X-prolyl Dipeptidyl Aminopeptidase Research Articles

Characterization, Semirational Design for pH Robustness, and the Application in Bioactive Peptide Production of a X-Prolyl Dipeptidyl Aminopeptidase from Lactococcus lactis MY-3.

PepXLcMY-3, an X-prolyl dipeptidyl aminopeptidase derived from Lactobacillus lactis MY-3, was screened and recombinantly expressed in Escherichia coli. The enzyme could exhibit about 40% activity within the pH range of 6.0-10. To further improve the pH robustness, site E396 located in the active pocket was discovered through alanine scanning. The mutant E396I displayed both developed activity and kcat/Km. The optimal pH of E396I shifted from 6.0 to 10 compared to WT, with the relative activity within the pH range of 6.0-10 significantly increased. The site K648 was then proposed by semirational design. The activity of mutant E396I/K648D reached 4.03 U/mg. The optimal pH was restored to 6.0, and the pH stability was further improved. E396I/K648D could totally hydrolyze β-casomorphin 7 within 30 min. The hydrolysate showed 64.5% inhibition on angiotensin I converting enzyme, which was more efficient than those produced by E396I and WT, 23.2 and 44.7%, respectively.

Transcription activity of lactic acid bacterial proteolysis-related genes during cheese maturation

The catabolism of milk protein in cheese is one way how the microorganisms influence the sensorial characteristics of the final product. In this investigation, we paid attention to four genes [prtP (cell-envelope proteinase gene), pepX (X-prolyl dipeptidyl aminopeptidase gene), pepN (aminopeptidase gene) and bcaT (branched chain aminotransferase gene)] responsible for the production of volatile aroma-active compounds from milk proteins by lactic acid bacteria (LAB). We studied the dynamics of these genes and their corresponding LAB host, during the maturation of a raw ewes’ milk-based cheese, using metagenomics and metatranscriptomics approaches. The transcriptome-oriented experiments included the analysis of total RNA (at three stages of cheese maturation) and also the construction of specific cDNA sub-libraries of the abovementioned genes. The proteolytic transcriptome analysis was supported by following the transcription activity of 16S rRNA gene and by metagenomic investigation. The combination of the described methods permitted to screen the dynamics of targeted genes throughout the cheese production. Lactococci were the major players in the LAB group, but the analysis provided also information on the role and properties of members of the genus Lactobacillus, such as Lb. rhamnosus, Lb. helveticus, Lb. pentosus, Lb. curvatus, Lb. parabuchneri, Lb. plantarum, Lb. brevis, Lb. delbrueckii, Lb. paracasei, Lb. fermentum and Lb. heilongjiangensis, proteolysis-related genes of which were active during cheese ripening.

Evaluation of a Commercial Sandwich Enzyme-Linked Immunosorbent Assay for the Quantification of Beta-Casomorphin 7 in Yogurt Using Solid-Phase Extraction Coupled to Liquid Chromatography-Tandem Mass Spectrometry as the "Gold Standard" Method.

This study investigated beta-casomorphin 7 (BCM7) in yogurt by means of LC-tandem MS (MS/MS) and enzyme-linked immunosorbent assay (ELISA) and use LC-MS/MS as the "gold standard" method to evaluate the applicability of a commercial ELISA. The level of BCM7 in milk obtained from ELISA analysis was much lower than that obtained by LC-MS/MS analysis and trended to increase during fermentation and storage of yogurt. Meanwhile, the results obtained from LC-MS/MS showed that BCM7 degraded during stages of yogurt processing, and its degradation may have been caused by X-prolyl dipeptidyl aminopeptidase activity. As a result, the commercial sandwich ELISA kit was not suitable for the quantification of BCM7 in fermented dairy milk.

