Monoclonal Antibodies Recognize Conformational Epitopes on Wild-type and Recombinant Mutant Amidases from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Hybridoma technology was used to raise monoclonal antibodies (MAbs) against wild-type amidase from Pseudomonas aeruginosa. Hybridoma clones secreting polyol-responsive MAbs (PR-MAbs) were screened that bind antigen tightly. but release under mild- and non-denaturing elution conditions, which can be used as ligands in immunoaffinity chromatography. Two of these hybridoma clones (C9E4 and B1E4) secreting MAbs against wild-type amidase were selected in order to check if they are PR-MAbs by using ELISA-elution assay. These hybridoma cell lines secreted MAbs of IgG class which were purified in a single step by Protein A-Sepharose CL-4B chromatography, which revealed two protein bands on SDS-PAGE. Specificity studies of MAb C9E4 revealed that it recognized a common epitope on wild-type and mutant T103I amidases as determined by direct ELISA, as well as by Western blotting under native conditions. This MAb exhibited a higher-affinity constant (K) for the mutant T103I amidase than for the wild-type enzyme. However, this MAb did not recognize either wild-type or mutant T103I enzymes under denaturing conditions suggesting that it binds to a conformation-sensitive epitope on amidase molecule. On the other hand, it also does not recognize either native or denatured forms of mutant C91A amidase suggesting that this substitution disrupted the conformational epitope present on amidase molecule. Furthermore, MAb C9E4 inhibited about 80% of wild-type amidase activity, whereas it activated about 80% of mutant amidase (T103I) activity. However, this MAb did not affect mutant C91A amidase activity which is in agreement with other results presented in this work. The data presented in this work suggest that this MAb acts as a powerful probe to detect conformational changes in native and denatured amidases as well as to differentiate wild-type and mutant (T103I and C91A) amidases.     

Substitutions of Thr-103-Ile and Trp-138-Gly in amidase from Pseudomonas aeruginosa are responsible for altered kinetic properties and enzyme instability

Pseudomonas aeruginosa Ph1 is a mutant strain derived from strain AI3. The strain AI3 is able to use acetanilide as a carbon source through a mutation (T103I) in the amiE gene that encodes an aliphatic amidase (EC 3.5.1.4). The mutations in the amiE gene have been identified (Thr103Ile and Trp138Gly) by direct sequencing of PCR-amplified mutant gene from strain Ph1 and confirmed by sequencing the cloned PCR-amplified gene. Site-directed mutagenesis was used to alter the wild-type amidase gene at position 138 for Gly. The wild-type and mutant amidase genes (W138G, T103I-W138G, and T103I) were cloned into an expression vector and these enzymes were purified by affinity chromatography on epoxy-activated Sepharose 6B-acetamide/phenylacetamide followed by gel filtration chromatography. Altered amidases revealed several differences in kinetic properties, namely, in substrate specificity, sensitivity to urea, optimum pH, and enzyme stability, compared with the wild-type enzyme. The W138G enzyme acted on acetamide, acrylamide, phenylacetamide, and p-nitrophenylacetamide, whereas the double mutant (W138G and T103I) amidase acted only on p-nitrophenylacetamide and phenylacetamide. On the other hand, the T103I enzyme acted on p-nitroacetanilide and acetamide. The heat stability of altered enzymes revealed that they were less thermostable than the wild-type enzyme, as the mutant (W138G and W138G-T103I) enzymes exhibited t _1/2 values of 7.0 and 1.5 min at 55°C, respectively. The double substitution T103I and W138G on the amidase molecule was responsible for increased instabiliby due to a conformational change in the enzyme molecule as detected by monoclonal antibodies. This conformational change in altered amidase did not alter its M _r value and monoclonal antibodies reacted differently with the active and inactive T103I-W138G amidase.     

