Structure of peptidase T from <i>Salmonella typhimurium</i>

Håkansson, Kjell; Miller, Charles G.

doi:10.1046/j.0014-2956.2001.02665.x

Cited by 52 publications

(50 citation statements)

References 32 publications

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“…In each of the crystal structures determined for homodimeric members the subunits display an extended open conformation (16,21,22). For Sk␤AS, AcyI, and others, it has been suggested that a few residues from the dimerization domain participate in substrate binding and catalysis (16,23).…”

mentioning

confidence: 99%

Crystal Structures of Yeast β-Alanine Synthase Complexes Reveal the Mode of Substrate Binding and Large Scale Domain Closure Movements

Lundgren

Andersen

Piškur

et al. 2007

Journal of Biological Chemistry

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␤-Alanine synthase is the final enzyme of the reductive pyrimidine catabolic pathway, which is responsible for the breakdown of uracil and thymine in higher organisms. The fold of the homodimeric enzyme from the yeast Saccharomyces kluyveri identifies it as a member of the AcyI/M20 family of metallopeptidases. Its subunit consists of a catalytic domain harboring a di-zinc center and a smaller dimerization domain. The present site-directed mutagenesis studies identify Glu 159 and Arg 322 as crucial for catalysis and His 262 and His 397 as functionally important but not essential. We determined the crystal structures of wild-type ␤-alanine synthase in complex with the reaction product ␤-alanine, and of the mutant E159A with the substrate N-carbamyl-␤-alanine, revealing the closed state of a dimeric AcyI/M20 metallopeptidase-like enzyme. Subunit closure is achieved by a ϳ30°rigid body domain rotation, which completes the active site by integration of substrate binding residues that belong to the dimerization domain of the same or the partner subunit. Substrate binding is achieved via a salt bridge, a number of hydrogen bonds, and coordination to one of the zinc ions of the di-metal center.

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mentioning

confidence: 99%

Crystal Structures of Yeast β-Alanine Synthase Complexes Reveal the Mode of Substrate Binding and Large Scale Domain Closure Movements

Lundgren

Andersen

Piškur

et al. 2007

Journal of Biological Chemistry

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“…In fact, the Arg and His residues are strictly conserved. Based on the literature available for homologous enzymes, the conserved Arg residue is or might be necessary for substrate binding in ␤-alanine synthase (4,37,38), EcAam (2), peptidase V (PepV) (31), carnosinase (55), IAA-amino acid hydrolase homolog 2 (ILL2) (7), peptidase D (PepD) (9), DapE (46), hAcy1 (35), peptidase T (PepT) (6,24), and metallopeptidase (Sapep) (20). Several homologous structures present different molecules bound to these residues, namely, the structures under PDB IDs 1VIX, 1FNO, 3IC1, 3IFE, and 2QYV.…”

Section: R369 H219(a) N260(a)mentioning

confidence: 99%

Mutational and Structural Analysis ofl-N-Carbamoylase Reveals New Insights into a Peptidase M20/M25/M40 Family Member

et al. 2012

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c N-Carbamoyl-L-amino acid amidohydrolases (L-carbamoylases) are important industrial enzymes used in kinetic resolution of racemic mixtures of N-carbamoyl-amino acids due to their strict enantiospecificity. In this work, we report the first L-carbamoylase structure belonging to Geobacillus stearothermophilus CECT43 (BsLcar), at a resolution of 2.7 Å. Structural analysis of BsLcar and several members of the peptidase M20/M25/M40 family confirmed the expected conserved residues at the active site in this family, and site-directed mutagenesis revealed their relevance to substrate binding. We also found an unexpectedly conserved arginine residue (Arg 234 in BsLcar), proven to be critical for dimerization of the enzyme. The mutation of this sole residue resulted in a total loss of activity and prevented the formation of the dimer in BsLcar. Comparative studies revealed that the dimerization domain of the peptidase M20/M25/M40 family is a "small-molecule binding domain," allowing further evolutionary considerations for this enzyme family. N-Carbamoyl-L-amino acid amidohydrolases (L-carbamoylases; EC 3.5.1.87) irreversibly hydrolyze the amide bond of the carbamoyl group in L-N-carbamoyl-amino acids. Due to their stereospecificity, L-carbamoylases are widely used in kinetic resolution for the production of optically pure amino acids (see reference 44 and references therein). Furthermore, their substrate promiscuity allows their use in different multienzymatic processes, increasing their economic and industrial relevance (48). L-Carbamoylases have been isolated and characterized from different sources, although most of the studies carried out have focused on biotechnological applications (44). Their relationship with other amidohydrolases and with the peptidase M20/M25/M40 family has been established based on sequence similarity (21,29,42). A closer relationship among L-carbamoylases and ␤-ureidopropionases is supported by the findings of two ␤-ureidopropionases that are able to hydrolyze N-carbamoyl-L-␣-amino acids (40,41,47).In a previous work, we were able to show the involvement in catalysis of several highly conserved residues in L-carbamoylases, as well as the relationship of these enzymes with several peptidases (42). Dimerization was also proved to be necessary for enzymatic activity, as the residues involved in catalysis come from both monomers. In this work, we determined the first crystal structure of an L-carbamoylase, belonging to Geobacillus stearothermophilus CECT43 (BsLcar). Mutagenesis studies of BsLcar confirmed the importance of conserved residues in this enzyme. Unexpectedly, a highly conserved arginine in L-carbamoylases has been proved critical for the dimerization of the enzyme, and thus for enzymatic activity. Comparative studies revealed striking characteristics of the different "dimerization" domains of the peptidase M20/M25/ M40 family, in agreement with previous evolutionary hypotheses on this family of enzymes (3, 37) and suggesting new evolutionary considerations. Finally, an alternative re...

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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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Structure of peptidase T from Salmonella typhimurium

Cited by 52 publications

References 32 publications

Crystal Structures of Yeast β-Alanine Synthase Complexes Reveal the Mode of Substrate Binding and Large Scale Domain Closure Movements

Crystal Structures of Yeast β-Alanine Synthase Complexes Reveal the Mode of Substrate Binding and Large Scale Domain Closure Movements

Mutational and Structural Analysis ofl-N-Carbamoylase Reveals New Insights into a Peptidase M20/M25/M40 Family Member

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

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