Crystal Structure of Prolyl Aminopeptidase from Serratia marcescens

Yoshimoto, Tadashi; Kabashima, Tsutomu; Uchikawa, Kouichirou; Inoue, Takahiko; Tanaka, Nobutada; Nakamura, Kazuo; Tsuru, Masato; Itō, Kiyoshi

doi:10.1093/oxfordjournals.jbchem.a022486

Cited by 47 publications

(52 citation statements)

References 19 publications

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“…Previously, PAPs have been broadly classified into two groups on the basis of their existence as monomeric or multimeric proteins (Polaina & MacCabe, 2007) and also tentatively by substrate specificity (Yoshimoto et al, 1999). This study definitively shows that PAPs occupy at least two subfamilies that correlate well with multimerization.…”

Section: Discussionmentioning

confidence: 61%

“…This study definitively shows that PAPs occupy at least two subfamilies that correlate well with multimerization. The 3D structures of only two PAPs have been solved (both of which are functional as monomers), from the Gramnegative bacteria Xanthomonas campestris (XcPIP; Medrano et al, 1998) and Serratia marcescens (Yoshimoto et al, 1999). These proteins are folded into two contiguous domains (Fig.…”

Section: Discussionmentioning

confidence: 99%

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Characterization of a multimeric, eukaryotic prolyl aminopeptidase: an inducible and highly specific intracellular peptidase from the non-pathogenic fungus Talaromyces emersonii

et al. 2009

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Fungi are capable of degrading proteins in their environment by secreting peptidases. However, the link between extracellular digestion and intracellular proteolysis has scarcely been investigated. Mycelial lysates of the filamentous fungus Talaromyces emersonii were screened for intracellular peptidase production. Five distinct proteolytic activities with specificity for the p-nitroanilide (pNA) peptides Suc-AAPF-pNA, Suc-AAA-pNA, K-pNA, F-pNA and P-pNA were identified. The native enzyme responsible for the removal of N-terminal proline residues was purified to homogeneity by ammonium sulfate fractionation followed by five successive chromatographic steps. The enzyme, termed Talaromyces emersonii prolyl aminopeptidase (TePAP), displayed a 50-fold specificity for cleaving N-terminal Pro-X (k cat /K m 52.1¾10 6 M "1 s "1 ) compared with Ala-X or Val-X bonds. This intracellular aminopeptidase was optimally active at pH 7.4 and 50 6C. Peptide sequencing facilitated the design of degenerate oligonucleotides from homologous sequences encoding putative fungal proline aminopeptidases, enabling subsequent cloning of the gene. TePAP was shown to be relatively uninhibited by classical serine peptidase inhibitors and to be sensitive to selected cysteine-and histidine-modifying reagents, yet gene sequence analysis identified the protein as a serine peptidase with an a/b hydrolase fold. Northern analysis indicated that Tepap mRNA levels were regulated by the composition of the growth medium. Highest Tepap transcript levels were observed when the fungus was grown in medium containing glucose and the protein hydrolysate casitone. Interestingly, both the induction profile and substrate preference of this enzyme suggest potential co-operativity between extracellular and intracellular proteolysis in this organism. Gel filtration chromatography suggested that the enzyme exists as a 270 kDa homo-hexamer, whereas most bacterial prolyl aminopeptidases (PAPs) are monomers. Phylogenetic analysis of known PAPs revealed two diverse subfamilies that are distinguishable on the basis of primary and secondary structure and appear to correlate with the subunit composition of the native enzymes. Sequence comparisons revealed that PAPs with key conserved topological features are widespread in bacterial and fungal kingdoms, and this study identified many putative PAP candidates within sequenced genomes. This work represents, to our knowledge, the first detailed biochemical and molecular analysis of an inducible PAP from a eukaryote and the first intracellular peptidase isolated from the thermophilic fungus T. emersonii.Abbreviations: AEBSF, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride; DEPC, diethylpyrocarbonate; E-64, trans-epoxy-succinyl-L-leucylamido-(4-guanidino)-butane; NEM, N-ethylmaleimide; PAP, prolyl aminopeptidase; PCMB, p-chloromercuribenzoate; pNA, p-nitroanilide; Pro-AMC, L-proline-7-amino-4-methylcoumarin; TLCK, N-tosyl-L-lysine chloromethyl ketone; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone.3These authors contrib...

