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Tadashi Yoshimoto - One of the best experts on this subject based on the ideXlab platform.
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Biochemistry and structural biology of microbial enzymes and their medical applications
Yakugaku Zasshi-journal of The Pharmaceutical Society of Japan, 2007Co-Authors: Tadashi YoshimotoAbstract:Microbial enzymes were studied from two medicinal viewpoints. First, we examined proline-specic peptidases from pathogenic microorganisms. We found several proline-specic peptidases in pathogenic bacteria. Among them, Prolyl tripeptidyl Aminopeptidase from Porphylomonas gingivals and Prolyl Aminopeptidase from Serratia marcescens were crystallized. The complex structures of those enzymes and inhibitors were claried in X-ray crystallography. Aminopeptidase N, which has wide specicity for amino acids, was distributed in the pathogens. The crystal structure of the Aminopeptidase N elucidated the reasons for its wide substrate specicity but inertness to the X-Pro bond. It was also revealed that proline-specic peptidases and Aminopeptidase N cooperatively degrade collagen for the uptake of amino acids as nutrition when these bacteria infect cells. Second, we applied enzymes from microorganisms to diagnostic ana- lyses. We found a series of creatinine-metabolizing enzymes in Pseudomonas putida. Creatininase, creatinase, and sar- cosine oxidase were coupled and have been developed for a diagnostic analysis kit that examines renal function. The structures of the native and the Mn 2+ -activated creatininases were determined in X-ray crystallography. Based on the structure, the activated enzyme was used for an improved assay kit. The structure of D-3-hydroxybutyrate de- hydrogenase from Pseudomonas fragi was also claried in crystallography. The enzyme is useful for diagnostic analysis of diabetes mellitus while monitoring ketone bodies. Key words―structural biology; proline-specic peptidase; microbial enzyme; diagnostic analysis
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unusual extra space at the active site and high activity for acetylated hydroxyproline of Prolyl Aminopeptidase from serratia marcescens
Journal of Bacteriology, 2006Co-Authors: Yoshitaka Nakajima, Makoto Sakata, Yue Xu, Kanako Nakashima, Futoshi Matsubara, Susumi Hatakeyama, Tadashi YoshimotoAbstract:The Prolyl Aminopeptidase complexes of Ala-TBODA [2-alanyl-5-tert-butyl-(1, 3, 4)-oxadiazole] and Sar-TBODA [2-sarcosyl-5-tert-butyl-(1, 3, 4)-oxadiazole] were analyzed by X-ray crystallography at 2.4 A resolution. Frames of alanine and sarcosine residues were well superimposed on each other in the pyrrolidine ring of proline residue, suggesting that Ala and Sar are recognized as parts of this ring of proline residue by the presence of a hydrophobic proline pocket at the active site. Interestingly, there was an unusual extra space at the bottom of the hydrophobic pocket where proline residue is fixed in the Prolyl Aminopeptidase. Moreover, 4-acetyloxyproline-βNA (4-acetyloxyproline β-naphthylamide) was a better substrate than Pro-βNA. Computer docking simulation well supports the idea that the 4-acetyloxyl group of the substrate fitted into that space. Alanine scanning mutagenesis of Phe139, Tyr149, Tyr150, Phe236, and Cys271, consisting of the hydrophobic pocket, revealed that all of these five residues are involved significantly in the formation of the hydrophobic proline pocket for the substrate. Tyr149 and Cys271 may be important for the extra space and may orient the acetyl derivative of hydroxyproline to a preferable position for hydrolysis. These findings imply that the efficient degradation of collagen fragment may be achieved through an acetylation process by the bacteria.
