|Hirayama A, Miyanaga A, Kudo F, Eguchi T
|Mechanism-Based Trapping of the Quinonoid Intermediate by Using the K276R Mutant of PLP-Dependent 3-Aminobenzoate Synthase PctV in the Biosynthesis of Pactamycin
|PLP-dependent enzymes; biosynthesis; natural products; pactamycin; quinonoid
|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.