Identification of Amino Acid Residues Essential for the Catalytic Reaction of Bacillus kaustophilus Leucine Aminopeptidase

The functional significance of amino acid residues Lys-265, Asp-270, Lys-277, Asp-288, Asp-347, Glu-349, and Arg-351 of Bacillus kaustophilus leucine aminopeptidase was explored by site-directed mutagenesis. Variants with an apparent molecular mass of approximately 54 kDa were overexpressed in Escherichia coli and purified to homogeneity by nickel-chelate chromatography. The purified mutant enzymes had no LAP activity, implying that these residues are important for the catalytic reaction of the enzyme.     

Site-directed mutagenesis of the conserved Ala348 and Gly350 residues at the putative active site of Bacillus kaustophilus leucine aminopeptidase

Leucine aminopeptidase (LAP) is an exopeptidase that catalyzes the hydrolysis of amino acid residues from the amino terminus of proteins and peptides. Sequence alignment shows that the conserved Ala348 and Gly350 residues of Bacillus kaustophilus LAP (BkLAP) are located right next to a coordinated ligand. We further investigated the roles of these two residues by performing computer modeling and site-directed mutagenesis. Based on the modeling, the carbonyl group of Ala348 interacts with Asn345 and Asn435, and that of Gly350 with Ile353 and Leu354, where these interactions might maintain the zinc-coordinated residues at their correct positions. Replacement of Ala348 with Arg resulted in a dramatic reduction in LAP activity. A complete loss of the activity was also observed in A348E, A348V, and the Gly350 variants. Measurement of intrinsic tryptophan fluorescence revealed alteration of the microenvironment of aromatic amino acid residues, while circular dichroism spectra were nearly identical for wild-type and all mutant enzymes. Protein modeling and site-directed mutagenesis suggest that residues Ala348 and Gly350 are essential for BkLAP in maintaining a stable active-site environment for the catalytic reaction.     

Inactivation of Bacillus stearothermophilus Leucine Aminopeptidase II by Hydrogen Peroxide and Site-Directed Mutagenesis of Methionine Residues on the Enzyme

Leucine aminopeptidases (LAPs) are exopeptidases that remove the N-terminal L-leucine from peptide substrates. Oxidative stability assay showed that the recombinant Bacillus stearothermophilus LAP II (rLAPII) was sensitive to oxidative damage by hydrogen peroxide at the elevated temperature. The H2O2-treated enzyme experienced obvious changes in the secondary structure when the oxidant concentration increased to 300 mM. To investigate the role of methionine residues on the oxidative inactivation, each of the five methionine residues in the rLAPII was replaced with leucine by site-directed mutagenesis. The mutant enzymes with an apparent Mr of approximately 44.5 kDa were overexpressed in Escherichia coli and were purified to homogeneity by nickel-chelate chromatography. The specific activities for Met82Leu, Met88Leu, Met254Leu, and Met382Leu were similar to that of the wild-type enzyme, whereas a reduced activity was observed in Met136Leu. The 50% decrease in the catalytic efficiency (kcat/Km) for Met136Leu was caused by 47% decrease in kcat value. As compared with the wild-type enzyme, all mutant proteins were more sensitive to the oxidant, implying that the methionine residues of B. stearothermophilus LAP II are important for the protection of the enzyme from oxidative inactivation.     

Residues Arg114 and Arg337 are critical for the proper function of Escherichia coli gamma-glutamyltranspeptidase

To evaluate the importance of conserved Arg114 and Arg337 residues of Escherichia coli γ-glutamyltranspeptidase (EcGGT), Lys, Leu, or Asp-substituted mutants were constructed by site-directed mutagenesis. The wild-type and mutant enzymes were overexpressed in the recombinant E. coli M15 and purified by nickel-chelate chromatography to near homogeneity. With the exception of R114K, all the other mutants significantly lost GGT activity, confirming the importance of these two residues in EcGGT. Kinetic analysis of R114L, R114D, R337K, and R337L revealed a significant increase in K_m with a minor change in k_cat, leading to more than an 8-fold decrease in k_cat/K_m values. Mutations of Arg337 impaired the capability of autocatalytic processing of the enzyme. In vitro maturation experiments revealed that EcGGT precursor mutants, pro-R337K and pro-R337L, could precede a time-dependent autocatalytic process to generate the small and large subunits, while no autocatalytic processing was observed in pro-R337D. Computer modeling showed that the critical bonding distance of Gln390 O-Thr391 HG1 and Gln390 C-Thr391 OG1 are significantly increased in Arg337 replacements, implying that these distance changes might be responsible for the lack of enzyme maturation.     

