Crystal Structure of N-Carbamoyl-D-Amino-Acid Amidohydrolase Reveals a Homotetramer and Insight into a Catalytic Cleft

 

Wen-Ching Wang1*, Wen-Hwei Hsu2*, Fan-Tso Chien1, and Cheng-Yu Chen1

1Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan,

2Institute of Molecular Biology, National Chung Hsing University, Taichung, Taiwan,

 

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 Å resolution, to an R-factor of 18.6%.  The crystal structure shows a four-layer a/ß fold with two six-stranded ß sheets packed on either side by two a 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. 

 

 

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