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novel inhibitor for Prolyl Aminopeptidase from serratia marcescens and studies on the mechanism of substrate recognition of the enzyme using the inhibitor
Archives of Biochemistry and Biophysics, 2003Co-Authors: Takahiko Inoue, Nobutada Tanaka, Kazuo T Nakamura, Susumi Hatakeyama, Tomohiro Tozaka, Tadashi YoshimotoAbstract:Abstract Prolyl Aminopeptidase from Serratia marcescens hydrolyzed x–β-naphthylamides (x=Prolyl, alanyl, sarcosinyl, l -α-aminobutylyl, and norvalyl), which suggested that the enzyme has a pocket for a five-member ring. Based on the substrate specificity, novel inhibitors of Pro, Ala, and Sar having 2- tert -butyl-[1,3,4]oxadiazole (TBODA) were synthesized. The K i value of Pro–TBODA, Ala–TBODA, and Sar–TBODA was 0.5 μM, 1.6 μM, and 12 mM, respectively. The crystal structure of enzyme–Pro–TBODA complex was determined. Pro–TBODA was located at the active site. Four electrostatic interactions were located between the enzyme and the amino group of Pro inhibitors (Glu204:0E1-N:Inh, Glu204:0E2-N:Inh, Glu232:0E1-N:Inh, and Gly46:O-N:Inh), and the residue of the inhibitors was inserted into the hydrophobic pocket composed of Phe139, Leu141, Leu146, Tyr149, Tyr150, and Phe236. The roles of Phe139, Tyr149, and Phe236 in the hydrophobic pocket and Glu204 and Glu232 in the electrostatic interactions were confirmed by site-directed mutagenesis, which indicated that the molecular recognition of proline is achieved through four electrostatic interactions and an insertion in the hydrophobic pocket of the enzyme.
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substrate recognition mechanism of Prolyl Aminopeptidase from serratia marcescens
Journal of Biochemistry, 2000Co-Authors: Takahiko Inoue, Tsutomu Kabashima, Huashan Huang, Naota Kanada, Nik Azmi, Essam Azab, Tadashi YoshimotoAbstract:: Molecular cloning of the gene and the crystal structure of the Prolyl Aminopeptidase [EC 3.4.11.5] from Serratia marcescens have been studied by us [J. Biochem. 122, 601-605 (1997); ibid. 126, 559-565 (1999)]. Through these studies, Phe139, Tyr149, Glu204, and Arg136 were estimated to be concerned with substrate recognition. To elucidate the details of the mechanism for the substrate specificity, the site-directed mutagenesis method was applied. The F139A mutant showed an 80-fold decrease in catalytic efficiency (k(cat)/K(m)), but the Y149A mutant did not show a significant change in catalytic efficiency. The catalytic efficiency of the E204Q mutant was about 4% of that of the wild type. The peptidase activity of the mutant (R136A) was markedly decreased, however, arylamidase activity with Pyr-bNA was retained as in the wild-enzyme. From these results, it was clarified that the pyrrolidine ring and the amino group of proline at the S1 site were recognized by Phe139 and Glu204, respectively. P1' of a substrate was recognized by Arg136. On the other hand, the enzyme had two cysteine residues. Mutants C74A and C271A were inhibited by PCMB, but the double mutated enzyme (C74/271A) was resistant to it.
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Two Continuous Spectrophotometric Assays for Methionine Aminopeptidase
Analytical Biochemistry, 2000Co-Authors: Ying Zhou, Tian Yi, Tadashi YoshimotoAbstract:Abstract Two spectrophotometric assays have been developed for methionine Aminopeptidases (MetAPs). The first method employs a thioester substrate which, upon enzymatic removal of the N-terminal methionine, generates a free thiol group. The released thiol is quantitated using Ellman's reagent. The MetAP reaction is conveniently monitored on a UV-VIS spectrophotometer in a continuous fashion, with the addition of an excess of Ellman's reagent into the assay reaction. Two tripeptide analogues were synthesized and found to be excellent substrates of both Escherichia coli MetAP and human MetAP2 ( k cat / K M = 2.8 × 10 5 M −1 s −1 for the most reactive substrate). In the second assay method, the MetAP reaction is coupled to a Prolyl Aminopeptidase reaction using Met-Pro- p -nitroanilide as substrate. MetAP-catalyzed cleavage of the N-terminal methionine produces Prolyl- p -nitroanilide, which is rapidly hydrolyzed by the Prolyl Aminopeptidase from Bacillus coagulans to release a chromogenic product, p -nitroaniline. This allows the MetAP reaction to be continuously monitored at 405 nm on a UV-VIS spectrophotometer. The assays have been applied to determine the pH optima and kinetic constants for the E. coli and human MetAPs as well as to screen MetAP inhibitors. These results demonstrate that the current assays are convenient, rapid, and sensitive methods for kinetic studies of MetAPs and effective tools for screening MetAP inhibitors.
Wieslaw Bielawski - One of the best experts on this subject based on the ideXlab platform.
