Publications

• Crystal structure of N-carbamyl-D-amino acid amidohydrolase with a novel catalytic framework common to amidohydrolases.

  Nakai T., Hasegawa T., Yamashita E., Yamamoto M., Kumasaka T., Ueki T., Nanba H., Ikenaka Y., Takahashi S., Sato M., Tsukihara T.

  <h4>Background</h4>N-carbamyl-D-amino acid amidohydrolase (DCase) catalyzes the hydrolysis of N-carbamyl-D-amino acids to the corresponding D-amino acids, which are useful intermediates in the preparation of beta-lactam antibiotics. To understand the catalytic mechanism of N-carbamyl-D-amino acid ... >> More

  <h4>Background</h4>N-carbamyl-D-amino acid amidohydrolase (DCase) catalyzes the hydrolysis of N-carbamyl-D-amino acids to the corresponding D-amino acids, which are useful intermediates in the preparation of beta-lactam antibiotics. To understand the catalytic mechanism of N-carbamyl-D-amino acid hydrolysis, the substrate specificity and thermostability of the enzyme, we have determined the structure of DCase from Agrobacterium sp. strain KNK712.<h4>Results</h4>The crystal structure of DCase has been determined to 1.7 A resolution. The enzyme forms a homotetramer and each monomer consists of a variant of the alpha + beta fold. The topology of the enzyme comprises a sandwich of parallel beta sheets surrounded by two layers of alpha helices, this topology has not been observed in other amidohydrolases such as the N-terminal nucleophile (Ntn) hydrolases.<h4>Conclusions</h4>The catalytic center could be identified and consists of Glu46, Lys126 and Cys171. Cys171 was found to be the catalytic nucleophile, and its nucleophilic character appeared to be increased through general-base activation by Glu46. DCase shows only weak sequence similarity with a family of amidohydrolases, including beta-alanine synthase, aliphatic amidases and nitrilases, but might share highly conserved residues in a novel framework, which could provide a possible explanation for the catalytic mechanism for this family of enzymes. << Less

  Structure 8:729-737(2000) [PubMed] [EuropePMC]

• Crystallographic and fluorescence studies of ligand binding to N-carbamoylsarcosine amidohydrolase from Arthrobacter sp.

  Zajc A., Romao M.J., Turk D., Huber R.

  Crystal structures of N-carbamoylsarcosine amidohydrolase (CSHase; EC 3.5.1.59) have been analyzed by X-ray diffraction methods with two different inhibitors bound to the active site at 2.28 A and 2.37 A resolution. The catalytic center of the enzyme could be identified on the basis of these struc ... >> More

  Crystal structures of N-carbamoylsarcosine amidohydrolase (CSHase; EC 3.5.1.59) have been analyzed by X-ray diffraction methods with two different inhibitors bound to the active site at 2.28 A and 2.37 A resolution. The catalytic center of the enzyme could be identified on the basis of these structures. The four substrate binding sites are situated at the intersubunit interfaces of the compact dimers AB and CD of the homotetrameric enzyme. Both inhibitors inactivate the enzyme irreversibly through covalent binding of their aldehyde groups to the thiol group of the active-site cysteine residue Cys177. Within the identified substrate binding sites a number of residues from different subunits are involved in hydrogen bonding of the inhibitors. Two residues (Ala172 and Thr173) that form an unusual cis-peptide bond at the binding site are important components in fixing the examined inhibitors by hydrogen bonds. An electrochemical enzyme assay for CSHase was used to test the effect of inhibitors and substrate analogs on the enzyme's activity, revealing the high substrate specificity of CSHase. The intrinsic tryptophan fluorescence of CSHase increases strongly upon substrate and inhibitor binding. As most of the tryptophyl residues are located at the active sites, they are thus considerably affected by ligand binding. Fluorescence-detected stopped-flow measurements have been used to study the kinetics of glyoxylate and substrate binding to CSHase. Substrate and inhibitor binding could clearly be distinguished in the stopped-flow experiments. Inhibitor binding reveals at least three different elementary processes, whereas substrate binding is much faster and contains phases with different signs in amplitude. << Less

  J. Mol. Biol. 263:269-283(1996) [PubMed] [EuropePMC]

• Crystal structure analysis, refinement and enzymatic reaction mechanism of N-carbamoylsarcosine amidohydrolase from Arthrobacter sp. at 2.0-A resolution.

  Romao M.J., Turk D., Gomis-Rueth F.-X., Huber R.

  N-carbamoylsarcosine amidohydrolase from Arthrobacter sp., a tetramer of polypeptides with 264 amino acid residues each, has been crystallized and its structure solved and refined at 2.0 A resolution, to a crystallographic R-factor of 18.6%. The crystals employed in the analysis contain one tetram ... >> More

  N-carbamoylsarcosine amidohydrolase from Arthrobacter sp., a tetramer of polypeptides with 264 amino acid residues each, has been crystallized and its structure solved and refined at 2.0 A resolution, to a crystallographic R-factor of 18.6%. The crystals employed in the analysis contain one tetramer of 116,000 M(r) in the asymmetric unit. The structure determination proceeded by multiple isomorphous replacement, followed by solvent-flattening and density averaging about the local diads within the tetramer. In the final refined model, the root-mean-square deviation from ideality is 0.01 A for bond distances and 2.7 degrees for bond angles. The asymmetric unit consists of 7853 protein atoms, 431 water molecules and four sulfate ions bound into the putative active site clefts in each subunit. One subunit contains a central six-stranded parallel beta-pleated sheet packed by helices on both sides. On one side, two helices face the solvent, while two of the helices on the other side are buried in the tight intersubunit contacts. The catalytic center of the enzyme, tentatively identified by inhibitor binding, is located at the interface between two subunits and involves residues from both. It is suggested that the nucleophilic group involved in hydrolysis of the substrate is the thiol group of Cys117 and a nucleophilic addition-elimination mechanism is proposed. << Less

  J. Mol. Biol. 226:1111-1130(1992) [PubMed] [EuropePMC]