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

Nakai, T.; Hasegawa, Tomokazu; Yamashita, Eiki; Yamamoto, Masaki; Kumasaka, Takashi; Ueki, Tatzuo; Nanba, Hirokazu; Ikenaka, Yasuhiro; Takahashi, Satoshi; Sato, Mamoru; Tsukihara, Tomitake

doi:10.1016/s0969-2126(00)00160-x

Cited by 119 publications

(116 citation statements)

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“…The crystal structures of nitrilase superfamily members (PDB: 1ems), worm NitFit (Pace and Brenner 2001), and bacterial (PDB: 1erz) Ncarbamyl-D-amino acid amidohydrolase (Nakai et al 2000) indicate that these enzymes were multimeric α-β-β-α sandwich fold proteins and have a conserved Glu, Lys, Cys catalytic triad responsible for covalent catalysis (Pace et al 2000;Brenner 2002).…”

Section: Introductionmentioning

confidence: 99%

A novel thermostable nitrilase superfamily amidase from Geobacillus pallidus showing acyl transfer activity

Makhongela

Głowacka

Agarkar

et al. 2007

Appl Microbiol Biotechnol

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An amidase (EC 3.5.1.4) in branch 2 of the nitrilase superfamily, from the thermophilic strain Geobacillus pallidus RAPc8, was produced at high expression levels (20 U/mg) in small-scale fermentations of Escherichia coli. The enzyme was purified to 90% homogeneity with specific activity of 1,800 U/mg in just two steps, namely, heat-treatment and gel permeation chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electron microscopic (EM) analysis of the homogenous enzyme showed the native enzyme to be a homohexamer of 38 kDa subunits. Analysis of the biochemical properties of the amidase showed that the optimal temperature and pH for activity were 50 and 7.0°C, respectively. The amidase exhibited high thermal stability at 50 and 60°C, with half-lives greater than 5 h at both temperatures. At 70 and 80°C, the half-life values were 43 and 10 min, respectively. The amidase catalyzed the hydrolysis of low molecular weight aliphatic amides, with D-selectivity towards lactamide. Inhibition studies showed activation/inhibition data consistent with the presence of a catalytically active thiol group. Acyl transfer reactions were demonstrated with acetamide, propionamide, isobutyramide, and acrylamide as substrates and hydroxylamine as the acyl acceptor; the highest reaction rate being with isobutyramide. Immobilization by entrapment in polyacrylamide gels, covalent binding on Eupergit C beads at 4°C and on Amberlite-XAD57 resulted in low protein binding and low activity, but immobilization on Eupergit C beads at 25°C with cross-linking resulted in high protein binding yield and high immobilized specific activity (80% of non-immobilized activity). Characterization of Eupergit C-immobilized preparations showed that the optimum reaction temperature was unchanged, the pH range was somewhat broadened, and stability was enhanced giving half-lives of 52 min at 70°C and 30 min at 80°C. The amidase has potential for application under high temperature conditions as a biocatalyst for D-selective amide hydrolysis producing enantiomerically pure carboxylic acids and for production of novel amides by acyl transfer.

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Section: Introductionmentioning

confidence: 99%

A novel thermostable nitrilase superfamily amidase from Geobacillus pallidus showing acyl transfer activity

Makhongela

Głowacka

Agarkar

et al. 2007

Appl Microbiol Biotechnol

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“…As expected, utilization of a subunit of yeast AS (PDB code 1r3n) as the search model remained unsuccessful. In subsequent trials, the crystal structures of N-carbamyl-d-amino-acid amidohydrolase (PDB code 1erz; 24% amino-acid sequence identity; Nakai et al, 2000) and of the hypothetical protein PH0642 from Pyrococcus horikoshii (PDB code 1j31; 28% identity; Sakai et al, 2004) served as search models in the monomeric, dimeric or tetrameric state and at diverse levels of sequence adjustment and truncation. However, no clear molecularreplacement solution has yet been obtained.…”

Section: Resultsmentioning

confidence: 99%

“…Of proteins of known crystal structure, the primary sequence of the homotetrameric N-carbamyl-d-amino-acid amidohydrolase shows the highest homology to fruit-fly AS (24% identity, 43% homology). Its subunit structure comprises a sandwich of parallel -sheets surrounded by two layers of -helices (Nakai et al, 2000). Like AS, N-carbamyl-d-amino-acid amidohydrolase catalyzes the hydrolytic cleavage of an N-carbamyl group from its substrate.…”

