title: Mechanism-Based Trapping of the Quinonoid Intermediate by Using the K276R Mutant of PLP-Dependent 3-Aminobenzoate Synthase PctV in the Biosynthesis of Pactamycin.
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.
authors.affiliation: Department of Chemistry and Materials Science, Tokyo Institute of Technology, 2-12-1 O-okayama, Meguro-ku, Tokyo, 152-8551, Japan.
authors.forename: Akane
authors.initials: A
authors.lastname: Hirayama
authors.affiliation: Department of Chemistry, Tokyo Institute of Technology, 2-12-1 O-okayama, Meguro-ku, Tokyo, 152-8551, Japan.
authors.forename: Akimasa
authors.initials: A
authors.lastname: Miyanaga
authors.affiliation: Department of Chemistry, Tokyo Institute of Technology, 2-12-1 O-okayama, Meguro-ku, Tokyo, 152-8551, Japan. fkudo@chem.titech.ac.jp.
authors.forename: Fumitaka
authors.initials: F
authors.lastname: Kudo
authors.affiliation: Department of Chemistry and Materials Science, Tokyo Institute of Technology, 2-12-1 O-okayama, Meguro-ku, Tokyo, 152-8551, Japan. eguchi@cms.titech.ac.jp.
authors.forename: Tadashi
authors.initials: T
authors.lastname: Eguchi
mesh_terms: D001665:Binding Sites; D055162:Biocatalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010142:Pactamycin / Q000096:biosynthesis* / Q000737:chemistry; D011732:Pyridoxal Phosphate / Q000737:chemistry; D012765:Shikimic Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D062368:meta-Aminobenzoates / Q000737:chemistry / Q000378:metabolism
chemical_list: D062368:meta-Aminobenzoates; D010142:Pactamycin; C096387:3-dehydroshikimate; D012765:Shikimic Acid; D011732:Pyridoxal Phosphate; D010088:Oxidoreductases; C019768:benzoate synthase; C044855:3-aminobenzoic acid
keywords: PLP-dependent enzymes; biosynthesis; natural products; pactamycin; quinonoid