pactamycin and 3-dehydroshikimate

Mutational analysis of the pyridoxal 5'-phosphate (PLP)-dependent enzyme PctV was carried out to elucidate the multi-step reaction mechanism for the formation of 3-aminobenzoate (3-ABA) from 3-dehydroshikimate (3-DSA). Introduction of mutation K276R led to the accumulation of a quinonoid intermediate with an absorption maximum at 580 nm after the reaction of pyridoxamine 5'-phosphate (PMP) with 3-DSA. The chemical structure of this intermediate was supported by X-ray crystallographic analysis of the complex formed between the K276R mutant and the quinonoid intermediate. These results clearly show that a quinonoid intermediate is involved in the formation of 3-ABA. They also indicate that Lys276 (in the active site of PctV) plays multiple roles, including acid/base catalysis during the dehydration reaction of the quinonoid intermediate.

Topics: Binding Sites; Biocatalysis; Catalytic Domain; Crystallography, X-Ray; Kinetics; meta-Aminobenzoates; Molecular Dynamics Simulation; Mutagenesis, Site-Directed; Oxidoreductases; Pactamycin; Pyridoxal Phosphate; Shikimic Acid; Spectrophotometry, Ultraviolet

Natural amino donation: A PLP-dependent aminotransferase PctV, encoded in the pactamycin biosynthetic gene cluster, was found to catalyze the formation of 3-aminobenzoate from 3-dehydroshikimate with L-glutamate as the amino donor. The PctV reaction comprises a transamination and two dehydration reactions. This is the first report of a simple 3-ABA synthase in nature.

Topics: Biological Products; Catalysis; Magnetic Resonance Spectroscopy; meta-Aminobenzoates; Models, Molecular; Pactamycin; Shikimic Acid; Streptomyces; Substrate Specificity