title: Substrate Distortion and the Catalytic Reaction Mechanism of 5-Carboxyvanillate Decarboxylase.
abstract: 5-Carboxyvanillate decarboxylase (LigW) catalyzes the conversion of 5-carboxyvanillate to vanillate in the biochemical pathway for the degradation of lignin. This enzyme was shown to require Mn(2+) for catalytic activity and the kinetic constants for the decarboxylation of 5-carboxyvanillate by the enzymes from Sphingomonas paucimobilis SYK-6 (kcat = 2.2 s(-1) and kcat/Km = 4.0 × 10(4) M(-1) s(-1)) and Novosphingobium aromaticivorans (kcat = 27 s(-1) and kcat/Km = 1.1 × 10(5) M(-1) s(-1)) were determined. The three-dimensional structures of both enzymes were determined in the presence and absence of ligands bound in the active site. The structure of LigW from N. aromaticivorans, bound with the substrate analogue, 5-nitrovanillate (Kd = 5.0 nM), was determined to a resolution of 1.07 Å. The structure of this complex shows a remarkable enzyme-induced distortion of the nitro-substituent out of the plane of the phenyl ring by approximately 23°. A chemical reaction mechanism for the decarboxylation of 5-carboxyvanillate by LigW was proposed on the basis of the high resolution X-ray structures determined in the presence ligands bound in the active site, mutation of active site residues, and the magnitude of the product isotope effect determined in a mixture of H2O and D2O. In the proposed reaction mechanism the enzyme facilitates the transfer of a proton to C5 of the substrate prior to the decarboxylation step.
authors.affiliation: Department of Chemistry, Texas A&M University , College Station, Texas 77843, United States.
authors.forename: Anna
authors.initials: A
authors.lastname: Vladimirova
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.
authors.forename: Yury
authors.initials: Y
authors.lastname: Patskovsky
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Fedorov
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.
authors.forename: Jeffrey B
authors.initials: JB
authors.lastname: Bonanno
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.
authors.forename: Elena V
authors.initials: EV
authors.lastname: Fedorov
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.
authors.forename: Rafael
authors.initials: R
authors.lastname: Toro
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.
authors.forename: Brandan
authors.initials: B
authors.lastname: Hillerich
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.
authors.forename: Ronald D
authors.initials: RD
authors.lastname: Seidel
authors.affiliation: Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis , Indianapolis, Indiana 46202, United States.
authors.forename: Nigel G J
authors.initials: NG
authors.lastname: Richards
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.
authors.forename: Steven C
authors.initials: SC
authors.lastname: Almo
authors.affiliation: Department of Chemistry, Texas A&M University , College Station, Texas 77843, United States.
authors.forename: Frank M
authors.initials: FM
authors.lastname: Raushel
mesh_terms: D055162:Biocatalysis; D002262:Carboxy-Lyases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D042301:Sphingomonadaceae / Q000201:enzymology; D020578:Sphingomonas / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; C466306:5-carboxyvanillate decarboxylase; D002262:Carboxy-Lyases