Effect of high pressure on structural modifications and enzymatic activity of a purified X-prolyl dipeptidyl aminopeptidase from Streptococcus thermophilus

PepX aminopeptidase from Streptococcus thermophilus ACA DC 0022, used in Greek Feta cheese manufacturing, was purified. PepX comprises two subunits of equal molecular mass estimated, using SDS-PAGE and native-PAGE electrophoresis, to be 86 kDa.The effects of high pressure processing (100–450 MPa, combined with 20–40 °C) on purified PepX activity and structure were studied. Activation of the enzyme was observed after processing at 100–200 MPa and 20–30 °C. More intense processing conditions led to enzyme inactivation. PepX HP-induced conformational changes were also investigated through application of Circular Dichroism spectroscopy (CD). Pressures up to 200 MPa resulted in a structurally molten globule-like state where PepX maintained its secondary structure but the tertiary structure was substantially affected and enzyme activity increased. Both secondary and tertiary structures were affected severely by higher pressures (450 MPa), which reduced enzyme activity.

Statistical optimization of cell disruption techniques for releasing intracellular X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis spp. lactis

X-prolyl dipeptidyl aminopeptidase (PepX) is an intracellular enzyme from the Gram-positive bacterium Lactococcus lactis spp. lactis NRRL B-1821, and it has commercial importance. The objective of this study was to compare the effects of several cell disruption methods on the activity of PepX. Statistical optimization methods were performed for two cavitation methods, hydrodynamic (high-pressure homogenization) and acoustic (sonication), to determine the more appropriate disruption method. Two level factorial design (2FI), with the parameters of number of cycles and pressure, and Box–Behnken design (BBD), with the parameters of cycle, sonication time, and power, were used for the optimization of the high-pressure homogenization and sonication methods, respectively. In addition, disruption methods, consisting of lysozyme, bead milling, heat treatment, freeze–thawing, liquid nitrogen, ethylenediaminetetraacetic acid (EDTA), Triton-X, sodium dodecyl sulfate (SDS), chloroform, and antibiotics, were performed and compared with the high-pressure homogenization and sonication methods. The optimized values of high-pressure homogenization were one cycle at 130MPa providing activity of 114.47mUml−1, while sonication afforded an activity of 145.09mUml−1 at 28min with 91% power and three cycles.In conclusion, sonication was the more effective disruption method, and its optimal operation parameters were manifested for the release of intracellular enzyme from a L. lactis spp. lactis strain, which is a Gram-positive bacterium.

Characterization of the recombinant exopeptidases PepX and PepN from Lactobacillus helveticus ATCC 12046 important for food protein hydrolysis.

The proline-specific X-prolyl dipeptidyl aminopeptidase (PepX; EC 3.4.14.11) and the general aminopeptidase N (PepN; EC 3.4.11.2) from Lactobacillus helveticus ATCC 12046 were produced recombinantly in E. coli BL21(DE3) via bioreactor cultivation. The maximum enzymatic activity obtained for PepX was 800 µkatH-Ala-Pro-pNA L−1, which is approx. 195-fold higher than values published previously. To the best of our knowledge, PepN was expressed in E. coli at high levels for the first time. The PepN activity reached 1,000 µkatH-Ala-pNA L−1. After an automated chromatographic purification, both peptidases were biochemically and kinetically characterized in detail. Substrate inhibition of PepN and product inhibition of both PepX and PepN were discovered for the first time. An apo-enzyme of the Zn2+-dependent PepN was generated, which could be reactivated by several metal ions in the order of Co2+>Zn2+>Mn2+>Ca2+>Mg2+. PepX and PepN exhibited a clear synergistic effect in casein hydrolysis studies. Here, the relative degree of hydrolysis (rDH) was increased by approx. 132%. Due to the remarkable temperature stability at 50°C and the complementary substrate specificities of both peptidases, a future application in food protein hydrolysis might be possible.

