Reconstitution and characterization of the Vibrio cholerae vibriobactin synthetase from VibB, VibE, VibF, and VibH

Vibriobactin [N-1-(2,3-dihydroxybenzoyl)-N-5,N-9-bis[2-(2,3-dihydroxyphenyl )-5-methyloxazolinyl- 4-carboxamido]norspermidine], is an iron chelator fro m the cholera-causing bacterium Vibrio cholerae. The six-domain, 270 kDa no nribosomal peptide synthetase (NRPS) VibF, a component of vibriobactin synt hetase, has been heterologously expressed in Escherichia coli and purified. VibF has an unusual NRPS domain organization: cyclization-cyclization-aden ylation-condensation-peptidyl carrier protein-condensation (Cy-1-Cy-2-A-C-1 -PCP-C-2). VibF activates and covalently loads its PCP with L-threonine, an d together with vibriobactin synthetase proteins VibE (adenylation) and Vib B (aryl carrier protein) condenses and heterocyclizes 2,3-dihydroxybenzoyl- VibB with L-Thr to 2-dihydroxyphenyl-5-methyloxazolinyl-4-carboxy-vibF in t he first demonstration of oxazoline formation by an NRPS cyclization domain . This enzyme-bound aryl oxazoline can be transferred by VibF to various am ine accepters but most efficiently to N-1-(2,3-dihydroxybenzoyl)norspermidi ne (k(cat) 122 min(-1), K-m = 1.7 muM), the product of 2,3-dihydroxybenzoyl -VibB, norspermidine, and VibH. This diacylated product undergoes a second aryl oxazoline acylation on its remaining secondary amine, also catalyzed b y VibF, to yield vibriobactin. Vibriobactin biosynthesis in vitro has thus been accomplished from four proteins, VibE, VibB, VibF, and VibH, with the substrates 2,3-dihydroxybenzoic acid, L-Thr, norspermidine, and ATP. Vibrio bactin synthetase is an unusual NRPS in that all intermediates are not cova lently tethered as PCP thioesters and in that it represents an NRPS pathway with two branch points.