Substitution of Glu-59 by val in amidase from Pseudomonas aeruginosa results in a catalytically inactive enzyme

A mutant strain, KLAM59, of Pseudomonas aeruginosa has been isolated that synthesizes a catalytically inactive amidase. The mutation in the amidase gene has been identified (Glu59Val) by direct sequencing of PCR-amplified mutant gene and confirmed by sequencing the cloned PCR-amplified gene. The wild-type and altered amidase genes were cloned into an expression vector and both enzymes were purified by affinity chromatography on epoxy-activated Sepharose 6B-acetamide followed by gel filtration chromatography. The mutant enzyme was catalytically inactive, and it was detected in column fractions by monoclonal antibodies previously raised against the wild-type enzyme using an ELISA sandwich method. The recombinant wild-type and mutant enzymes were purified with a final recovery of enzyme in the range of 70–80%. The wild-type and mutant enzymes behaved differently on the affinity column as shown by their elution profiles. The molecular weights of the purified wild-type and mutant amidases were found to be 210,000 and 78,000 Dalton, respectively, by gel filtration chromatography. On the other hand, the mutant enzyme ran as a single protein band on SDS-PAGE and native PAGE with a M _r of 38,000 and 78,000 Dalton, respectively. These data suggest that the substitution Glu59Val was responsible for the dimeric structure of the mutant enzyme as opposed to the hexameric form of the wild-type enzyme. Therefore, the Glu59 seems to be a critical residue in the maintenance of the native quaternary structure of amidase.     

Immobilized metal affinity chromatography of monoclonal immunoglobulin M against mutant amidase from Pseudomonas aeruginosa

The chromatographic behavior of monoclonal antibodies (MAbs) of immunoglobulin (Ig) M class against mutant (T103I) amidase from Pseudomonas aeruginosa was investigated on immobilized metal chelates. The effect of ligand concentration, the length of spacer arm, and the nature of metal ion were investigated in immobilized metal affinity chromatography (IMAC). The MAbs against mutant amidase adsorbed to Cu(II), Ni(II), Zn(II), Co(II), and Ca(II)-iminodiacetic acid (IDA) agarose columns. The increase in ligand concentration (epichlorohydrin: 30–60 and 1,4-butanediol-diglycidyl ether: 16–36) resulted in higher adsorption to IgM into immobilized metal chelates. The length of spacer arm was found to affect protein adsorption, as longer spacer arm (i.e., 1,4-butanediol-diglycidyl ether) increased protein adsorption of immobilized metal chelates. The adsorption of IgM onto immobilized metal chelates was pH dependent because an increase in the binding of IgM was observed as the pH varied from 6.0 to 8.0. The adsorption of IgM to immobilized metal chelates was the result of coordination of histidine residues to metal chelates that are available in the third constant domain of heavy chain (CH3) of immunoglobulins, as the presence of imidazole (5 mM) in the equilibration buffer abolished the adsorption of IgM to the column. The combination of tailor-made stationary phases for IMAC and a correct design of the adsorption parameters permitted to devise a one-step purification procedure for IgM. Culture supernatants containing IgM against mutant amidase (T103I) were purified either by IMAC on EPI-60-IDA-Co (II) column or by gel filtration chromatography on Sephacryl S-300HR. The specific content of IgM and final recovery of antibody activity exhibited similar values for both purification schemes. The purified preparations of IgM obtained by both schemes were apparently homogeneous on native polyacrylamide gel electrophoresis with a M _r of 851,000 Da. The results presented in this work strongly suggest that one-step purification of IgM by IMAC is a cost-effective and process-compatible alternative to other types of chromatography.     

Screening of suitable immobilized metal chelates for adsorption of monoclonal antibodies against mutant amidase from Pseudomonas aeruginosa

The chromatographic behaviour of monoclonal antibodies (MAbs) of IgM class against mutant (T103I) amidase from Pseudomonas aeruginosa was investigated. The effect of ligand concentration, the length of spacer arm and the nature of metal ion were investigated on immobilized metal ion affinity chromatography (IMAC). MAbs against mutant amidase adsorbed to Cu (II), Ni (II), Zn (II), Co (II) and Ca (II)-IDA agarose columns. The adsorption of MAbs onto immobilized metal chelates was pH dependent because an increase in the binding of MAbs was observed as the pH was raised from 6.0 to 8.0. The adsorption of MAbs to metal chelates was due to coordination of histidine residues which are available in the 3rd constant domain of heavy chain (CH3) of immunoglobulins since the presence of imidazole in the equilibration buffer abolished the adsorption of MAbs to the column packed with commercial IDA-Zn(II) agarose at pH 8.0. The combination of tailor-made stationary phases for IMAC and a correct choice of the adsorption conditions permitted to design a one-step purification procedure for MAbs of IgM class. Culture supernatants containing MAbs of IgM class against mutant amidase (T103I) were chromatographed by IMAC Co (II) column at pH 8.0. The results strongly suggest that one-step purification of MAbs of IgM class by IMAC is a cost-effective and process-compatible alternative to the other purification procedures.     