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tspap1 encodes a novel plant Prolyl Aminopeptidase whose expression is induced in response to suboptimal growth conditions
Biochemical and Biophysical Research Communications, 2012Co-Authors: Urszula Szawlowska, Edyta Zdunekzastocka, Agnieszka Grabowska, Wieslaw BielawskiAbstract:Highlights: Black-Right-Pointing-Pointer A cDNA encoding a novel plant Prolyl Aminopeptidase, TsPAP1, was obtained from triticale. Black-Right-Pointing-Pointer The cloned TsPAP1 cDNA is 1387 bp long and encodes a protein of 390 amino acids. Black-Right-Pointing-Pointer The deduced TsPAP1 protein revealed characteristics of the monomeric bacterial PAPs. Black-Right-Pointing-Pointer The TsPAP1 mRNA level increased under drought, salinity and in the presence of metal ions. -- Abstract: A triticale cDNA encoding a Prolyl Aminopeptidase (PAP) was obtained by RT-PCR and has been designated as TsPAP1. The cloned cDNA is 1387 bp long and encodes a protein of 390 amino acids with a calculated molecular mass of 43.9 kDa. The deduced TsPAP1 protein exhibits a considerable sequence identity with the biochemically characterized bacterial and fungal PAP proteins of small molecular masses ({approx}35 kDa). Moreover, the presence of conserved regions that are characteristic for bacterial monomeric PAP enzymes (the GGSWG motif, the localization of the catalytic triad residues and the segment involved in substrate binding) has also been noted. Primary structure analysis and phylogenetic analysis revealed that TsPAP1 encodes a novel plant PAP protein that is distinct from the multimeric proteins that have thus far been characterized in plants and whose counterparts have been recognized only inmore » bacteria and fungi. A significant increase in the TsPAP1 transcript level in the shoots of triticale plants was observed under drought and saline conditions as well as in the presence of cadmium and aluminium ions in the nutrient medium. This paper is the first report describing changes in the transcript levels of any plant PAP in response to suboptimal growth conditions.« less
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biochemical characterisation of Prolyl Aminopeptidase from shoots of triticale seedlings and its activity changes in response to suboptimal growth conditions
Plant Physiology and Biochemistry, 2011Co-Authors: Urszula Szawlowska, Edyta Zdunekzastocka, Wieslaw BielawskiAbstract:Abstract Prolyl Aminopeptidase (PAP) was isolated from the shoots of three-day-old triticale seedlings and was purified using a five-step purification procedure (acid precipitation, gel filtration, anion-exchange chromatography, hydrophobic chromatography and rechromatography). The enzyme was purified 460-fold with a recovery of 6%. Prolyl Aminopeptidase appears to be a tetramer consisting of four subunits, each with a molecular weight of approximately 54 kDa. Its pH and temperature optimum are pH 7.5 and 37 °C, respectively. The enzyme prefers substrates with Pro and Hyp at the N-terminus, but is also capable of hydrolysing β -naphthylamides ( β -NA) of Ala, Phe, and Leu. The K m value of PAP against Pro- β -NA was the lowest among the substrates tested and it was 1.47 × 10 −5 M. The activity of PAP was not inhibited by EDTA, 1,10-phenantroline, or pepstatin A. The most effective inhibitors were DFP, Pefabloc, and PMSF, which are serine protease inhibitors. However, significant inhibition was also observed in the presence of E-64, which modifies sulfhydryl groups. A significant increase of the Aminopeptidase activity against Pro- β -NA was observed in shoots of triticale plants grown under salinity, drought stress, and in the presence of cadmium and aluminium ions in the nutrient solution.
Takahiko Inoue - One of the best experts on this subject based on the ideXlab platform.