A New Binding Site Involving the C-terminal Domain to Design Specific Inhibitors of PepX

PepX is a X-prolyl dipeptidyl aminopeptidase of S15 family that cleaves dipeptides from the N-terminus of polypeptides having a proline or alanine residue at the second position. Involved in bacterial nutrition and in peptide maturation, this serine exopeptidase, counterpart of the mammalian DDP-4, has been proposed to play a role in pathogenicity for Streptococci and to be a promising target against trypanosomes. Searching for specific inhibitors, we undertook docking simulations on the whole surface of PepX from Lactococcus lactis, type example of the S15 family, which revealed a new putative binding site in connection with the active site and involving the C-terminal domain. Accordingly to the results of the computations, we synthesized two peptidomimetics of low molecular weight: the valinephenylpiperazine and the valine-isopropylpiperazine that can accommodate to this putative binding site. Experiments revealed that the valine-phenylpiperazine was an uncompetitive inhibitor whereas the valine-isopropylpiperazine showed to be an activator of the enzyme activity. The valine-phenylpiperazine is interacting with ASN 379, GLU 383, GLU 474, residues in connection with the specificity and active sites, and with the residues from the C-terminal domain LEU 693, GLU 710 and GLN 712. These results point out a role of the C-terminal domain in controlling access to the active site of enzymes of the S15 family, like PepX, the cocaine esterase or the alpha-amino acid ester hydrolase, and could have applications in human health giving new perspectives to struggle against streptococci or trypanosomes by designing inhibitors specific to the S15 family of enzymes.

Extracellular Production and Characterization of Streptomyces X-prolyl Dipeptidyl Aminopeptidase

X-prolyl dipeptidyl aminopeptidases (X-PDAPs) are useful in various food industries. In this study, we performed sequence-based screening to obtain a stable X-PDAP enzyme from thermophilic Streptomyces strains. We found three genes that encoded X-PDAP from Streptomyces thermoluteus subsp. fuscus NBRC 14270 (14270 X-PDAP), Streptomyces thermocyaneoviolaceus NBRC 14271 (14271 X-PDAP), and Streptomyces thermocoerulescens NBRC 14273, which were subsequently cloned and sequenced. The deduced amino acid sequences of these genes showed high similarity, with ~80% identity with each other. The isolated X-PDAPs and an X-PDAP from Streptomyces coelicolor were expressed in Streptomyces lividans using a hyperexpression vector: pTONA5a. Among these genes, only 14270 and 14271 X-PDAPs caused overexpression and extracellular production without artificial signal peptides. We also characterized the biochemical properties of purified 14271 X-PDAP. In addition, we found that, in peptide synthesis via an aminolysis reaction, this enzyme recognized D-amino acid derivatives as acyl acceptors, similar to L-amino acid derivatives.

Influence of fermentation temperature and autolysis on ACE-inhibitory activity and peptide profiles of milk fermented by selected strains of Lactobacillus helveticus and Lactococcus lactis

Bovine milk was fermented with single strains of Lc. lactis or Lb. helveticus at three temperatures around the optimum for each species. Fermentation was stopped at pH 4.6 and 4.3/4.0, and the products were stored at 5 °C for 1 and 7 days. The extent of lysis was examined by release of X-prolyldipeptidyl aminopeptidase activity, and the angiotensin-I-converting enzyme- (ACE-) inhibitory activity and peptide profiles of the products were determined. Fermentation temperature significantly influenced the bacterial growth, extent of lysis, and ACE-inhibitory activity. ACE-inhibitory activity was high at all temperatures, and slightly higher at the optimal growth temperature, whereas the extent of lysis was highest at a suboptimal growth temperature. Peptide profiles were marginally affected by temperature and cell lysis. The major peptides were identified, including a number of known ACE-inhibitory peptides. Our results suggest that the cell wall proteinase was the primary catalyst in release of ACE-inhibitory peptides.