Human lipoprotein lipase: relationship of activity, heparin affinity, and conformation as studied with monoclonal antibodies.

The objective of this study was to investigate how a conformational change in lipoprotein lipase (LPL) affects its molecular functions. Monoclonal antibodies (MAbs) were raised against purified bovine milk lipoprotein lipase. MAb 5D2 bound to human and bovine LPL both before and after denaturation of LPL. MAb 5F9 also recognized LPL from both species, but only after denaturation of the antigen, suggesting that a conformational change led to exposure of a previously hidden epitope. The MAbs were used in two sandwich enzyme-linked immunosorbent assays (ELISAs). One ELISA used the same MAb (5D2) to coat the plate and detect the bound antigen. This ELISA thus required the same epitope to be present in duplicate for detection (as would be the case with a dimeric antigen). The second ELISA used MAb 5F9 to coat the plate and MAb 5D2 to detect the antigen. This ELISA detected LPL only after it had been denatured. By measuring the same sample before and after denaturation with guanidine hydrochloride (GuHCl) in the 5F9 ELISA, and subtracting one from the other, a measure of native LPL was obtained. In inactivation experiments using human LPL, activity and the measure of LPL mass obtained in the 5D2 ELISA decreased and were related inversely to the measured mass obtained in the 5F9 ELISA which increased, indicating that loss of activity is closely linked to dimer dissociation and loss of native conformation. The effect of conformation and dimeric structure on LPL-heparin interaction was studied by heparin-Sepharose chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic properties of wild-type and altered recombinant amidases by the use of ion-selective electrode assay method

A novel assay method was investigated for wild-type and recombinant mutant amidases (EC 3.5.1.4) from Pseudomonas aeruginosa by ammonium ion-selective electrode (ISE). The initial velocity is proportional to the enzyme concentration by using the wild-type enzyme. The specific activities of the purified amidase were found to be 88.2 and 104.2 U mg protein(-1) for the linked assay and ISE methods, respectively. The kinetic constants--Vmax, Km, and Kcat--determined by Michaelis-Menten plot were 101.13 U mg protein(-1), 1.12x10(-2) M, and 64.04 s(-1), respectively, for acrylamide as the substrate. On the other hand, the lower limit of detection and range of linearity of enzyme concentration were found to be 10.8 and 10.8 to 500 ng, respectively, for the linked assay method and 15.0 and 15.0 to 15,000 ng, respectively, for the ISE method. Hydroxylamine was found to act as an uncompetitive activator of hydrolysis reaction catalyzed by amidase given that there is an increase in Vmax and Km when acetamide was used as the substrate. However, the effect of hydroxylamine on the hydrolysis reaction was dependent on the type of amidase and substrate involved in the reaction mixture. The degrees of activation (epsilon(a)) of the wild-type and mutant (T103I and C91A) enzymes were found to be 2.54, 12.63, and 4.33, respectively, for acetamide as the substrate. However, hydroxylamine did not activate the reaction catalyzed by wild-type and altered (C91A and W138G) amidases by using acrylamide and acetamide, respectively, as the substrate. The activating effect of hydroxylamine on the hydrolysis of acetamide, acrylamide, and p-nitrophenylacetamide can be explained by the fact that additional formation of ammonium ions occurred due to the transferase activity of amidases. However, the activating effect of hydroxylamine on the hydrolysis of p-nitroacetanilide may be due to a change in conformation of enzyme molecule. Therefore, the use of ISE permitted the study of the kinetic properties of wild-type and mutant amidases because it was possible to measure initial velocity of the enzyme-catalyzed reaction in real time.     

pH-, temperature- and ion-dependent oligomerization of Sulfolobus solfataricus recombinant amidase: a study with site-specific mutants