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novel inhibitor for Prolyl Aminopeptidase from serratia marcescens and studies on the mechanism of substrate recognition of the enzyme using the inhibitor
Archives of Biochemistry and Biophysics, 2003Co-Authors: Takahiko Inoue, Nobutada Tanaka, Kazuo T Nakamura, Susumi Hatakeyama, Tomohiro Tozaka, Tadashi YoshimotoAbstract:Abstract Prolyl Aminopeptidase from Serratia marcescens hydrolyzed x–β-naphthylamides (x=Prolyl, alanyl, sarcosinyl, l -α-aminobutylyl, and norvalyl), which suggested that the enzyme has a pocket for a five-member ring. Based on the substrate specificity, novel inhibitors of Pro, Ala, and Sar having 2- tert -butyl-[1,3,4]oxadiazole (TBODA) were synthesized. The K i value of Pro–TBODA, Ala–TBODA, and Sar–TBODA was 0.5 μM, 1.6 μM, and 12 mM, respectively. The crystal structure of enzyme–Pro–TBODA complex was determined. Pro–TBODA was located at the active site. Four electrostatic interactions were located between the enzyme and the amino group of Pro inhibitors (Glu204:0E1-N:Inh, Glu204:0E2-N:Inh, Glu232:0E1-N:Inh, and Gly46:O-N:Inh), and the residue of the inhibitors was inserted into the hydrophobic pocket composed of Phe139, Leu141, Leu146, Tyr149, Tyr150, and Phe236. The roles of Phe139, Tyr149, and Phe236 in the hydrophobic pocket and Glu204 and Glu232 in the electrostatic interactions were confirmed by site-directed mutagenesis, which indicated that the molecular recognition of proline is achieved through four electrostatic interactions and an insertion in the hydrophobic pocket of the enzyme.
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substrate recognition mechanism of Prolyl Aminopeptidase from serratia marcescens
Journal of Biochemistry, 2000Co-Authors: Takahiko Inoue, Tsutomu Kabashima, Huashan Huang, Naota Kanada, Nik Azmi, Essam Azab, Tadashi YoshimotoAbstract:: Molecular cloning of the gene and the crystal structure of the Prolyl Aminopeptidase [EC 3.4.11.5] from Serratia marcescens have been studied by us [J. Biochem. 122, 601-605 (1997); ibid. 126, 559-565 (1999)]. Through these studies, Phe139, Tyr149, Glu204, and Arg136 were estimated to be concerned with substrate recognition. To elucidate the details of the mechanism for the substrate specificity, the site-directed mutagenesis method was applied. The F139A mutant showed an 80-fold decrease in catalytic efficiency (k(cat)/K(m)), but the Y149A mutant did not show a significant change in catalytic efficiency. The catalytic efficiency of the E204Q mutant was about 4% of that of the wild type. The peptidase activity of the mutant (R136A) was markedly decreased, however, arylamidase activity with Pyr-bNA was retained as in the wild-enzyme. From these results, it was clarified that the pyrrolidine ring and the amino group of proline at the S1 site were recognized by Phe139 and Glu204, respectively. P1' of a substrate was recognized by Arg136. On the other hand, the enzyme had two cysteine residues. Mutants C74A and C271A were inhibited by PCMB, but the double mutated enzyme (C74/271A) was resistant to it.
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crystal structure of Prolyl Aminopeptidase from serratia marcescens
Journal of Biochemistry, 1999Co-Authors: Tadashi Yoshimoto, Tsutomu Kabashima, Kouichirou Uchikawa, Takahiko Inoue, Nobutada Tanaka, Kazuo T Nakamura, Masato TsuruAbstract:: Prolyl Aminopeptidase from Serratia marcescens specifically catalyzes the removal of N-terminal proline residues from peptides. We have solved its three-dimensional structure at 2.3 A resolution by the multiple isomorphous replacement method. The enzyme consists of two contiguous domains. The larger domain shows the general topology of the alpha/beta hydrolase fold, with a central eight-stranded beta-sheet and six helices. The smaller domain consists of six helices. The catalytic triad (Ser113, His296, and Asp268) is located near the large cavity at the interface between the two domains. Cys271, which is sensitive to SH reagents, is located near the catalytic residues, in spite of the fact that the enzyme is a serine peptidase. The specific residues which make up the hydrophobic pocket line the smaller domain, and the specificity of the exo-type enzyme originates from this smaller domain, which blocks the N-terminal of P1 proline.
Edyta Zdunekzastocka - One of the best experts on this subject based on the ideXlab platform.
