Crystal structure and site-directed mutagenesis studies of N-carbamoyl-D-amino-acid amidohydrolase from Agrobacterium radiobacter reveals a homotetramer and insight into a catalytic cleft.
The N-carbamoyl-D-amino-acid amidohydrolase (D-NCAase) is used on an industrial scale for the production of D-amino acids. The crystal structure of D-NCAase was solved by multiple isomorphous replacement with anomalous scattering using xenon and gold derivatives, and refined to 1.95 A resolution, to an R-factor of 18.6 %. The crystal structure shows a four-layer alpha/beta fold with two six-stranded beta sheets packed on either side by two alpha helices. One exterior layer faces the solvent, whereas the other one is buried and involved in the tight intersubunit contacts. A long C-terminal fragment extends from a monomer to a site near a dyad axis, and associates with another monomer to form a small and hydrophobic cavity, where a xenon atom can bind. Site-directed mutagenesis of His129, His144 and His215 revealed strict geometric requirements of these conserved residues to maintain a stable conformation of a putative catalytic cleft. A region located within this cleft involving Cys172, Glu47, and Lys127 is proposed for D-NCAase catalysis and is similar to the Cys-Asp-Lys site of N-carbamoylsarcosine amidohydrolase. The homologous active-site framework of these enzymes with distinct structures suggests convergent evolution of a common catalytic mechanism.
Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan. lswwc@dragon.nchu.edu.tw
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D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D017124:Conserved Sequence / Q000235:genetics; D018360:Crystallography, X-Ray; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D012231:Rhizobium / Q000201:enzymology*; D016415:Sequence Alignment; D014978:Xenon / Q000378:metabolism
D021122:Protein Subunits; D014978:Xenon; D006639:Histidine; D000581:Amidohydrolases; C080477:N-carbamoyl-D-amino acid amidohydrolase