Recombinant amidase from Sulfolobus solfataricus occurred as a dimer of 110 kDa comprising identical subunits. Only dimers were present at pHs above 7.0, but with decreasing pH, dimers associated into octamers, with complete oligomerization occurring at pH 3.0. Oligomerization showed reversible temperature-dependence, with octamer formation increasing with temperature from 36 °C to between 70 and 80° C. Increasing salt concentrations, favored dissociation of the octamers. Among the three investigated factors affecting the dimer–octamer equilibrium, the most important was pH. Among four mutants obtained by site-specific mutagenesis and selection for pH and temperature sensitivity, the T319I and D487N mutant amidases, like that of the native Sulfolobus solfataricus, responded to changes in pH and temperature with a conformational change affecting the dimer–octamer equilibrium. The Y41C and L34P mutant amidases were unaffected by pH and temperature, remaining always in the dimeric state. The differences among mutants in protein conformation must be related to the position of the introduced mutation. Although the L34P and Y41C mutations are located in the helical region 33–48 (LLKLQLESYERLDSLP), which is close to the amino-terminal segment of the protein, the T319I mutation is located in a strand on the surface of the protein, which is far from, and opposite to, the amino-terminal segment. The D487N mutation is located in the center of the protein, far distant from the 33–48 segment. These observations suggest that the segment of the protein closest to the amino-terminus plays a key role in the association of dimers into octamers.     

针对炭疽保护性抗原不同结构域的中和性单克隆抗体的筛选和鉴定

炭疽保护性抗原（PA）是炭疽毒素的重要组分,同时也是现有炭疽疫苗的主要有效成分,在炭疽杆菌的致病与免疫中发挥关键作用。以重组PA为免疫原,采用B淋巴细胞杂交瘤技术,结合炭疽毒素敏感细胞的毒性中和试验,大量筛选抗PA单克隆抗体,获得了9株炭疽毒素中和性单抗。进一步分析表明这些单抗以IgG1亚类为主,分别识别PA 3个结构域的4个不同中和表位区。针对结构域2的4株单抗识别同一表位区,其中3株单抗的中和活性强于抗PA多抗;针对结构域4的4株单抗识别两个不同表位区;另有1株单抗识别位于结构域3的表位。实验结果提示PA具有多个中和表位,分别位于其不同结构域,其中结构域2、4包含主要中和表位。实验中获得的针对不同表位的中和性单抗为深入研究PA的免疫保护机理提供了工具,也为研制针对炭疽毒素的被动免疫制剂和治疗药物打下基础。     

Selection of amidases with novel substrate specificities from penicillin amidase of Escherichia coli

To obtain amidases with novel substrate specificity, the cloned gene for penicillin amidase of Escherichia coli ATCC 11105 was mutagenized and mutants were selected for the ability to hydrolyze glutaryl-(L)-leucine and provide leucine to Leu- host cells. Cells with the wild-type enzyme did not grow in minimal medium containing glutaryl-(L)-leucine as a sole source of leucine. The growth rates of Leu- cells that expressed these mutant amidases increased as the glutaryl-(L)-leucine concentration increased or as the medium pH decreased. Growth of the mutant strains was restricted by modulation of medium pH and glutaryl-(L)-leucine concentration, and successive generations of mutants that more efficiently hydrolyzed glutaryl-(L)-leucine were isolated. The kinetics of glutaryl-(L)-leucine hydrolysis by purified amidases from two mutants and the respective parental strains were determined. Glutaryl-(L)-leucine hydrolysis by the purified mutant amidases occurred most rapidly between pH 5 and 6, whereas hydrolysis by wild-type penicillin amidase at this pH was negligible. The second-order rate constants for glutaryl-(L)-leucine hydrolysis by two "second-generation" mutant amidases, 48 and 77 M-1 s-1, were higher than the rates of hydrolysis by the respective parental amidases. The increased rates of glutaryl-(L)-leucine hydrolysis resulted from both increases in the molecular rate constants and decreases in apparent Km values. The results show that it is possible to deliberately modify the substrate specificity of penicillin amidase and successively select mutants with amidases that are progressively more efficient at hydrolyzing glutaryl-(L)-leucine.     

Production and Characterization of Monoclonal Antibodies Specific to Closterovirus-like Particles Associated with Grapevine Leafroll Disease

Three hybridoma lines secreting monoclonal antibodies to a closterovirus-like particle (GLRV 3) associated with grapevine leafroll disease were produced. One of the antibody (MAbt) reacted with one extremity of the filamentous virus particle whereas the other two (MAb2 and 3) reacted with the entire surface of the virus particle. Using MAb2 in ELISA it is possible to detect 46 of the 50 GLRV-3 isolates. In order to detect all the 50 isolates, it is necessary to use MAb1 in ELISA in conjunction with polyclonal antibodies. In immunoblotting, the three MAbs recognized a 43 Kd viral protein.     

Selection of amidases with novel substrate specificities from penicillin amidase of Escherichia coli.

