1. 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.
- Author
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Wang WC, Hsu WH, Chien FT, and Chen CY
- Subjects
- Amidohydrolases genetics, Amidohydrolases metabolism, Amino Acid Sequence, Binding Sites, Conserved Sequence genetics, Crystallography, X-Ray, Histidine genetics, Histidine metabolism, Models, Molecular, Molecular Sequence Data, Mutation genetics, Protein Structure, Quaternary, Protein Subunits, Sequence Alignment, Xenon metabolism, Amidohydrolases chemistry, Mutagenesis, Site-Directed genetics, Rhizobium enzymology
- Abstract
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.
- Published
- 2001
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