Vibriobactin [N(1)-(2,3-dihydroxybenzoyl)-N(5),N(9)-bis[2-(2, 3-dihydroxyphenyl)-5-methyloxazolinyl-4-carboxamido]
norspermidine] , is an
iron chelator from the
cholera-causing bacterium Vibrio cholerae. The six-domain, 270 kDa
nonribosomal peptide synthetase (NRPS) VibF, a component of
vibriobactin synthetase, has been heterologously expressed in Escherichia coli and purified. VibF has an unusual NRPS domain organization: cyclization-cyclization-adenylation-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, and together with
vibriobactin synthetase proteins VibE (adenylation) and VibB (aryl
carrier protein) condenses and heterocyclizes 2, 3-dihydroxybenzoyl-VibB with L-Thr to 2-dihydroxyphenyl-5-methyloxazolinyl-4-carboxy-VibF in the first demonstration of oxazoline formation by an NRPS cyclization domain. This
enzyme-bound aryl oxazoline can be transferred by VibF to various
amine acceptors but most efficiently to N(1)-(2, 3-dihydroxybenzoyl)norspermidine (k(cat) = 122 min(-1), K(m) = 1.7 microM), 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 by 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.
Vibriobactin synthetase is an unusual NRPS in that all intermediates are not covalently tethered as PCP thioesters and in that it represents an NRPS pathway with two branch points.