To obtain amidases with novel substrate specificity, the cloned gene for penicillin amidase of Escherichia coli ATCC 11105 was mutagenized and mutants were selected for the ability to hydrolyze glutaryl-(L)-leucine and provide leucine to Leu- host cells. Cells with the wild-type enzyme did not grow in minimal medium containing glutaryl-(L)-leucine as a sole source of leucine. The growth rates of Leu- cells that expressed these mutant amidases increased as the glutaryl-(L)-leucine concentration increased or as the medium pH decreased. Growth of the mutant strains was restricted by modulation of medium pH and glutaryl-(L)-leucine concentration, and successive generations of mutants that more efficiently hydrolyzed glutaryl-(L)-leucine were isolated. The kinetics of glutaryl-(L)-leucine hydrolysis by purified amidases from two mutants and the respective parental strains were determined. Glutaryl-(L)-leucine hydrolysis by the purified mutant amidases occurred most rapidly between pH 5 and 6, whereas hydrolysis by wild-type penicillin amidase at this pH was negligible. The second-order rate constants for glutaryl-(L)-leucine hydrolysis by two "second-generation" mutant amidases, 48 and 77 M-1 s-1, were higher than the rates of hydrolysis by the respective parental amidases. The increased rates of glutaryl-(L)-leucine hydrolysis resulted from both increases in the molecular rate constants and decreases in apparent Km values. The results show that it is possible to deliberately modify the substrate specificity of penicillin amidase and successively select mutants with amidases that are progressively more efficient at hydrolyzing glutaryl-(L)-leucine.     

Molecular basis of altered enzyme specificities in a family of mutant amidases from Pseudomonas aeruginosa.

A family of mutant amidases has been derived by experimental evolution of the aliphatic amidase of Pseudomonas aeruginosa strain PAC1. Mutation amiE16, in the structural gene for the enzyme, results in the production of the mutant B amidase by strain B6. This strain, unlike the wild-type, can utilize butyramide for growth. Strain B6 gave rise by a single mutational event to strain V9, utilizing valeramide, and strain PhB3, utilizing phenylacetamide. Strain V9 was not itself able to utilize phenylacetamide but gave rise by mutation to the phenylacetamide-utilizing mutant PhV1. Peptide 108 was isolated from chymotryptic digests of mutant amidases from strains B6, PhB3 and PhV1, but could not be detected in chymotryptic digests of the wild-type amidase. The sequence of peptide 108 was established as Met-Arg-His-Gly-Asp-Ile-Phe. Thermolytic digests of mutant amidases from strains B6, PhB3, PhV1 and V9 were compared with digests of the wild-type amidase. A peptide of the composition Met, Arg, His, Gly2, Asp3, Ile, Ser3, Thr, Val was found in the digest of the wild-type amidase and was replaced in the digests of the mutant amidases by a peptide of the composition Met, Arg, His, Gly2, Asp3, Ile, Ser3, Thr, Val, Phe. Mutation amiE16 is common to the four mutant enzymes and can be accounted for by the mutation Ser leads to Phe. The sequence of the chymotryptic peptide corresponds with the N-terminal sequence of the amidase protein, and can also be related to the thermolysin peptides. It is concluded that mutation amiE16 is a Ser leads to Phe change at position 7 from the N-terminus and the effect of this on the enzyme conformation is discussed.     

Immobilized metal affinity chromatography of monoclonal immunoglobulin M against mutant amidase from Pseudomonas aeruginosa

