Mechanism-Based trapping of the quinonoid intermediate by using the k276r mutant of plp-Dependent 3-Aminobenzoate synthase pctv in the biosynthesis of pactamycin.
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.