Generating oxidation-resistant variants of <Emphasis Type="Italic">Bacillus kaustophilus</Emphasis> leucine aminopeptidase by substitution of the critical methionine residues with leucine

Bacillus kaustophilus leucine aminopeptidase (bkLAP) was sensitive to oxidative damage by hydrogen peroxide. To improve its oxidative stability, the oxidation-sensitive methionine residues in the enzyme were replaced with leucine by site-directed mutagenesis. The variants, each with an apparent molecular mass of approximately 54 kDa, were overexpressed in recombinant Escherichia coli M15 cells and purified to homogeneity by nickel-chelate chromatography. The specific activity for M282L, M285L, M289L and M321L decreased by more than 43%, while M400L, M426L, M445L, and M485L showed 191, 79, 313, and 103%, respectively, higher activity than the wild-type enzyme. Although the mutations did not cause significant changes in the K _m value, more than 67.8% increase in the value of k _cat/K _m was observed in the M400L, M426L, M445L and M485L. In the presence of 50 mM H₂O₂, most variants were more stable with respect to the wild-type enzyme, indicating that the oxidative stability of the enzyme can be improved by engineering the methionine residues. This revised version was published online in June 2006 with corrections to the Cover Date.     

Histidines 345 and 378 of <Emphasis Type="Italic">Bacillus stearotheromophilus</Emphasis> leucine aminopeptidase II are essential for the catalytic activity of the enzyme

The conserved histidine residues, His-191, His-227, His-345, and His-378, in Bacillus stearothermophilus leucine aminopeptidase II (LAPII) were replaced with leucine by site-directed mutagenesis. The overexpressed wild-type and mutant enzymes have been purified by nickel-chelate chromatography and their molecular masses were approximately 44.5 kDa. Under assay conditions, no LAP activity was detected in H345L and H378L. Although the K_m value for H191L increased more than 30% with respect to the wild-type LAPII, alteration in this residue did not lead to a significant change on the catalytic efficiency. The 39% decrease in K_cat/K_m for H227L was partly caused by a 3.9-fold increase in K_m value. Based on these results, it is suggested that His-345 and His-378 play a crucial role in the catalytic reaction of B. stearothermophilus LAPII.     

Identification of glutamate residues important for catalytic activity of Bacillus stearothermophilus leucine aminopeptidase II

Each of four conserved glutamate residues of Bacillus stearothermophilus leucine aminopeptidase II (BsLAPII) was replaced with aspartate, lysine, and leucine respectively by site-directed mutagenesis. The over-expressed wild-type and mutant enzymes were purified to homogeneity by nickel-chelate chromatography and the molecular mass of the subunit was determined to be 44.5 kDa by SDS-PAGE. The specific activity for the Glu-316 and Glu-340 mutants was completely abolished, while Glu-249 mutants showed comparable activity to that of the wild-type BsLAPII. Compared with the wild-type enzyme, the E250D and E250L mutant enzymes retained less than 18% of the enzyme activity and exhibited a dramatic decrease in the value of k _cat/K _m. These observations indicate that Glu-250, Glu-316, and Glu-340 residues are critical for the catalytic activity of BsLAPII.     

Purification and characterization of leucine aminopeptidase from kidney bean cotyledons

A leucine aminopeptidase (EC 3,4,11.1) was purified from cotyledons of resting kidney beans ( Phaseolus vulgaris L. cv. Processor) by acidic extraction, ammonium sulfate fractionation and chromatography on DEAE-Sephacel, Sephacryl S-300, Mono Q HPLC and Superose HPLC columns. The yield of the 317-fold purified enzyme was 9%. On gel filtrations on Sephacryl S-300 and Superose HPLC the elution volumes of the enzyme corresponded to an M, of 360 000. The enzyme gave one band on native gel electrophoresis and an electrophoretic titration in an immobilized pH gradient gave a single curve with a pI of 4.8. Two bands were observed in an SDS-gel electrophoresis with M_r values of 58 000 and 60 000 both with and without reduction by 2-mercaptoethanol, indicating that subunits of the enzyme are not linked by disulphide bridges. The purified enzyme most rapidly liberated Leu and Ala of the N-termini of di-and oligopeptides, optimally at pH 9.0 ± 0.5. The enzyme was stable in the presence of glycerol, dithiothreitol and Mg²⁺, while the latter also had an activating effect. Bestatin inhibited the enzyme competitively with Leu-Gly-Gly with a Kⁱ-value of 1.5 nM . These observations indicate that the purified aminopeptidase from the cotyledons of resting kidney beans corresponds to the cytosolic leucine aminopeptidase of mammalian tissues (EC 3.4, 11.1). The high enzyme activity observed suggests that this aminopeptidase has an important role in the production of free amino acids during germination.     

