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|標題:||Structural basis for catalysis and substrate specificity of Agrobacterium radiobacter N-carbamoyl-D-amino acid amidohydrolase||作者:||Chen, C.Y.
|關鍵字:||x-ray-diffraction;crystal-structure;microbial transformation;protein;structures;refinement;purification;hydantoins;program;cloning;reveals||Project:||Journal of Biological Chemistry||期刊/報告no：:||Journal of Biological Chemistry, Volume 278, Issue 28, Page(s) 26194-26201.||摘要:||
N-Carbamoyl-D-amino acid amidohydrolase is an industrial biocatalyst to hydrolyze N-carbamoyl-D-amino acids for producing valuable D-amino acids. The crystal structure of N-carbamoyl-D-amino acid amidohydrolase in the unliganded form exhibits a alpha-beta-beta-alpha fold. To investigate the roles of Cys(172), Asn(173), Arg(175), and Arg(176) in catalysis, C172A, C172S, N173A, R175A, R176A, R175K, and R176K mutants were constructed and expressed, respectively. All mutants showed similar CD spectra and had hardly any detectable activity except for R173A that retained 5% of relative activity. N173A had a decreased value in k(cat) or K-m, whereas R175K or R176K showed high Km and very low kcat values. Crystal structures of C172A and C172S in its free form and in complex form with a substrate, along with N173A and R175A, have been determined. Analysis of these structures shows that the overall structure maintains its four-layer architecture and that there is limited conformational change within the binding pocket except for R175A. In the substrate-bound structure, side chains of Glu(47), Lys(127), and C172S cluster together toward the carbamoyl moiety of the substrate, and those of Asn(173), Arg(175), and Arg(176) interact with the carboxyl group. These results collectively suggest that a Cys(172)-Glu(47)-Lys(127) catalytic triad is involved in the hydrolysis of the carbamoyl moiety and that Arg(175) and Arg(176) are crucial in binding to the carboxyl moiety, hence demonstrating substrate specificity. The common (Glu/Asp)-Lys-Cys triad observed among N-carbamoyl-D-amino acid amidohydrolase, Nit-Fhit, and another carbamoylase suggests a conserved and robust platform during evolution, enabling it to catalyze the reactions toward a specific nitrile or amide efficiently.
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