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H Zhou - One of the best experts on this subject based on the ideXlab platform.
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short communication characteristics of proteolytic activities of endo and Exopeptidases in alfalfa herbage and their implications for proteolysis in silage
Journal of Dairy Science, 2012Co-Authors: H Zhou, D J Undersander, Aruna NandetyAbstract:The pH optimum and thermostability of both Exopeptidases and endopeptidases were investigated in this study to elucidate the possible role of plant proteases in proteolysis during ensiling of alfalfa herbage. Proteolytic activities of 4 classes of endopeptidases (i.e., serine, metallo, aspartic, and cysteine peptidase) and 5 classes of Exopeptidases (i.e., aminopeptidase, carboxypeptidase, dipeptidase, dipeptidyl-peptidase, and tripeptidyl-peptidase) were examined within pH values of 3 to 9, and within temperatures from 20 to 90°C. Serine and metalloproteases, the principal endopeptidases that hydrolyzed most of the protein to nonprotein nitrogen in alfalfa silage, had optimum activities at pH 4. Among the major Exopeptidases contributing protein degradation in ensiled alfalfa, dipeptidase and tripeptidyl-peptidase had stable activities between pH 4 and 6, and carboxypeptidase activity was optimal at pH 5. The optimum temperature for most peptidase activities was 40°C. Proteolytic activities of both endo- and Exopeptidases increased with the elevation of incubating temperature from 20 to 40°C. The pH value in well-preserved alfalfa silage is often above 4.0, and the temperatures in the ensiled mass range from 25 to 40°C. Therefore, high proteolytic activities between pH 4 and 6 and the temperature range of ensiled alfalfa suggest that plant peptidases play a role in hydrolyzing protein during prolonged storage.
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contribution of Exopeptidases to formation of nonprotein nitrogen during ensiling of alfalfa
Journal of Dairy Science, 2011Co-Authors: H Zhou, R J Long, W ChengAbstract:The experiment was conducted to investigate the exopeptidase classes in alfalfa (Medicago sativa L.) leaves, and to determine their contribution to the formation of nonprotein nitrogen (NPN) components during ensiling. Six classes of inhibitors that included bestatin (aminopeptidase inhibitor), potato carboxypeptidase inhibitor (PCI, carboxypeptidase inhibitor), 1,10-phenanthroline (dipeptidase inhibitor), diprotin A (dipeptidyl-peptidase inhibitor), butabindide (tripeptidyl-peptidase inhibitor), and dipeptide Phe-Arg (peptidyl-dipeptidase inhibitor) were used. To determine the contribution of each exopeptidase to the formation of NPN products, aqueous extracts of fresh alfalfa were fermented to imitate the proteolytic process of ensiled alfalfa and to ensure that each class of exopeptidase inhibitor would have immediate contact with the proteases in the alfalfa extract. Five classes of Exopeptidases; namely, aminopeptidase, carboxypeptidase, dipeptidase, dipeptidyl-peptidase, and tripeptidyl-peptidase, were shown to be present in alfalfa leaves, each playing a different role in alfalfa protein degradation. Aminopeptidase, carboxypeptidase, and dipeptidase were the main Exopeptidases contributing to the formation of NH3-N. Among the 5 Exopeptidases, tripeptidyl-peptidase appeared to be the principal exopeptidase in hydrolyzing forage protein into peptides, whereas carboxypeptidase and dipeptidase appeared to be more important in contributing to the formation of amino acid-N. Dipeptidyl-peptidase and tripeptidyl-peptidase did not play a role in the formation of NH3-N or amino acid-N. Dipeptidase, carboxypeptidase, and tripeptidyl-peptidase were the principal Exopeptidases for hydrolyzing forage protein into NPN during ensilage, and treatment with a mixture of the 5 inhibitors reduced the total NPN concentration in the fermented alfalfa extract to about 45% of that in the control after 21 d of fermentation.
Aruna Nandety - One of the best experts on this subject based on the ideXlab platform.
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short communication characteristics of proteolytic activities of endo and Exopeptidases in alfalfa herbage and their implications for proteolysis in silage
Journal of Dairy Science, 2012Co-Authors: H Zhou, D J Undersander, Aruna NandetyAbstract:The pH optimum and thermostability of both Exopeptidases and endopeptidases were investigated in this study to elucidate the possible role of plant proteases in proteolysis during ensiling of alfalfa herbage. Proteolytic activities of 4 classes of endopeptidases (i.e., serine, metallo, aspartic, and cysteine peptidase) and 5 classes of Exopeptidases (i.e., aminopeptidase, carboxypeptidase, dipeptidase, dipeptidyl-peptidase, and tripeptidyl-peptidase) were examined within pH values of 3 to 9, and within temperatures from 20 to 90°C. Serine and metalloproteases, the principal endopeptidases that hydrolyzed most of the protein to nonprotein nitrogen in alfalfa silage, had optimum activities at pH 4. Among the major Exopeptidases contributing protein degradation in ensiled alfalfa, dipeptidase and tripeptidyl-peptidase had stable activities between pH 4 and 6, and carboxypeptidase activity was optimal at pH 5. The optimum temperature for most peptidase activities was 40°C. Proteolytic activities of both endo- and Exopeptidases increased with the elevation of incubating temperature from 20 to 40°C. The pH value in well-preserved alfalfa silage is often above 4.0, and the temperatures in the ensiled mass range from 25 to 40°C. Therefore, high proteolytic activities between pH 4 and 6 and the temperature range of ensiled alfalfa suggest that plant peptidases play a role in hydrolyzing protein during prolonged storage.
