Crystal structure of carboxypeptidase G2, a bacterial enzyme with applications in cancer therapy

Rowsell, Siân; Pauptit, Richard A.; Tucker, Alec D.; Melton, Roger G.; Blow, D. M.; Brick, P.

doi:10.1016/s0969-2126(97)00191-3

Cited by 193 publications

(249 citation statements)

References 51 publications

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“…On the other hand, alignments of the human ASPA sequence against carboxypeptidase A, carboxypeptidase G, and aminopeptidase have recently been reported (Makarova & Grishin 1999). Two zinc ions were reported to bind to aminopeptidase (Berman et al 2000,Chevrier et al 1994) and carboxypeptidase G2 (Berman et al 2000,Rowsell et al 1997. In these peptidases, the ligands were shared between the two zinc ions.…”

Section: Homology-based Modeling Of Aspamentioning

confidence: 99%

Mutational analysis of aspartoacylase: Implications for Canavan Disease

et al. 2007

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Mutations that result in undetectable activity of aspartoacylase, which catalyzes the deacetylation of N-acetyl-L-aspartate, correlate with Canavan Disease, a neurodegenerative disorder usually fatal during childhood. The underlying biochemical mechanisms of how these mutations ablate activity are poorly understood. Therefore, we developed and tested a three-dimensional homology model of aspartoacylase based on zinc dependent carboxypeptidase A. Mutations of the putative zinc-binding residues (H21G, E24D/G, and H116G), the general proton donor (E178A), and mutants designed to switch the order of the zinc-binding residues (H21E/E24H and E24H/H116E) yielded wild-type aspartoacylase protein levels and undetectable ASPA activity. Mutations that affect substrate carboxyl binding (R71N) and transition state stabilization (R63N) also yielded wild-type aspartoacylase protein levels and undetectable aspartoacylase activity. Alanine substitutions of Cys124 and Cys152, residues indicated by homology modeling to be in close proximity and in the proper orientation for disulfide bonding, yielded reduced ASPA protein and activity levels. Finally, expression of several previously tested (E24G, D68A, C152W, E214X, D249V, E285A, and A305E) and untested (H21P, A57T, I143T, P183H, M195R, K213E/G274R, G274R, and F295S) Canavan Disease mutations resulted in undetectable enzyme activity, and only E285A and P183H showed wild-type aspartoacylase protein levels. These results show that aspartoacylase is a member of the caboxypeptidase A family and offer novel explanations for most loss-of-function aspartoacylase mutations associated with Canavan Disease.

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Section: Homology-based Modeling Of Aspamentioning

confidence: 99%

Mutational analysis of aspartoacylase: Implications for Canavan Disease

et al. 2007

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“…In contrast, carboxypeptidase G2 (CPG2; Pseudomonas sp.) (35) and peptidases T (PepT; Salmonella typhimurium) (36) and V (PepV, Lactobacillus delbrueckii) (37) consist of two domains, which are both structurally related to their counterparts in Sk␤AS. The arrangement of the two domains relative to each other does, however, vary between the proteins.…”

Section: Structural Homology To Dizinc-dependent Exopeptidasesmentioning

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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“…strain RS-16 for which the three-dimensional structure is available (Rowsell et al, 1997) has a distant sequence similarity (17% amino-acid identity) to the T. litoralis l-aminoacylase. The overall sequence identity between these two enzymes is low; however, reiterative BLAST search (Altschul et al, 1997) analysis suggests they are both members of the same protein family, peptidase M20.…”

Section: Introductionmentioning

confidence: 99%

Crystallization and preliminary X-ray diffraction analysis ofL-aminoacylase from the hyperthermophilic archaeonThermococcus litoralis

Hollingsworth

Isupov

Littlechild

2002

Acta Crystallogr D Biol Cryst

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The enzyme l-aminoacylase catalyses the hydrolysis of N-acyll-amino acids from peptides or proteins. The recombinant enzyme from the hyperthermophilic archaeon Thermococcus litoralis has been puri®ed to homogeneity. This zinc-containing enzyme has been crystallized from ammonium sulfate using the sitting-drop vapourdiffusion method. The crystals diffract to 2.8 A Ê resolution and belong to the rhombohedral space group R32, with unit-cell parameters a = b = 102.4, c = 178.5 A Ê , = 120 in a hexagonal lattice setting. The asymmetric unit contains one enzyme monomer, containing a single zinc ion. Two synchrotron data sets have been collected at a remote wavelength and at the maximum f H wavelength for zinc. This has allowed the position of the metal to be identi®ed in anomalous Patterson maps.

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Crystal structure of carboxypeptidase G2, a bacterial enzyme with applications in cancer therapy

Cited by 193 publications

References 51 publications

Mutational analysis of aspartoacylase: Implications for Canavan Disease

Mutational analysis of aspartoacylase: Implications for Canavan Disease

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

Crystallization and preliminary X-ray diffraction analysis ofL-aminoacylase from the hyperthermophilic archaeonThermococcus litoralis

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