Vibriobactin biosynthesis in Vibrio cholerae: VibH is an amide synthase homologous to nonribosomal peptide synthetase condensation domains

The Vibrio cholerae siderophore vibriobactin is biosynthesized from three m olecules of 2,3-dihydroxybenzoate (DHB), two molecules of L-threonine, and one of norspermidine. Of the four genes positively implicated in vibriobact in biosynthesis, we have here expressed, purified, and assayed the products of three: vibE, vibB, and vibH. All three are homologous to nonribosomal p eptide synthetase (NRPS) domains: VibE is a 2,3-dihydroxybenzoate-adenosyl monophosphate Ligase, VibB is a bifunctional isochorismate lyase-aryl carri er protein (ArCP), and VibH is a novel amide synthase that represents a fre e-standing condensation (C) domain. VibE and VibB are homologous to EntE an d EntB from Escherichia coli enterobactin synthetase; VibE activates DHB as the acyl adenylate and then transfers it to the free thiol of the phosphop antetheine arm of VibB's ArCP domain. VibH then condenses this DHB thioeste r (the donor) with the small molecule norspermidine (the acceptor), forming N-1-(2,3-dihydroxybenzoyl)norspermidine (DHB-NSPD) with a k(cat) of 600 mi n(-1) and a K-m for acyl-VibB of 0.88 muM and for norspermidine of 1.5 mM. Exclusive monoacylation of a primary amine of norspermidine was observed. V ibH also tolerates DHB-acylated EntB and 1,7-diaminoheptane, octylamine, an d hexylamine as substrates, albeit at lowered catalytic efficiencies. DHB-N SPD possesses one of three acylations required for mature vibriobactin, and its formation confirms VibH's role in vibriobactin biosynthesis. VibH is a unique NRPS condensation domain that acts upon an upstream carrier-protein -bound donor and a downstream amine, turning over a soluble amide product, in contrast to an archetypal NRPS-embedded C domain that condenses two carr ier protein thioesters.