Vladimirova Anna, Patskovsky Yury, Fedorov Alexander A, Bonanno Jeffrey B, Fedorov Elena V, Toro Rafael, Hillerich Brandan, Seidel Ronald D, Richards Nigel G J, Almo Steven C, Raushel Frank M
Department of Chemistry, Texas A&M University , College Station, Texas 77843, United States.
Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.
J Am Chem Soc. 2016 Jan 27;138(3):826-36. doi: 10.1021/jacs.5b08251. Epub 2016 Jan 12.
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.
5-羧基香草酸脱羧酶（LigW）在木质素降解的生化途径中催化5-羧基香草酸转化为香草酸。已证明该酶的催化活性需要Mn(2+)，并测定了少动鞘氨醇单胞菌SYK-6（kcat = 2.2 s(-1)，kcat/Km = 4.0×10(4) M(-1) s(-1)）和芳香新鞘氨醇菌（kcat = 27 s(-1)，kcat/Km = 1.1×10(5) M(-1) s(-1)）的酶对5-羧基香草酸脱羧反应的动力学常数。在活性位点存在和不存在配体结合的情况下，测定了这两种酶的三维结构。测定了与底物类似物5-硝基香草酸结合的芳香新鞘氨醇菌LigW的结构（Kd = 5.0 nM），分辨率为1.07 Å。该复合物的结构显示，硝基取代基在酶的作用下从苯环平面扭曲约23°。基于在活性位点存在配体结合时测定的高分辨率X射线结构、活性位点残基的突变以及在H2O和D2O混合物中测定的产物同位素效应的大小，提出了LigW催化5-羧基香草酸脱羧的化学反应机制。在所提出的反应机制中，酶在脱羧步骤之前促进质子向底物C5的转移。
