Selectivity of the yersiniabactin synthetase adenylation domain in the two-step process of amino acid activation and transfer to a hole-carrier protein domain

The adenylation (A) domain of the Yersinia pestis nonribosomal peptide synt hetase that biosynthesizes the siderophore yersiniabactin (Ybt) activates t hree molecules of L-cysteine and covalently aminoacylates the phosphopantet heinyl (P-pant) thiols on three peptidyl carrier protein (PCP) domains embe dded in the two synthetase subunits, two in cis (PCP1, PCP2) in subunit HMW P2 and one in trans (PCP3) in subunit HMWP1. This two-step process of activ ation and loading by the A domain is analogous to the operation of the amin oacyl-tRNA synthetases in ribosomal peptide synthesis. Adenylation domain s pecificity for the first step of reversible aminoacyl adenylate formation w as assessed with the amino acid-dependent [P-32]-PPi-ATP exchange assay to show that S-2-aminobutyrate and beta-chloro-L-alanine were alternate substr ates. The second step of A domain catalysis, capture of the bound aminoacyl adenylate by the P-pant-SH of the PCP domains, was assayed both by catalyt ic release of PPi and by covalent aminoacylation of radiolabeled substrates on either the PCP1 fragment of HMWP2 or the PCP3-thioesterase double domai n fragment of HMWP1. There was little selectivity for capture of each of th e three adenylates by PCP3 in the second step, arguing against any hydrolyt ic proofreading of incorrect substrates by the A domain. The holo-PCP3 doma in accelerated PPi release and catalytic turnover by 100-200- fold over the leak rate (<1 min(-1)) of aminoacyl adenylates into solution while PCP1 in trans had only about a 5-fold effect. Free pantetheine could capture cyste inyl adenylate with a 25-50-fold increase in k(cat) while CoA was 10-fold l ess effective. The K-m of free pantetheine (30-50 mM) was 3 orders of magni tude larger than that of PCP3-TE (10-25 mu M), indicating a net 10(4) great er catalytic efficiency for transfer to the P-pant arm of PCP3 by the Ybt s ynthetase A domain, relative to P-pant alone.