Structure of proline iminopeptidase from Xanthomonas campestris pv. citri: a prototype for the prolyl oligopeptidase family

The membrane-bound glycoprotein dipeptidyl peptidase IV (DP IV, CD26) is a unique multifunctional protein, acting as receptor, binding and proteolytic molecule. We have determined the sequence and 1.8 Å crystal structure of native DP IV prepared from porcine kidney. The crystal structure reveals a 2-2-2 symmetric tetrameric assembly which depends on the natively glycosylated ␤-propeller blade IV. The crystal structure indicates that tetramerization of DP IV is a key mechanism to regulate its interaction with other components. Each subunit comprises two structural domains, the N-terminal eight-bladed ␤-propeller with open Velcro topology and the C-terminal ␣͞␤-hydrolase domain. Analogy with the structurally related POP and tricorn protease suggests that substrates access the buried active site through the ␤-propeller tunnel while products leave the active site through a separate side exit. A dipeptide mimicking inhibitor complexed to the active site discloses key determinants for substrate recognition, including a Glu-Glu motif that distinguishes DP IV as an aminopeptidase and an oxyanion trap that binds and activates the P 2-carbonyl oxygen necessary for efficient postproline cleavage. We discuss active and nonactive site-directed inhibition strategies of this pharmaceutical target protein.serine protease ͉ oxyanion hole ͉ substrate channeling ͉ drug design ͉ diabetes mellitus

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“…2 and 3. The ␤-sheet exerts a significant twist of more than 90°, in line with observations on related ␣͞␤-hydrolases (33)(34)(35).…”

Section: Resultssupporting

confidence: 87%

“…The sequential and threedimensional arrangement of the catalytic residues Ser-630, His-740, Asp-708 corresponds to that of related ␣͞␤-hydrolases (33,35,36). The oxyanion hole is formed by the amide Tyr-631 and the hydroxyl O of Tyr-547 is occupied by a water molecule in the uninhibited structure.…”

Section: Resultsmentioning

confidence: 99%

The crystal structure of dipeptidyl peptidase IV (CD26) reveals its functional regulation and enzymatic mechanism

Engel

Hoffmann

Wagner

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

293

251

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“…In the aminopeptidase from Xanthomonas campestris pv. citri (28) and the tricorn-interacting aminopeptidase F1 from Thermoplasma acidophilum (12), the corresponding segment constitutes a separate "upper" domain, capping the "lower" ␣/␤ hydrolase domain. The respective active sites of this enzyme, containing the catalytic triad, are located at the interface between the two domains.…”

Section: Resultsmentioning

confidence: 99%

Characterization of a Novel Intracellular Endopeptidase of the α/β Hydrolase Family from Streptomyces coelicolor A3(2)

Nagy

Banerjee²,

Tamura

et al. 2003

J Bacteriol

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In a proteasome-lacking mutant of Streptomyces coelicolor A3(2), an intracellular enzyme with chymotrypsinlike activity, absent from the wild type, was detected. Complementation that restored proteasome function did not suppress expression of the endopeptidase. Since the enzyme was not found in two other S. coelicolor proteasome mutants, its expression probably resulted from a secondary mutation arisen in the proteasome mutant. Purification of the endopeptidase revealed its identity to SCO7095, a putative hydrolase encoded by the S. coelicolor A3(2) genome with no known homologue. Based on the prediction of a Ser-Asp-His catalytic triad and an ␣/␤ hydrolase fold, SCO7095 was assigned to peptidase clan SC. N-terminally His-tagged SCO7095 was efficiently expressed in Escherichia coli cells and purified for further characterization. Although SCO7095 is distantly related to several proline iminopeptidases, including Thermoplasma acidophilum tricorn-interacting F1, no aminopeptidase activity was detected. On synthetic substrates, the monomeric enzyme exhibited not only chymotrypsin-like activity but also thrombin-like activity.

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“…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%

“…3a). A secondary structure prediction of each protein identified 12 a-helices and eight b-strands that correlate with the known structure of XcPIP (Medrano et al, 1998). An additional four helices (Ma1-4) are present in TePAP, PapA and AsPAP, and are predicted to be unique to the multimeric PAPs.…”

Section: Sequence Analysis and Secondary Structure Predictionmentioning

confidence: 95%

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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Structure of proline iminopeptidase from Xanthomonas campestris pv. citri: a prototype for the prolyl oligopeptidase family

Cited by 72 publications

References 45 publications

The crystal structure of dipeptidyl peptidase IV (CD26) reveals its functional regulation and enzymatic mechanism

The crystal structure of dipeptidyl peptidase IV (CD26) reveals its functional regulation and enzymatic mechanism

Characterization of a Novel Intracellular Endopeptidase of the α/β Hydrolase Family from Streptomyces coelicolor A3(2)

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

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