Optimization of sour milk fermentation for the production of ACE-inhibitory peptides and purification of a novel peptide from whey protein hydrolysate

The effect of fermentation conditions on the production of angiotensin-I converting enzyme (ACE) inhibitory peptide in sour milk fermented by Lactobacillus helveticus LB10 was investigated using response-surface methodology. Optimal conditions to produce the maximum production of ACE-inhibitory peptides were found to be 4% (v/w) inoculum, 7.5 initial pH of medium and 39.0 °C. The fermented milk resulted in 75.46% inhibition in ACE activity. The cell-envelope proteinase, assisted by X-prolyldipeptidyl aminopeptidase of Lb. helveticus LB10 produced the ACE-inhibitory peptides. A novel ACE-inhibitory peptide from whey protein hydrolysate produced by crude proteinases of Lb. helveticus LB10 was purified. The separations were performed with Sephadex ® G-75 and Sephadex G-15 gel filtration chromatography and reversed-phase, high-performance liquid chromatography. The peptide with the RLSFNP sequence was isolated from β-lactoglobulin hydrolysate and its IC 50 while inhibiting ACE activity was 177.39 μ m.

Mechanism of Degradation of Immunogenic Gluten Epitopes from Triticum turgidum L. var. durum by Sourdough Lactobacilli and Fungal Proteases

As shown by R5 antibody-based sandwich and competitive enzyme-linked immunosorbent assay (ELISA), selected sourdough lactobacilli, in combination with fungal proteases, hydrolyzed gluten (72 h at 37 degrees C) of various cultivars of Triticum turgidum L. var. durum to less than 20 ppm. Complementary electrophoretic, chromatography, and mass spectrometry techniques were used to characterize the gluten and epitope hydrolysis. Nine peptidases were partially purified from the pooled cytoplasmic extract of the sourdough lactobacilli and used to hydrolyze the 33-mer epitope, the most immunogenic peptide generated during digestion of Triticum species. At least three peptidases (general aminopeptidase type N [PepN], X-prolyl dipeptidyl aminopeptidase [PepX], and endopeptidase PepO) were necessary to detoxify the 33-mer without generation of related immunogenic epitopes. After 14 h of incubation, the combination of all or at least six different peptidases totally hydrolyzed the 33-mer (200 mM) into free amino acids. The same results were found for other immunogenic epitopes, such as fragments 57-68 of alpha 9-gliadin, 62-75 of A-gliadin, and 134-153 of gamma-gliadin. When peptidases were used for fermentation of durum wheat semolina, they caused the hydrolysis of gluten to ca. 2 ppm. The in vivo digestion was simulated, and proteins/peptides extracted from pepsin-trypsin (PT) digestion of durum wheat semolina fermented with selected sourdough lactobacilli induced the expression of gamma interferon and interleukin 2 at levels comparable to those of the negative control. Durum wheat semolina fermented with sourdough lactobacilli was freeze-dried and used for making Italian-type pasta. The scores for cooking and sensory properties for this pasta were higher that those of conventional gluten-free pasta.

A Model to Assess Lactic Acid Bacteria Aminopeptidase Activities in Parmigiano Reggiano Cheese During Ripening

The aim of this work was to investigate in which phases of ripening of Parmigiano Reggiano cheese lactic acid bacteria aminopeptidases present in cheese extract could be involved in release of free amino acids and to better understand the behavior of these enzymes in physical-chemical conditions that are far from their optimum. In particular, we evaluated 6 different substrates to reproduce broad-specificity aminopeptidase N, broad-specificity aminopeptidase C, glutamyl aminopeptidase A, peptidase with high specificity for leucine and alanine, proline iminopeptidase, and X-prolyl dipeptidyl aminopeptidase activities releasing different N-terminal amino acids. The effects of pH, NaCl concentration, and temperature on the enzyme activities of amino acid β-naphthylamide (βNA)-substrates were determined by modulating the variables in 19 different runs of an experimental design, which allowed the building of mathematical models able to assess the effect on aminopeptidases activities over a range of values, obtained with bibliographic data, covering different environmental conditions in different zones of the cheese wheel at different aging times. The aminopeptidases tested in this work were present in cell-free Parmigiano Reggiano cheese extract after a 17-mo ripening and were active when tested in model system. The modeling approach shows that to highlight the individual and interactive effects of chemical-physical variables on enzyme activities, it is helpful to determine the true potential of an amino-peptidase in cheese. Our results evidenced that the 6 different lactic acid bacteria peptidases participate in cheese proteolysis and are induced or inhibited by the cheese production parameters that, in turn, depend on the cheese dimension. Generally, temperature and pH exerted the more relevant effects on the enzymatic activities, and in many cases, a relevant interactive effect of these variables was observed. Increasing salt concentration slowed down broad-specificity amino-peptidase C, glutamyl aminopeptidase A, proline iminopeptidase, and peptidase with high specificity for leucine and alanine. Interestingly, this variable did not affect broad-specificity aminopeptidase N and positively affected X-prolyl dipeptidyl aminopeptidase. The models elaborated varying pH, temperatures, and salt concentration and were a useful, low cost, and fast tool to understand the role of the main peptidases in the different phases of cheese ripening in relation to the major environmental factors influencing enzyme activity.

