Ta. Keating et al., Vibriobactin biosynthesis in Vibrio cholerae: VibH is an amide synthase homologous to nonribosomal peptide synthetase condensation domains, BIOCHEM, 39(50), 2000, pp. 15513-15521
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.