Structure-based approach to alter the substrate specificity of Bacillus subtilis aminopeptidase

Aminopeptidases can selectively catalyze the cleavage of the N-terminal amino acid residues from peptides and proteins. Bacillus subtilis aminopeptidase (BSAP) is most active toward p-nitroanilides (pNAs) derivatives of Leu, Arg, and Lys. The BSAP with broad substrate specificity is expected to improve its application. Based on an analysis of the predicted structure of BSAP, four residues (Leu 370, Asn 385, Ile 387, and Val 396) located in the substrate binding region were selected for saturation mutagenesis. The hydrolytic activity toward different aminoacyl-pNAs of each mutant BSAP in the culture supernatant was measured. Although the mutations resulted in a decrease of hydrolytic activity toward Leu-pNA, N385L BSAP exhibited higher hydrolytic activities toward Lys-pNA (2.2-fold) and Ile-pNA (9.1-fold) than wild-type BSAP. Three mutant enzymes (I387A, I387C and I387S BSAPs) specially hydrolyzed Phe-pNA, which was undetectable in wild-type BSAP. Among these mutant BSAPs, N385L and I387A BSAPs were selected for further characterized and used for protein hydrolysis application. Both of N385L and I387A BSAPs showed higher hydrolysis efficiency than the wild-type BASP and a combination of the wild-type and N385L and I387A BSAPs exhibited the highest hydrolysis efficiency for protein hydrolysis. This study will greatly facilitate studies aimed on change the substrate specificity and our results obtained here should be useful for BSAP application in food industry.     

Electrophoretic polymorphism of erythrocyte leucine aminopeptidase in the wild boar, Sus scrofa

Summary. An erythrocyte leucine aminopeptidase (Rbc LAP) electrophoretic polymorphism was detected in Italian wild boars, Sus scrofa. Such a polymorphism has not previously been reported in the domestic pig. It is suggested that this locus could be a marker for genetic differences between the domestic and the wild forms of Sus scrofa.     

Identification of amino acid residues essential for the catalytic reaction of Bacillus kaustophilus leucine aminopeptidase

The functional significance of amino acid residues Lys-265, Asp-270, Lys-277, Asp-288, Asp-347, Glu-349, and Arg-351 of Bacillus kaustophilus leucine aminopeptidase was explored by site-directed mutagenesis. Variants with an apparent molecular mass of approximately 54 kDa were overexpressed in Escherichia coli and purified to homogeneity by nickel-chelate chromatography. The purified mutant enzymes had no LAP activity, implying that these residues are important for the catalytic reaction of the enzyme.     

MHJ_0461 is a multifunctional leucine aminopeptidase on the surface of Mycoplasma hyopneumoniae

Aminopeptidases are part of the arsenal of virulence factors produced by bacterial pathogens that inactivate host immune peptides. Mycoplasma hyopneumoniae is a genome-reduced pathogen of swine that lacks the genetic repertoire to synthesize amino acids and relies on the host for availability of amino acids for growth. M. hyopneumoniae recruits plasmin(ogen) onto its cell surface via the P97 and P102 adhesins and the glutamyl aminopeptidase MHJ_0125. Plasmin plays an important role in regulating the inflammatory response in the lungs of pigs infected with M. hyopneumoniae. We show that recombinant MHJ_0461 (rMHJ_0461) functions as a leucine aminopeptidase (LAP) with broad substrate specificity for leucine, alanine, phenylalanine, methionine and arginine and that MHJ_0461 resides on the surface of M. hyopneumoniae. rMHJ_0461 also binds heparin, plasminogen and foreign DNA. Plasminogen bound to rMHJ_0461 was readily converted to plasmin in the presence of tPA. Computational modelling identified putative DNA and heparin-binding motifs on solvent-exposed sites around a large pore on the LAP hexamer. We conclude that MHJ_0461 is a LAP that moonlights as a multifunctional adhesin on the cell surface of M. hyopneumoniae.     