The chromatographic behavior of monoclonal antibodies (MAbs) of immunoglobulin (Ig) M class against mutant (T103I) amidase from Pseudomonas aeruginosa was investigated on immobilized metal chelates. The effect of ligand concentration, the length of spacer arm, and the nature of metal ion were investigated in immobilized metal affinity chromatography (IMAC). The MAbs against mutant amidase adsorbed to Cu(II), Ni(II), Zn(II), Co(II), and Ca(II)-iminodiacetic acid (IDA) agarose columns. The increase in ligand concentration (epichlorohydrin: 30-60 and 1,4-butanediol-diglycidyl ether: 16-36) resulted in higher adsorption to IgM into immobilized metal chelates. The length of spacer arm was found to affect protein adsorption, as longer spacer arm (i.e., 1,4-butanediol-diglycidyl ether) increased protein adsorption of immobilized metal chelates. The adsorption of IgM onto immobilized metal chelates was pH dependent because an increase in the binding of IgM was observed as the pH varied from 6.0 to 8.0. The adsorption of IgM to immobilized metal chelates was the result of coordination of histidine residues to metal chelates that are available in the third constant domain of heavy chain (CH3) of immunoglobulins, as the presence of imidazole (5 mM) in the equilibration buffer abolished the adsorption of IgM to the column. The combination of tailor-made stationary phases for IMAC and a correct design of the adsorption parameters permitted to devise a one-step purification procedure for IgM. Culture supernatants containing IgM against mutant amidase (T103I) were purified either by IMAC on EPI-60-IDA-Co (II) column or by gel filtration chromatography on Sephacryl S-300HR. The specific content of IgM and final recovery of antibody activity exhibited similar values for both purification schemes. The purified preparations of IgM obtained by both schemes were apparently homogeneous on native polyacrylamide gel electrophoresis with a Mr of 851,000 Da. The results presented in this work strongly suggest that one-step purification of IgM by IMAC is a cost-effective and processcompatible alternative to other types of chromatography.     

Fine mapping of antigenic site II of herpes simplex virus glycoprotein D.

Glycoprotein D (gD) is a virion envelope component of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) which plays an important role in viral infection and pathogenesis. Previously, anti-gD monoclonal antibodies (MAbs) were arranged into groups which recognize distinct type-common and type-specific sites on HSV-1 gD (gD-1) and HSV-2 gD (gD-2). Several groups recognize discontinuous epitopes which are dependent on tertiary structure. Three groups, VII, II, and V, recognize continuous epitopes present in both native and denatured gD. Previously, group II consisted of a single MAb, DL6, whose epitope was localized between amino acids 268 and 287. In the study reported here, we extended our analysis of the antigenic structure of gD, concentrating on continuous epitopes. The DL6 epitope was localized with greater precision to residues 272 to 279. Four additional MAbs including BD78 were identified, each of which recognizes an epitope within residues 264 to 275. BD78 and DL6 blocked each other in binding to gD. In addition, a mutant form of gD was constructed in which the proline at 273 was replaced by serine. This change removes a predicted beta turn in gD. Neither antibody reacted with this mutant, indicating that the BD78 and DL6 epitopes overlap and constitute an antigenic site (site II) within residues 264 to 279. A separate antigenic site (site XI) was recognized by MAb BD66 (residues 284 to 301). This site was only six amino acids downstream of site II, but was distinct as demonstrated by blocking studies. Synthetic peptides mimicking these and other regions of gD were screened with polyclonal antisera to native gD-1 or gD-2. The results indicate that sites II, V, VII, and XI, as well as the carboxy terminus, are the major continuous antigenic determinants on gD. In addition, the results show that the region from residues 264 through 369, except the transmembrane anchor, contains a series of continuous epitopes.     

Alteration of the catalytic efficiency of penicillin amidase from Escherichia coli

Ampicillin and cephalexin are beta-lactam antibiotics that are synthesized by the condensation of D-(-)-alpha-aminophenylacetic acid with 6-aminopenicillanic acid or 7-aminodeacetoxycephalosporanic acid, respectively. The rates at which the penicillin amidase of Escherichia coli catalyzes these reactions are too low to be of practical use. The objective of this study was to determine whether it is possible to alter the substrate specificity of penicillin amidase and select enzymes that efficiently hydrolyze substrates with alpha-aminophenylacetyl moieties at low pH, at which the alpha-amino group is nearly completely protonated. In this study, D-(-)-alpha-aminophenylacetyl-(L)-leucine (APAL) was used as a substrate analog of ampicillin and cephalexin. The gene for the penicillin amidase of E. coli ATCC 11105 was cloned and transferred to a leucine auxotroph of E. coli; numerous amidase mutants were selected by their ability to cleave APAL and provide leucine for growth in low-pH medium. The plasmid encoding one of the mutant amidases (pA135) was used to transform naive cells, and transformants that expressed the mutant amidase were shown to grow more rapidly in medium at pH 6.5 containing 0.1 mM APAL as the sole leucine source than did cells with the wild-type amidase. The mutant amidase was purified, and the second-order rate constant (kcat/Km) for APAL hydrolysis at pH 6.5 was found to be 10-fold greater than the rate observed with the wild-type enzyme. The difference between the rates of APAL hydrolysis by the mutant and wild-type amidases increased as the pH of the reactions decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alteration of the catalytic efficiency of penicillin amidase from Escherichia coli.