Angiotensin I-converting enzyme inhibitory activity in milk fermented by wild-type and peptidase-deletion derivatives of Lactobacillus helveticus CNRZ32

Development of molecular methods has enabled detailed studies of the proteolytic system of Lactobacillus helveticus, which has a central role in the release of bioactive peptides during the fermentation of milk. The impact of general aminopeptidase (PepN) and X-prolyl dipeptidyl aminopeptidase (PepX) activity of L. helveticus on the level of angiotensin I-converting enzyme (ACE) inhibitory activity in fermented milk was elucidated by taking advantage of peptidase-negative derivatives of L. helveticus CNRZ32. According to the results, increased level of ACE-inhibitory activity was attained in milk fermented by the peptidase-deficient mutants. The ACE-inhibitory activity determined with the PepN-deficient strain decreased towards the end of fermentation, suggesting that PepN gives rise to a provisional accumulation of ACE-inhibitory peptides before their hydrolysis to shorter peptides or free amino acids. The ACE-inhibitory activity determined with the PepX-deficient strain increased throughout the fermentation, indicating specific blocking of the further hydrolysis of ACE-inhibitory peptides.

Enzymatic activities of lactic acid bacteria isolated from Cornetto di Matera sourdoughs

Forty-one strains of lactic acid bacteria (LAB) isolated from Cornetto di Matera sourdoughs were screened for their enzymatic activities, to elucidate their possible roles during the fermentation process. Urease, peptidase, phytase, phosphatase and β-glucosidase activities were measured spectrophotometrically using synthetic substrates. Proteolytic activities were examined in model doughs, using neutral and acidified sterile doughs as controls. All strains had low urease, glutamyl aminopeptidase and iminopeptidase activities, whereas differences within species were observed for the other enzymatic activities. Leuconostoc mesenteroides and Lactobacillus curvatus strains generally showed high aminopeptidase, X-prolyl dipeptidyl aminopeptidase, β-glucosidase and phytase activities, while the enzymatic activities of Lactobacillus plantarum, Lactobacillus pentosus and Weissella cibaria varied between strains. In order to classify the strains on the basis of similar enzymatic profiles, a hierarchical cluster analysis was carried out. Several strains of L. plantarum, L. curvatus and Leuc. mesenteroides showed an interesting combination of proteolytic, peptidase, β-glucosidase and phytase activities, suggesting their possible usefulness as a mixed starter culture in bread-making processes.

Enzymatic ability of Bifidobacterium animalis subsp. lactis to hydrolyze milk proteins: identification and characterization of endopeptidase O.