A new variant of serum leucine aminopeptidase in the mouse: Its development and possible regulation

Serum leucine aminopeptidase (LAP) isozymes were compared in four strains of inbred mice during postnatal development, adult life, and pregnancy. In pregnancy, no changes in the maternal serum LAP pattern were observed, in contrast to human studies. One strain, DD/S, differs from the other three in serum LAP. Polymorphism in serum LAP has not been previously described in the mouse. Neonatal DD/S mice exhibit a single band of serum LAP upon starch gel electrophoresis; however, between 14 and 18 days of age, two distinct bands appear, which persist throughout adult life. In the strains C57BL/6J, BALB/cJ, and DBA/2J there is a single band of activity at all stages. Crosses and backcrosses between DD/S and C57BL/6J show that the double-band variant is inherited as an autosomal recessive. The variant is independent of both the supernatant malic enzyme (Mod-1) and the intestinal LAP (Lap-1) loci, which are known to be linked on chromosome 9. The serum LAP variant is linked to an intestinal alkaline phosphatase variant. The presence of a separate structural gene is suggested by the genetic independence of the serum LAP variant from Lap-1. Also, the two serum LAP bands of DD/S are not interconverted by treatment with neuraminidase, -mercaptoethanol, or heat or by mixing the sera of DD/S and C57BL/6J prior to electrophoresis. The level of serum LAP activity in DD/S is approximately twice that in C57BL/6J. While these observations imply two structurally distinct proteins, the absence of any trace of the second LAP band in the heterozygote strongly suggests that the LAP variant protein is not the result of a separate structural gene. Intestinal LAP in DD/S migrates with the same electrophoretic mobility as the serum LAP variant, implying that the variant might originate in the intestine and its appearance in the serum be modulated by some factor at an unlinked locus.This work was supported by National Institutes of Health Grant RR08117.     

Purification and properties of an intracellular leucine aminopeptidase from the fungus,Penicillium citrinum strain IFO 6352

An intracellular leucine aminopeptidase (LAP) fromPenicillium citrinum (IFO 6352) was purified to homogeneity using three successive purification steps. The enzyme has a native molecular mass of 63 kDa using HPLC gel filtration analysis and a molecular mass of 65 kDa when using SDS-polyacrylamide gel electrophoresis. This monomeric aminopeptidase showed maximum enzyme activity at pH 8.5. An optimum temperature was 45–50°C whenl-Leu-p-nitroanilide (pNA) was the substrate, and enzyme activity drastically decreased above 60°C. The Michaelis-Menten constants forl-Leu-pNA andl-Met-pNA were 2.7 mM and 1.8 mM, respectively. When the enzyme reacted with biosynthetic methionyl human growth hormone, it showed high specificity for N-terminal methionine residue and recognized a stop sequence (Xaa-Pro). The aminopeptidase was inactivated by EDTA or 1,10-phenanthroline, indicating that it is a metallo-exoprotease. Enzyme activity was restored to 90% of maximal activity by addition of Co²⁺ ions. The activity of EDTA-treated enzyme was restored by addition of Zn²⁺, but reconstitution with Ca²⁺, Mg²⁺ or Mn²⁺ restored some enzyme activity. It is likely that Co²⁺ ions play an important role in the catalysis or stability of thePenicillium citrinum aminopeptidase, as zinc plays a similar function in other leucine aminopeptidases.     

Analysis of essential leucine residue for catalytic activity of novel thermostable chitosanase by site-directed mutagenesis

Bacterial chitosanases share weak amino acid sequence similarities at certain regions of each enzyme. These regions have been assumed to be important for catalytic activities of the enzyme. To verify this assumption, the functional importance of the conserved region in a novel thermostable chitosanase (TCH-2) from Bacillus coagulans CK108 was investigated. Each of the conserved amino acid residues (Leu64, Glu80, Glu94, Asp98, and Gly108) was changed to aspartate and glutamine or asparagine and glutamate by site-directed mutagenesis, respectively. Kinetic parameters for colloidal chitosan hydrolysis were determined with wild-type and 10 mutant chitosanases. The Leu64 Arg and Leu64 Gln mutations were essentially inactive and kinetic parameters such as V _max and k _cat were approximately 1/10⁷ of those of the wild-type enzyme. The Asp98 Asn mutation did not affect the K _m value significantly, but decreased k _cat to 15% of that of wild-type chitosanase. On the other hand, the Asp98 srarr; Glu mutation affected neither K _m nor k _cat. The observation that approximately 15% of activity remained after the substitution of Asp98 by Asn indicated that the carboxyl side chain of Asp98 is not absolutely required for catalytic activity. These results indicate that the Leu64 residue is directly involved in the catalytic activity of TCH-2.     