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biochemical characterization of the triticale tspap1 a new type of plant Prolyl Aminopeptidase and its impact on proline content and flowering time in transgenic arabidopsis plants
Plant Physiology and Biochemistry, 2017Co-Authors: Edyta Zdunekzastocka, Agnieszka Grabowska, Tomasz Branicki, Beata MichniewskaAbstract:Abstract Proline Aminopeptidase (PAP, EC 3.4.11.5) is the only enzyme that effectively releases proline from the N-termini of peptides. The amino acid sequence of the PAP from Triticosecale, TsPAP1, comprises conserved regions, characteristic of the monomeric forms of PAP found in bacteria but not yet identified in plants. Therefore, we aimed to obtain and biochemically characterize the TsPAP1 protein. The recombinant TsPAP1 protein was received through heterologous expression of the TsPAP1 coding sequence in a bacterial expression system and purified with affinity chromatography. Gel filtration chromatography and SDS electrophoresis revealed that TsPAP1 is a monomer with a molecular mass of 37.5 kDa. TsPAP1 prefers substrates with proline at the N-terminus but is also capable of hydrolyzing β-naphthylamides of hydroxyproline and alanine. Among the peptides tested, the most preferred were di- and tripeptides, especially those with glycine in the Y position. The use of diagnostic inhibitors indicated that TsPAP1 is a serine peptidase; however, further characterization revealed that the SH residues are also important for maintaining its activity. To examine the role of TsPAP1 under physiological conditions, we developed transgenic Arabidopsis plants overexpressing TsPAP1. Compared with wild-type plants, the transgenic lines accumulated more proline, flowered an average of 3.5 days earlier, and developed more siliques than did untransformed controls. Our paper is the first to describe the biochemical properties of a novel monomeric plant PAP and contributes to the functional characterization of PAP proteins in plants.
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tspap1 encodes a novel plant Prolyl Aminopeptidase whose expression is induced in response to suboptimal growth conditions
Biochemical and Biophysical Research Communications, 2012Co-Authors: Urszula Szawlowska, Edyta Zdunekzastocka, Agnieszka Grabowska, Wieslaw BielawskiAbstract:Highlights: Black-Right-Pointing-Pointer A cDNA encoding a novel plant Prolyl Aminopeptidase, TsPAP1, was obtained from triticale. Black-Right-Pointing-Pointer The cloned TsPAP1 cDNA is 1387 bp long and encodes a protein of 390 amino acids. Black-Right-Pointing-Pointer The deduced TsPAP1 protein revealed characteristics of the monomeric bacterial PAPs. Black-Right-Pointing-Pointer The TsPAP1 mRNA level increased under drought, salinity and in the presence of metal ions. -- Abstract: A triticale cDNA encoding a Prolyl Aminopeptidase (PAP) was obtained by RT-PCR and has been designated as TsPAP1. The cloned cDNA is 1387 bp long and encodes a protein of 390 amino acids with a calculated molecular mass of 43.9 kDa. The deduced TsPAP1 protein exhibits a considerable sequence identity with the biochemically characterized bacterial and fungal PAP proteins of small molecular masses ({approx}35 kDa). Moreover, the presence of conserved regions that are characteristic for bacterial monomeric PAP enzymes (the GGSWG motif, the localization of the catalytic triad residues and the segment involved in substrate binding) has also been noted. Primary structure analysis and phylogenetic analysis revealed that TsPAP1 encodes a novel plant PAP protein that is distinct from the multimeric proteins that have thus far been characterized in plants and whose counterparts have been recognized only inmore » bacteria and fungi. A significant increase in the TsPAP1 transcript level in the shoots of triticale plants was observed under drought and saline conditions as well as in the presence of cadmium and aluminium ions in the nutrient medium. This paper is the first report describing changes in the transcript levels of any plant PAP in response to suboptimal growth conditions.« less
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biochemical characterisation of Prolyl Aminopeptidase from shoots of triticale seedlings and its activity changes in response to suboptimal growth conditions
Plant Physiology and Biochemistry, 2011Co-Authors: Urszula Szawlowska, Edyta Zdunekzastocka, Wieslaw BielawskiAbstract:Abstract Prolyl Aminopeptidase (PAP) was isolated from the shoots of three-day-old triticale seedlings and was purified using a five-step purification procedure (acid precipitation, gel filtration, anion-exchange chromatography, hydrophobic chromatography and rechromatography). The enzyme was purified 460-fold with a recovery of 6%. Prolyl Aminopeptidase appears to be a tetramer consisting of four subunits, each with a molecular weight of approximately 54 kDa. Its pH and temperature optimum are pH 7.5 and 37 °C, respectively. The enzyme prefers substrates with Pro and Hyp at the N-terminus, but is also capable of hydrolysing β -naphthylamides ( β -NA) of Ala, Phe, and Leu. The K m value of PAP against Pro- β -NA was the lowest among the substrates tested and it was 1.47 × 10 −5 M. The activity of PAP was not inhibited by EDTA, 1,10-phenantroline, or pepstatin A. The most effective inhibitors were DFP, Pefabloc, and PMSF, which are serine protease inhibitors. However, significant inhibition was also observed in the presence of E-64, which modifies sulfhydryl groups. A significant increase of the Aminopeptidase activity against Pro- β -NA was observed in shoots of triticale plants grown under salinity, drought stress, and in the presence of cadmium and aluminium ions in the nutrient solution.
