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Biosynthetic mechanism of the nonribosomal peptide AMB in Pseudomonas aeruginosa

Nonribosomal peptide synthetases (NRPSs) are enzymes found in bacteria and fungi that synthesize a class of natural products called non‐ribosomal peptides. The biosynthesis of the toxin AMB (L‐2‐amino‐4‐trans‐3‐butenoic acid) by Pseudomonas aeruginosa is directed by the amb gene cluster, which encodes two NRPSs (AmbB and AmbE) and two hydroxylases (AmbC and AmbD). Based on sequence homology and previous results, we predicted that the biosynthesis of AMB occurs through the action of the two hydroxylases on an Ala‐Glu‐Ala substrate tethered to AmbE. The modular, linear structure of NRPSs also allows us to predict the function of individual domains in a similar fashion. However, we do not know the regioselectivity or timing of these hydroxylases within the larger synthetic pathway. Sequence homology also indicates that the hydroxylated substrate may be subsequently dehydrated through a novel dehydratase function by the C‐domain. Here, we report evidence that AmbC is a β‐hydroxylase and that AmbD is a ɣ‐hydroxylase. We observed that both enzymes act on a substrate attached to the T1 domain of the AmbE NRPS. Our approach used a mutated AmbE construct containing only the first four (X‐A‐MT‐T) domains of the NRPS. We incubated the truncated construct with a Glu substrate, AmbC, AmbD, and SAM, which is the methyl source for the MT (methylation) domain in vivo. Analysis using HPLC showed a product that is consistent with hydroxylation of the Glu substrate. Confirmation of the predicted activity of AmbC and AmbD combined with a deeper understanding of the timing and regioselectivity of these enzymes will be useful in deciphering whether the C‐domain harbors novel dehydratase activity.