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.