Tsutomu Kabashima - One of the best experts on this subject based on the ideXlab platform.
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substrate recognition mechanism of Prolyl Aminopeptidase from serratia marcescens
Journal of Biochemistry, 2000Co-Authors: Takahiko Inoue, Tsutomu Kabashima, Huashan Huang, Naota Kanada, Nik Azmi, Essam Azab, Tadashi YoshimotoAbstract:: Molecular cloning of the gene and the crystal structure of the Prolyl Aminopeptidase [EC 3.4.11.5] from Serratia marcescens have been studied by us [J. Biochem. 122, 601-605 (1997); ibid. 126, 559-565 (1999)]. Through these studies, Phe139, Tyr149, Glu204, and Arg136 were estimated to be concerned with substrate recognition. To elucidate the details of the mechanism for the substrate specificity, the site-directed mutagenesis method was applied. The F139A mutant showed an 80-fold decrease in catalytic efficiency (k(cat)/K(m)), but the Y149A mutant did not show a significant change in catalytic efficiency. The catalytic efficiency of the E204Q mutant was about 4% of that of the wild type. The peptidase activity of the mutant (R136A) was markedly decreased, however, arylamidase activity with Pyr-bNA was retained as in the wild-enzyme. From these results, it was clarified that the pyrrolidine ring and the amino group of proline at the S1 site were recognized by Phe139 and Glu204, respectively. P1' of a substrate was recognized by Arg136. On the other hand, the enzyme had two cysteine residues. Mutants C74A and C271A were inhibited by PCMB, but the double mutated enzyme (C74/271A) was resistant to it.
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crystal structure of Prolyl Aminopeptidase from serratia marcescens
Journal of Biochemistry, 1999Co-Authors: Tadashi Yoshimoto, Tsutomu Kabashima, Kouichirou Uchikawa, Takahiko Inoue, Nobutada Tanaka, Kazuo T Nakamura, Masato TsuruAbstract:: Prolyl Aminopeptidase from Serratia marcescens specifically catalyzes the removal of N-terminal proline residues from peptides. We have solved its three-dimensional structure at 2.3 A resolution by the multiple isomorphous replacement method. The enzyme consists of two contiguous domains. The larger domain shows the general topology of the alpha/beta hydrolase fold, with a central eight-stranded beta-sheet and six helices. The smaller domain consists of six helices. The catalytic triad (Ser113, His296, and Asp268) is located near the large cavity at the interface between the two domains. Cys271, which is sensitive to SH reagents, is located near the catalytic residues, in spite of the fact that the enzyme is a serine peptidase. The specific residues which make up the hydrophobic pocket line the smaller domain, and the specificity of the exo-type enzyme originates from this smaller domain, which blocks the N-terminal of P1 proline.
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Prolyl Aminopeptidase from serratia marcescens cloning of the enzyme gene and crystallization of the expressed enzyme
Journal of Biochemistry, 1997Co-Authors: Tsutomu Kabashima, Ana Kitazono, Atsuko Kitano, Tadashi YoshimotoAbstract:: We cloned and sequenced the Serratia marcescens Prolyl Aminopeptidase (SPAP) gene. Nucleotide sequence analysis revealed an open reading frame of 951 bp, encoding a protein of 317 amino acids with a predicted molecular weight of 36,083. The expressed enzyme was purified about 90-fold on columns of Toyopearl HW65C and DEAE-Toyopearl, with an activity recovery of 30%. The apparent molecular weight of the purified enzyme was 36,000 and 38,000 as estimated by SDS-PAGE and gel filtration, respectively. The enzyme was not inhibited by diisopropyl phosphofluoridate (DFP) or phenylmethylsulfonyl fluoride (PMSF), but was markedly inhibited by 3,4-dichloroisocoumarin (DCIC). Crystals of the enzyme were grown by the hanging drop vapor diffusion method using PEG6000 as a precipitant at pH 6.5. The crystals are tetragonal with cell dimensions a= b =65.6 A, and c=169.8 A, a space group P4(1)2(1)2 or P4(3)2(1)2, and probably contain one monomer in the asymmetric unit. They diffract to at least 2.22 A resolution.