Kazunori Ohta - One of the best experts on this subject based on the ideXlab platform.
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s46 peptidases are the first Exopeptidases to be members of clan pa
Scientific Reports, 2015Co-Authors: Yasumitsu Sakamoto, Yoshiyuki Suzuki, I Iizuka, C Tateoka, S Roppongi, Mayu Fujimoto, Koji Inaka, Hiroaki Tanaka, Mika Masaki, Kazunori OhtaAbstract:The dipeptidyl aminopeptidase BII (DAP BII) belongs to a serine peptidase family, S46. The amino acid sequence of the catalytic unit of DAP BII exhibits significant similarity to those of clan PA endopeptidases, such as chymotrypsin. However, the molecular mechanism of the exopeptidase activity of family S46 peptidase is unknown. Here, we report crystal structures of DAP BII. DAP BII contains a peptidase domain including a typical double β-barrel fold and previously unreported α-helical domain. The structures of peptide complexes revealed that the α-helical domain covers the active-site cleft and the side chain of Asn330 in the domain forms hydrogen bonds with the N-terminus of the bound peptide. These observations indicate that the α-helical domain regulates the exopeptidase activity of DAP BII. Because S46 peptidases are not found in mammals, we expect that our study will be useful for the design of specific inhibitors of S46 peptidases from pathogens.
Yoshiyuki Suzuki - One of the best experts on this subject based on the ideXlab platform.
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s46 peptidases are the first Exopeptidases to be members of clan pa
Scientific Reports, 2015Co-Authors: Yasumitsu Sakamoto, Yoshiyuki Suzuki, I Iizuka, C Tateoka, S Roppongi, Mayu Fujimoto, Koji Inaka, Hiroaki Tanaka, Mika Masaki, Kazunori OhtaAbstract:The dipeptidyl aminopeptidase BII (DAP BII) belongs to a serine peptidase family, S46. The amino acid sequence of the catalytic unit of DAP BII exhibits significant similarity to those of clan PA endopeptidases, such as chymotrypsin. However, the molecular mechanism of the exopeptidase activity of family S46 peptidase is unknown. Here, we report crystal structures of DAP BII. DAP BII contains a peptidase domain including a typical double β-barrel fold and previously unreported α-helical domain. The structures of peptide complexes revealed that the α-helical domain covers the active-site cleft and the side chain of Asn330 in the domain forms hydrogen bonds with the N-terminus of the bound peptide. These observations indicate that the α-helical domain regulates the exopeptidase activity of DAP BII. Because S46 peptidases are not found in mammals, we expect that our study will be useful for the design of specific inhibitors of S46 peptidases from pathogens.
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Identification of the Catalytic Triad of Family S46 Exopeptidases, Closely Related to Clan PA Endopeptidases
Scientific Reports, 2014Co-Authors: Yoshiyuki Suzuki, Yasumitsu Sakamoto, Nobutada Tanaka, Hirofumi Okada, Yasushi Morikawa, Wataru OgasawaraAbstract:The Exopeptidases of family S46 are exceptional, as the closest homologs of these enzymes are the endopeptidases of clan PA. The three-dimensional structure of S46 enzymes is unknown and only one of the catalytic residues, the serine, has been identified. The catalytic histidine and aspartate residues are not experimentally identified. Here we present phylogenetic and experimental data that identify all residues of the catalytic triad of S46 peptidase, dipeptidyl aminopeptidase BII (DAP BII) from Pseudoxanthomonas mexicana WO24. Phylogenetic comparison with the protein and S46 peptidases, revealed His-86, Ser-657 and five aspartate residues as possible catalytic residues. Mutation studies identified the catalytic triad of DAP BII as His-86, Asp-224 and Ser-657, while secondary structure analysis predicted an extended alpha-helical domain in between Asp-224 and Ser-657. This domain is unique for family S46 Exopeptidases and its absence from the endopeptidases of clan PA might be key to their different hydrolysis activities.
Doreen Dobritzsch - One of the best experts on this subject based on the ideXlab platform.
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yeast beta alanine synthase shares a structural scaffold and origin with dizinc dependent Exopeptidases
Journal of Biological Chemistry, 2003Co-Authors: Stina Lundgren, Jure Piskur, Zoran Gojkovic, Doreen DobritzschAbstract:Abstract β-Alanine synthase (βAS) 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, βASs belong to two subfamilies, which exhibit a low degree of sequence similarity. We determined the structure of βAS from Saccharomyces kluyveri to a resolution of 2.7 A. 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 βAS, and a smaller domain mediating the majority of the intersubunit contacts. Both domains exhibit a mixed α/β-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 βAS 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.