligated cysteine of phosphopantothenoylcysteine by phosphopantothenoylcysteine decarboxylase (PPCDC; EC 4.1.1.36; coaC) gives 4’-phosphopantetheine. In bacterial systems elucidated so far, the second and third chemical steps – the ligation and decarboxylation of cysteine by PPCS and PPCDC – are catalyzed by a single bifunctional polypeptide (9, 11). Furthermore, CTP, rather than ATP, is required by the bifunctional PPCS (7, 9). Scheme 1 PPCS from few species have been characterized. The proposed mechanism of PPCS involves the formation of an acyl cytidylate intermediate, 4’-phosphopantothenoyl cytidylate, between the carboxylate moiety of d-pantothenate and the α-phosphate of CTP, with the concomitant release of pyrophosphate. Formation of the product occurs by transfer of the nucleotide-activated acyl group to the amino group of l-cysteine, yielding phosphopantothenoylcysteine and CMP (Scheme 2). This proposed mechanism is supported by structural studies on the E. coli N210D PPCS domain, where the acyl cytidylate intermediate was observed in the active site when the crystal of the mutated PPCS was soaked with CTP and PPA (12). However, there has been no complete study on the kinetic mechanism of PPCS. In this paper, we overexpress, purify, and characterize PPCS from Enterococcus faecalis, which unlike most other bacterial PPCS, is encoded as a monofunctional protein and has similarly distant homology from both the monofunctional human PPCS protein and bifunctional bacterial PPCS domains (5, 10). In this study we use initial velocity and product competition experiments to elucidate the kinetic mechanism of PPCS from E. faecalis, and report for the first time the chemical mechanism and nucleotide specificity of a monofunctional bacterial PPCS. Scheme 2