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Prolyl Aminopeptidase gene from flavobacterium meningosepticum cloning purification of the expressed enzyme and analysis of its sequence
Archives of Biochemistry and Biophysics, 1996Co-Authors: Ana Kitazono, Tsutomu Kabashima, Huashan Huang, Tadashi YoshimotoAbstract:Abstract In spite of the numerous studies regarding Prolyl Aminopeptidase, little is known about its mechanism and the significance of its similarity to a number of hydrolases of diverse specificity that belong to the α/β hydrolase-fold family ( Pseudomonas 2-hydroxymuconic semialdehyde hydrolase, atropinesterase, and 2-hydroxy-6-oxophenylhexa-2,4-dienoic acid hydrolase; human and rat epoxide hydrolases). We report the cloning and sequencing of the novel Prolyl Aminopeptidase gene from Flavobacterium meningosepticum (FPAP) which allowed a more comprehensive sequence comparison. FPAP was found to be a 35-kDa monomeric enzyme, releasing N-terminal proline but not hydroxyproline residues from small peptides and naphthylamide esters. Using the unweighted pair group method with arithmetic mean method, an evolutionary tree that depicts the probable relationship between the Prolyl Aminopeptidases and the α/β hydrolase-fold enzymes was constructed. Since the α/β hydrolase-fold family might also include the members of the Prolyl oligopeptidase family (Prolyl oligopeptidase, dipeptidyl peptidase IV, and Prolyl carboxypeptidase), this proposal links all the known Pro-Y bond-cleaving proline-specific peptidases (Prolyl oligopeptidase family, Prolyl Aminopeptidases, and prolinase) as enzymes with similar scaffolds and hydrolytic mechanisms. On the other hand, the enzymes that cleave X-Pro bonds are metalloenzymes grouped within the “pita-bread” fold family (Aminopeptidase P and prolidase). Although the latter two enzymes show significant sequence homology, Prolyl Aminopeptidase, prolinase, and the members of the Prolyl oligopeptidase family do not, and might share the α/β hydrolase-fold scaffold. This rationale would explain the failure in finding a common “proline-recognizing motif” in the primary structures of these proline-specific peptidases.
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Prolyl Aminopeptidase is also present in enterobacteriaceae cloning and sequencing of the hafnia alvei enzyme gene and characterization of the expressed enzyme
Journal of Biochemistry, 1996Co-Authors: Ana Kitazono, Tsutomu Kabashima, Atsuko Kitano, Tadashi YoshimotoAbstract:: The Hafnia alvei Prolyl Aminopeptidase gene (hpap) was cloned and sequenced, the expressed enzyme (HPAP) was purified to homogeneity and thoroughly characterized. An open reading frame of 1,281 bp was found to code for the enzyme, resulting in a protein of 427 amino acids with a molecular weight of 48,577. HPAP resembles the Aeromonas sobria enzyme, having 45% identity and the same distinctive properties with respect to size and substrate specificities. Both enzyme show similar chromatographic behavior, and HPAP could be purified following the procedure previously described for the Aeromonas enzyme. HPAP was found to be resistant to diisopropylphosphofluoridate as are most of the Prolyl Aminopeptidases hitherto described. In spite of this similarity, no inhibition by 1 mM p-chloromercuribenzoate solution could be detected. Significant inhibition was, however, observed when the enzyme was incubated with 3,4-dichloroisocoumarin. This study confirms the presence of two types of Prolyl Aminopeptidases, of which the Hafnia and Aeromonas enzymes constitute one group and the Bacillus, Neisseria, and Lactobacillus enzymes the other, and describes the cloning of the first Prolyl Aminopeptidase gene from an Enterobacteriaceae.