The proteolytic system of Bifidobacterium animalis subsp. lactis was analyzed, and an intracellular endopeptidase (PepO) was identified and characterized. This work reports the first complete cloning, purification, and characterization of a proteolytic enzyme in Bifidobacterium spp. Aminopeptidase activities (general aminopeptidases, proline iminopeptidase, X-prolyl dipeptidylaminopeptidase) found in cell extracts of B. animalis subsp. lactis were higher for cells that had been grown in a milk-based medium than for those grown in MRS. A high specific proline iminopeptidase activity was observed in B. animalis subsp. lactis. Whole cells and cell wall-bound protein fractions showed no caseinolytic activity; however, the combined action of intracellular proteolytic enzymes could hydrolyze casein fractions rapidly. The endopeptidase activity of B. animalis subsp. lactis was examined in more detail, and the gene encoding an endopeptidase O in B. animalis subsp. lactis was cloned and overexpressed in Escherichia coli. The deduced amino acid sequence for B. animalis subsp. lactis PepO indicated that it is a member of the M13 peptidase family of zinc metallopeptidases and displays 67.4% sequence homology with the predicted PepO protein from Bifidobacterium longum. The recombinant enzyme was shown to be a 74-kDa monomer. Activity of B. animalis subsp. lactis PepO was found with oligopeptide substrates of at least 5 amino acid residues, such as met-enkephalin, and with larger substrates, such as the 23-amino-acid peptide alpha s1-casein(f1-23). The predominant peptide bond cleaved by B. animalis subsp. lactis PepO was on the N-terminal side of phenylalanine residues. The enzyme also showed a post-proline secondary cleavage site.

Partial purification and characterization of an X-prolyl dipeptidyl aminopeptidase from Lactobacillus sanfranciscensis CB1

An X-prolyl dipeptidyl aminopeptidase (PepX) from Lactobacillus sanfranciscensis CB1, a key sourdough lactic acid bacterium, was partially purified by five chromatographic steps. As estimated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration, the enzyme appeared to be a 49.2–56 kDa monomer. Optimal activity occurred at pH 6.0 and 30 °C, with K m of 0.6 mM and V max of 99.6 nmol mg −1 min −1. The D value, calculated at 45 °C, was ∼5.46 s. The enzyme hydrolyzed (almost exclusively) substrates with a X-Pro N-terminal sequence. It did not possess prolidase, aminopeptidase or endopeptidase activities. No hydrolysis of the Pro-rich 33-mer epitope (a potent inducer of gut-derived human T-cell lines in celiac patients) was found when it was treated with PepX alone. When the general aminopeptidase type N was combined with PepX, the hydrolysis of 33-mer peptide (0.2 mM) was complete after 24 h of incubation at 30 °C. Leucine and glutamine residues were liberated from the Pro-rich 33-mer peptide by aminopeptidase type N, thus favouring the subsequent PepX activity. PepX was inactivated by p-chloromercuribenzoate, 3,4-dichloroisocoumarin and phenanthroline which exerted a competitive mode of inhibition. Among divalent cations, only Zn 2+, Hg 2+ and Mn 2+ markedly decreased the enzyme activity. Quadratic response surface methodology was used to study the individual and interactive effects of temperature, pH and NaCl on the PepX activity. The enzyme maintained considerable activity under the environmental conditions which characterize the sourdough fermentation.

Impaired growth rates in milk of Lactobacillus helveticus peptidase mutants can be overcome by use of amino acid supplements.

To evaluate the contribution of intracellular peptidases to the growth of the 14-amino-acid (aa) auxotroph Lactobacillus helveticus CNRZ32, single- and multiple-peptidase-deletion mutants were constructed. Two broad-specificity aminopeptidases (PepC and PepN) and X-prolyl dipeptidyl aminopeptidase (PepX) were inactivated through successive cycles of chromosomal gene replacement mutagenesis. The inactivation of all three peptidases in JLS247 ((Delta)pepC (Delta)pepN (Delta)pepX) did not affect the growth rate in amino acid-defined medium. However, the peptidase mutants generally had decreased specific growth rates when acquisition of amino acids required hydrolysis of the proteins in milk, the most significant result being a 73% increase in generation time for JLS247. The growth rate deficiencies in milk were overcome by amino acid supplements with some specificity to each of the peptidase mutants. For example, milk supplementation with Pro resulted in the most significant growth rate increase for (Delta)pepX strains and a 7-aa supplement (Asn, Cys, Ile, Pro, Ser, Thr, and Val) resulted in a JLS247 growth rate indistinguishable from that of the wild type. Our results show that characterization of the activities of the broad-specificity aminopeptidases had little predictive value regarding the amino acid supplements found to enhance the milk growth rates of the peptidase mutant strains. These results represent the first determination of the physiological roles with respect to specific amino acid requirements for peptidase mutants grown in milk.