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Section: Discussionmentioning

confidence: 61%

Section: Discussionmentioning

confidence: 99%

Characterization of a multimeric, eukaryotic prolyl aminopeptidase: an inducible and highly specific intracellular peptidase from the non-pathogenic fungus Talaromyces emersonii

et al. 2009

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“…Recently, we have reported the crystal structure of prolyl aminopeptidase from Serratia marcescens (17). Because proline and pyroglutamic acid have structures in common with the pyrrolidone ring, a similar substrate recognition mechanism was expected.…”

Section: Discussionmentioning

confidence: 99%

The Mechanism of Substrate Recognition of Pyroglutamyl-peptidase I from Bacillus amyloliquefaciens as Determined by X-ray Crystallography and Site-directed Mutagenesis

Itō

Inoue

Takahashi

et al. 2001

Journal of Biological Chemistry

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Pyroglutamyl-peptidase is able to specifically remove the amino-terminal pyroglutamyl residue protecting proteins or peptides from aminopeptidases. To clarify the mechanism of substrate recognition for the unique structure of the pyrrolidone ring, x-ray crystallography and site-directed mutagenesis were applied. The crystal structure of pyroglutamyl-peptidase bound to a transition state analog inhibitor (Inh), pyroglutaminal, was determined. Two hydrogen bonds were located between the main chain of the enzyme and the inhibitor (71:O⅐⅐⅐H-N:Inh and Gln71:N-H⅐⅐⅐OE:Inh), and the pyrrolidone ring of the inhibitor was inserted into the hydrophobic pocket composed of Phe-10, Phe-13, Thr-45, Ile-92, Phe-142, and Val-143. To study in detail the hydrophobic pocket, Phe-10, Phe-13, and Phe-142 were selected for mutation experiments. The k cat value of the F10Y mutant decreased, but the two phenylalanine mutants F13Y and F142Y did not exhibit significant changes in kinetic parameters compared with the wild-type enzyme. The catalytic efficiencies (k cat /K m ) for the F13A and F142A mutants were less than 1000-fold that of the wild-type enzyme. The x-ray crystallographic study of the F142A mutant showed no significant change except for a minor one in the hydrophobic pocket compared with the wild type. These findings indicate that the molecular recognition of pyroglutamic acid is achieved through two hydrogen bonds and an insertion in the hydrophobic pocket. In the pocket, Phe-10 is more important to the hydrophobic interaction than is Phe-142, and furthermore Phe-13 serves as an "induced fit" mechanism.

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“…The enzyme is composed of ␣/␤-hydrolase fold and helix domains (24). The catalytic mechanism of the enzyme was studied using substrates and inhibitors that each contained a prolyl, alanyl, sarcosyl, L-␣-aminobutyryl, or norvalyl group (8,9).…”

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confidence: 99%

Unusual Extra Space at the Active Site and High Activity for Acetylated Hydroxyproline of Prolyl Aminopeptidase from Serratia marcescens

et al. 2006

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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 Å 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-␤NA (4-acetyloxyproline ␤-naphthylamide) was a better substrate than Pro-␤NA. 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.Prolyl aminopeptidase (proline iminopeptidase; EC 3.4.11.5) catalyzes the removal of N-terminal proline from the peptides. This enzyme was first identified from Escherichia coli by Sarid et al. (21) and was found in several bacteria thereafter. The enzyme genes have been cloned from Aeromonas sobria (13), Bacillus coagulans (14), Flavobacterium meningosepticum (11), Hafnia alvei (12), Lactobacillus delbrueckii subsp. bulgaricus (3), Neisseria gonorrhoeae (1), Thermoplasma acidophilum (22), Xanthomonas campestris pv. citri (2), and Serratia marcescens (10).Most bacteria that produce prolyl aminopeptidase are pathogenic, including S. marcescens, a gram-negative bacterium that causes opportunistic disease in humans. The role of prolyl aminopeptidase seems to break down peptides into amino acids as a nutrient. Prolyl activity is necessary because other aminopeptidases cannot release proline residue efficiently. This is especially important for the ability of some pathogenic bacteria to degrade collagen, which has a large number of Gly-X-Y repeat sequences, where X is often proline and Y is often hydroxyproline. Complete digestion of proline-containing peptides derived from collagen requires prolyl aminopeptidase as well as other aminopeptidases. These enzymes of pathogenic bacteria act especially on collagen degradation. Since many S. marcescens strains are resistant to multiple antibiotics, they represent a growing public health problem. Recently, many patients have been infected by this bacterium in hospitals, and some of them have died. New types of antibacterial drugs different from the ordinary antibiotics for S. marcescens are desi...

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Crystal Structure of Prolyl Aminopeptidase from Serratia marcescens

Cited by 47 publications

References 19 publications

Characterization of a multimeric, eukaryotic prolyl aminopeptidase: an inducible and highly specific intracellular peptidase from the non-pathogenic fungus Talaromyces emersonii

Characterization of a multimeric, eukaryotic prolyl aminopeptidase: an inducible and highly specific intracellular peptidase from the non-pathogenic fungus Talaromyces emersonii

The Mechanism of Substrate Recognition of Pyroglutamyl-peptidase I from Bacillus amyloliquefaciens as Determined by X-ray Crystallography and Site-directed Mutagenesis

Unusual Extra Space at the Active Site and High Activity for Acetylated Hydroxyproline of Prolyl Aminopeptidase from Serratia marcescens

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