A.Hirayama
et al.
(2015).
Mechanism-Based Trapping of the Quinonoid Intermediate by Using the K276R Mutant of PLP-Dependent 3-Aminobenzoate Synthase PctV in the Biosynthesis of Pactamycin.
Chembiochem,
16,
2484-2490.
PubMed id: 26426567
DOI: 10.1002/cbic.201500426
Mechanism-Based Trapping of the Quinonoid Intermediate by Using the K276R Mutant of PLP-Dependent 3-Aminobenzoate Synthase PctV in the Biosynthesis of Pactamycin.
A.Hirayama,
A.Miyanaga,
F.Kudo,
T.Eguchi.
ABSTRACT
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.