Purification and partial characterisation of X-prolyl dipeptidyl aminopeptidase of Lactobacillus helveticus ITG LH1

Abstract A X-prolyl dipeptidyl aminopeptidase (EC 3.4.14.5, XPDAP) from Lactobacillus helveticus ITG LH1, a strain used for Swiss-type cheese, was purified by ion exchange and affinity chromatographies. The enzyme appeared to be a 140 kDa monomer. Optimal activity occurred at pH 7 and 40°C, but it was rapidly inactivated above 50°C. The enzyme was activated by NaCl and KCl up to 50–200 m m but its activity levelled off at higher salt concentrations. Its complete inhibition was caused by 0.1 m m HgCl 2 , 1 m m SnCl 2 and 2.5 m m CuCl 2 . It was inactivated by reagents specific for serine proteases, such as phenylmethylsulfonyl fluoride and sulfhydryl group-blocking reagents. The enzyme hydrolysed p -nitroanilide-substituted X-Pro and X-Ala dipeptides, as well as β -casomorphin 1-4.

The Structural Basis for Catalysis and Specificity of the X-Prolyl Dipeptidyl Aminopeptidase from Lactococcus lactis

The X-prolyl dipeptidyl aminopeptidase (X-PDAP) from Lactococcus lactis is a dimeric enzyme catalyzing the removal of Xaa-Pro dipeptides from the N terminus of peptides. The structure of the enzyme was solved at 2.2 Å resolution and provides a model for the peptidase family S15. Each monomer is composed of four domains. The larger one presents an α/β hydrolase fold and comprises the active site serine. The specificity pocket is mainly built by residues from a small helical domain which is, together with the N-terminal domain, essential for dimerization. A C-terminal moiety probably plays a role in the tropism of X-PDAP toward the cellular membrane. These results give new insights for further exploration of the role of the enzymes of the SC clan.

Identification of the Catalytic Residues of α-Amino Acid Ester Hydrolase from Acetobacter turbidans by Labeling and Site-directed Mutagenesis

The alpha-amino acid ester hydrolase from Acetobacter turbidans ATCC 9325 is capable of hydrolyzing and synthesizing the side chain peptide bond in beta-lactam antibiotics. Data base searches revealed that the enzyme contains an active site serine consensus sequence Gly-X-Ser-Tyr-X-Gly that is also found in X-prolyl dipeptidyl aminopeptidase. The serine hydrolase inhibitor p-nitrophenyl-p'-guanidino-benzoate appeared to be an active site titrant and was used to label the alpha-amino acid ester hydrolase. Electrospray mass spectrometry and tandem mass spectrometry analysis of peptides from a CNBr digest of the labeled protein showed that Ser(205), situated in the consensus sequence, becomes covalently modified by reaction with the inhibitor. Extended sequence analysis showed alignment of this Ser(205) with the catalytic nucleophile of some alpha/beta-hydrolase fold enzymes, which posses a catalytic triad composed of a nucleophile, an acid, and a base. Based on the alignments, 10 amino acids were selected for site-directed mutagenesis (Arg(85), Asp(86), Tyr(143), Ser(156), Ser(205), Tyr(206), Asp(338), His(370), Asp(509), and His(610)). Mutation of Ser(205), Asp(338,) or His(370) to an alanine almost fully inactivated the enzyme, whereas mutation of the other residues did not seriously affect the enzyme activity. Circular dichroism measurements showed that the inactivation was not caused by drastic changes in the tertiary structure. Therefore, we conclude that the catalytic domain of the alpha-amino acid ester hydrolase has an alpha/beta-hydrolase fold structure with a catalytic triad of Ser(205), Asp(338), and His(370). This distinguishes the alpha-amino acid ester hydrolase from the Ntn-hydrolase family of beta-lactam antibiotic acylases.