Section: Introductionmentioning

confidence: 99%

Crystallization and preliminary X-ray data analysis of β-alanine synthase fromDrosophila melanogaster

Lundgren

Andersen²,

Piškur³

et al. 2007

Acta Cryst Sect F

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-Alanine synthase catalyzes the last step in the reductive degradation pathway for uracil and thymine, which represents the main clearance route for the widely used anticancer drug 5-fluorouracil. Crystals of the recombinant enzyme from Drosophila melanogaster, which is closely related to the human enzyme, were obtained by the hanging-drop vapour-diffusion method. They diffracted to 3.3 Å at a synchrotron-radiation source, belong to space group C2 (unit-cell parameters a = 278.9, b = 95.0, c = 199.3 Å , = 125.8 ) and contain 8-10 molecules per asymmetric unit.

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“…DCase from Agrobacterium sp. exists as an associated homotetramer in the crystal (12,13). Each subunit of ϳ34 kDa folds into a single domain comprising a sandwich of parallel ␤-sheets surrounded by two layers of ␣-helices.…”

Section: Evolution Of Amidohydrolasesmentioning

confidence: 99%

“…The third subfamily of amidohydrolases consists of bacterial and archaebacterial Ncarbamyl-D-amino acid amidohydrolases (DCases), with a pairwise sequence identity as low as 20%. With the recent determination of the crystal structures of the DCases from two Agrobacterium species, this subfamily has thus far been the only one for which structural models are available (12,13).…”

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confidence: 99%

Yeast β-Alanine Synthase Shares a Structural Scaffold and Origin with Dizinc-dependent Exopeptidases

Lundgren

Gojković

Piškur

et al. 2003

Journal of Biological Chemistry

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␤-Alanine synthase (␤AS) is the final enzyme of the reductive pyrimidine catabolic pathway, which is responsible for the breakdown of pyrimidine bases, including several anticancer drugs. In eukaryotes, ␤ASs belong to two subfamilies, which exhibit a low degree of sequence similarity. We determined the structure of ␤AS from Saccharomyces kluyveri to a resolution of 2.7 Å. The subunit of the homodimeric enzyme consists of two domains: a larger catalytic domain with a dizinc metal center, which represents the active site of ␤AS, and a smaller domain mediating the majority of the intersubunit contacts. Both domains exhibit a mixed ␣/␤-topology. Surprisingly, the observed high structural homology to a family of dizinc-dependent exopeptidases suggests that these two enzyme groups have a common origin. Alterations in the ligand composition of the metal-binding site can be explained as adjustments to the catalysis of a different reaction, the hydrolysis of an N-carbamyl bond by ␤AS compared with the hydrolysis of a peptide bond by exopeptidases. In contrast, there is no resemblance to the three-dimensional structure of the functionally closely related N-carbamyl-D-amino acid amidohydrolases. Based on comparative structural analysis and observed deviations in the backbone conformations of the eight copies of the subunit in the asymmetric unit, we suggest that conformational changes occur during each catalytic cycle.In most living organisms, the degradation of uracil and thymine is achieved by the three-step reaction sequence of the reductive pyrimidine catabolic pathway. The first and rate-limiting enzyme is dihydropyrimidine dehydrogenase (dihydrouracil dehydrogenase (NADP ϩ ), EC 1.3.1.2), which catalyzes the NADPHdependent reduction of uracil and thymine to the corresponding dihydropyrimidines (Scheme 1). In the second step, dihydropyrimidinase (EC 3.5.2.2) generates N-carbamyl-␤-alanine and N-carbamyl-␤-aminoisobutyric acid, respectively, via reversible

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

Cited by 119 publications

References 42 publications

A novel thermostable nitrilase superfamily amidase from Geobacillus pallidus showing acyl transfer activity

A novel thermostable nitrilase superfamily amidase from Geobacillus pallidus showing acyl transfer activity

Crystallization and preliminary X-ray data analysis of β-alanine synthase fromDrosophila melanogaster

Yeast β-Alanine Synthase Shares a Structural Scaffold and Origin with Dizinc-dependent Exopeptidases

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