The role of Glu196 in the environment around the substrate binding site of leucine aminopeptidase from Streptomyces griseus

To investigate the role of Glu196 of leucine aminopeptidase from Streptomyces griseus (SGAP) in SGAP activation by calcium and substrate specificity, we constructed E196X SGAP by saturation mutagenesis. Most mutations led to the abrogation of SGAP activation by calcium, and substitution with Lys led to a marked increase in activity toward Asp-p-nitroanilide (pNA) and a decrease in that toward Lys-pNA. A similar result was obtained from the investigation using non-calcium-activated enzyme from Streptomyces septatus (SSAP). These results indicate that Glu196 of SGAP is associated with the environment around the substrate binding site besides its role in SGAP activation by calcium.     

Reduced activity of the hypertension-associated Lys528Arg mutant of human adipocyte-derived leucine aminopeptidase (A-LAP)/ER-aminopeptidase-1

The adipocyte-derived leucine aminopeptidase (A-LAP)/ER aminopeptidase-1 is a multi-functional enzyme belonging to the M1 family of aminopeptidases. It was reported that the polymorphism Lys528Arg in the human A-LAP gene is associated with essential hypertension. In this study, the role of Lys528 in the enzymatic activity of human A-LAP was examined by site-directed mutagenesis. Among non-synonymous polymorphisms tested, only Lys528Arg reduced enzymatic activity. The replacement of Lys528 with various amino acids including Ala, Met, His and Arg caused a significant decrease in the enzymatic activity. Molecular modeling of the enzyme suggested that Lys528 is located near the entrance of the substrate pocket. These results suggest that Lys528 is important for maximal activity of A-LAP by maintaining the appropriate structure of the substrate pocket of the enzyme. The reduced enzymatic activity of A-LAP may cause high blood pressure and the observed association between the polymorphism and hypertension.     

The genetics of California populations of Geukensia demissa (Dillwyn) (Mollusca): further evidence on the selective importance of leucine aminopeptidase variation in salinity acclimation

A study of the population genetics of the estuarine bivalve Geukensia demissa (Dillwyn) in southern California marshes revealed significant spatial and temporal variation in leucine aminopeptidase-1 (LAP-1) gene frequencies which were correlated with environmental salinity. S California populations also generally show a deficit of heterozygotes at the LAP-1 locus with the degree of this deficit also correlated with environmental salinity. The results of this study are similar to previous studies on Mytilus edulis Linné where it has been proposed that antagonistic directional selection in low vs. high salinity environments acts to maintain the LAP-1 polymorphism. However, a general similarity between the genetic patterns for LAP-1 and other loci in G. demissa in S California makes a strict selectionist interpretation difficult. A similar study of G. demissa in San Francisco Bay failed to detect heterogeneity in allele frequencies or significant heterozygote deficits over gradients in environmental salinity.     

Adsorption-elution purification of chimeric <Emphasis Type="Italic">Bacillus stearothermophilus</Emphasis> leucine aminopeptidase II with raw-starch-binding activity

Summary A chimericBacillus stearothermophilus leucine aminopeptidase II (LAPsbd) has been constructed by introducing the raw-starch-binding domain of Bacillus sp. strain TS-23 α-amylase into the enzyme. LAPsbd was adsorbed onto raw starch and the adsorbed enzyme could be eluted from the adsorbent by soluble starch in 20 mM Tris–HCl buffer (pH 8.0). The adsorption of LAPsbd onto raw starch was affected by raw starch concentration, pH, and temperature, while the temperature and incubation time had no obvious effects on the elution of adsorbed enzyme. The molecular weight of purified enzyme was estimated to be 61 kDa. About 84% of LAPsbd in the cell free extract was recovered through one adsorption–elution cycle with a purification of 20-fold. The high quantity and purity of the recovered enzyme coupled with the easy performance make the adsorption–elution procedure suitable for industrial applications.