Heterocycle formation in vibriobactin biosynthesis: Alternative substrate utilization and identification of a condensed intermediate

The iron-chelating peptide vibriobactin of the pathogenic Vibrio cholerae i s assembled by a four-subunit nonribosomal peptide synthetase complex, VibE , VibB, VibH, and VibF, using 2,3-dihydroxybenzoate and L-threonine as prec ursors to two 2,3-dihydroxyphenyl- (DHP-) methyloxazolinyl groups in amide linkage on a norspermidine scaffold. We have tested the ability of the six- domain VibF subunit (Cy-Cy-A-C-PCP-C) to utilize various L-threonine analog ues and found the beta -functionalized amino acids serine and cysteine can function as alternate substrates in aminoacyl-AMP formation (adenylation or A domain), aminoacyl-S-enzyme formation (A domain), acylation by 2,3-dihyd robenzoyl- (DHB-) S-VibB (heterocyclization or Cy domain), heterocyclizatio n to DHP-oxazolinyl- and DHP-thiazolinyl-S-enzyme forms of VibF (Cy domain) as well as transfer to DHB-norspermidine at both N-5 and N-9 positions (co ndensation or C domain) to make the bis(oxazolinyl) and bis(thiazolinyl) an alogues of vibriobactin. When L-threonyl-S-pantetheine or L-threonyi-S-(N-a cetyl)cysteamine was used as a small-molecule thioester analogue of the thr eonyl-S-VibF acyl enzyme intermediate, the Cy domain(s) of a CyCyA fragment of VibF generated DHB-threonyl-thioester products of the condensation step but not the methyloxazolinyl thioesters of the heterocyclization step. Thi s clean separation of condensation from cyclization validates a two-stage m echanism for threonyl, seryl, and cysteinyl heterocyclization domains in si derophore and antibiotic synthetases. Full heterocyclization activity could be restored by providing CyCyA with the substrate L-threonyl-S-peptidyl ca rrier protein (PCP)-C2, suggesting an important role for the protein scaffo ld component of the heterocyclization acceptor substrate. We also examined heterocyclization donor substrate specificity at the level of acyl group an d protein scaffold and observed intolerance for substitution at either posi tion.