Substrate Recognition Mechanism of Prolyl Aminopeptidase from Serrratia marcescens

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 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.

296

251

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“…Site-directed mutagenesis confirmed the contributions of Phe139, Phe236, and Tyr149 to the hydrophobic pocket, as well as those of Glu204 and Glu232 to the salt bridge formation (8,9). The present study focuses on the interface between the enzyme and the substrate.…”

Section: Discussionsupporting

confidence: 51%

“…The cloned plasmids were then used to transform E. coli DH1 or DH5␣ for expression. Other mutants were reported previously (9).…”

Section: Methodsmentioning

confidence: 87%

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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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“…The S1 specificity pocket ensures a hydrophobic environment (Trp-595, Phe-476, Val-644, Val-580, and Tyr-599) and a snug fit for the proline residue, and the specificity for proline residue was enhanced by the stacking between the indole ring of Trp-595 and the pyrrolidine ring of the substrate proline residue (distance, 3.4 to ϳ3.8 Å). From the superposition of both active sites, Phe-139 of prolyl aminopeptidase was estimated to have the same role as Trp-595 of prolyl endopeptidase (19). However, no remarkable hydrogen bonding between the proline residue of the inhibitor and prolyl oligopeptidase was found.…”

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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Substrate Recognition Mechanism of Prolyl Aminopeptidase from Serrratia marcescens

Cited by 29 publications

References 7 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

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

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

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