Antimetabolite biosynthesis in Pseudomonas aeruginosa

Abstract

L-2-amino-4-methoxy-trans-3-butenoic acid (methoxyvinylglycine, or short as AMB) is a γ-substituted vinylglycine that was identified in 1972 as an inhibitor of pyridoxal 5’-phosphate-dependent enzymes, resulting in antibiotic activity. Recent studies indicate that the gene cluster ambABCDE is responsible for AMB biosynthesis in Pseudomonas aeruginosa. Among the products of this gene cluster, AmbC and AmbD, two α-ketoglutarate-dependent oxygenases are involved in the modification of L-glutamate as substrate for AMB synthesis. AMB is then synthetized from one modified glutamate and two alanine moieties condensed to a tripeptide by the non-ribosomal peptide synthetases (NRPS) AmbB and AmbE. However, exact timing of the tailoring reactions remains unclear so far. The structural research was primarily focused on AmbC and AmbD. Additionally, the structure of unknown domain in NRPS AmbE needs to be determined to gain information on its function in AMB synthesis. X-ray crystallography was mainly applied to obtain structures of targeted proteins, combining with preliminary mass spectrometry (MS) experiments to provide a model for biosynthesis of AMB in Pseudomonas aeruginosa. The structures of AmbD and AmbC in the native state as well as in the ligand-bound state complex with the co-factor α-ketoglutarate and analog N-oxalylglycine (NOG) were determined. Additionally, the structure of an uncharacterized domain of AmbE was obtained and submitted to PDBeFold and Dali server, suggesting it to be related to NRPS condensation domains. However, there is no conserved motif HHxxxDG that would be expected for condensation domains. Further, the two condensation domains within AmbB and AmbE are sufficient to synthesize a tripeptide product, such that a third condensation domain seems unnecessary.