Ampicillin and cephalexin are beta-lactam antibiotics that are synthesized by the condensation of D-(-)-alpha-aminophenylacetic acid with 6-aminopenicillanic acid or 7-aminodeacetoxycephalosporanic acid, respectively. The rates at which the penicillin amidase of Escherichia coli catalyzes these reactions are too low to be of practical use. The objective of this study was to determine whether it is possible to alter the substrate specificity of penicillin amidase and select enzymes that efficiently hydrolyze substrates with alpha-aminophenylacetyl moieties at low pH, at which the alpha-amino group is nearly completely protonated. In this study, D-(-)-alpha-aminophenylacetyl-(L)-leucine (APAL) was used as a substrate analog of ampicillin and cephalexin. The gene for the penicillin amidase of E. coli ATCC 11105 was cloned and transferred to a leucine auxotroph of E. coli; numerous amidase mutants were selected by their ability to cleave APAL and provide leucine for growth in low-pH medium. The plasmid encoding one of the mutant amidases (pA135) was used to transform naive cells, and transformants that expressed the mutant amidase were shown to grow more rapidly in medium at pH 6.5 containing 0.1 mM APAL as the sole leucine source than did cells with the wild-type amidase. The mutant amidase was purified, and the second-order rate constant (kcat/Km) for APAL hydrolysis at pH 6.5 was found to be 10-fold greater than the rate observed with the wild-type enzyme. The difference between the rates of APAL hydrolysis by the mutant and wild-type amidases increased as the pH of the reactions decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

One-step affinity purification of amidase from mutant strains of Pseudomonas aeruginosa

Amidases (acylamide amidohydrolase EC 3.5.1.4) from mutant strains (i.e., B6, AI3, AIU1N, OUCH 4 and L10) of Pseudomonas aeruginosa were purified in one-step by ligand affinity chromatography using Epoxy-activated Sepharose 4B-acetamide. The yields of the purified enzymes were about 90% for all mutant strains with purification factors of about 10 and were apparently homogeneous when analysed by SDS-PAGE and native PAGE. The protein bands on native PAGE coincided with the stained band of enzyme activity for all amidase preparations. Affinity columns had a maximum binding capacity of 0.5 mg amidase protein/ml of sedimented gel and could be regenerated and reused several times without any loss of binding capacity and resolution. Affinity gels containing either semicarbazide or urea were also found useful for the isolation of amidase. The differences in substrate specificity of these amidases reported previously were also observed in the elution behaviour of these enzymes from the affinity columns.     

Identification of envelope protein epitopes that are important in the attenuation process of wild-type yellow fever virus.

Monoclonal antibodies (MAbs) have been prepared against vaccine and wild-type strains of yellow fever (YF) virus, and envelope protein epitopes specific for vaccine (MAbs H5 and H6) and wild-type (MAbs S17, S18, S24, and S56) strains of YF virus have been identified. Wild-type YF virus FVV, Dakar 1279, and B4.1 were each given six passages in HeLa cells. FVV and B4.1 were attenuated for newborn mice following passage in HeLa cells, whereas Dakar 1279 was not. Examination of the envelope proteins of the viruses with 87 MAbs showed that attenuated viruses gained only the vaccine epitope recognized by MAb H5 and lost wild-type epitopes recognized by MAbs S17, S18, and S24 whereas the nonattenuated Dakar 1279 HeLa p6 virus did not gain the vaccine epitope, retained the wild-type epitopes, and showed no other physical epitope alterations. MAb neutralization-resistant (MAbr) escape variants generated by using wild-type-specific MAbs S18 and S24 were found to lose the epitopes recognized by MAbs S18 and S24 and to acquire the epitope recognized by vaccine-specific MAb H5. In addition, the MAbr variants became attenuated for mice. Thus, the data presented in this paper indicate that acquisition of vaccine epitopes and loss of wild-type epitopes on the envelope protein are directly involved in the attenuation process of YF virus and suggest that the envelope protein is one of the genes encoding determinants of YF virus pathogenicity.     

Hemagglutinin-neuraminidase-independent fusion activity of simian virus 5 fusion (F) protein: difference in conformation between fusogenic and nonfusogenic F proteins on the cell surface

The fusion (F) protein of simian virus 5 (SV5) strain W3A is known to induce cell fusion in the absence of hemagglutinin-neuraminidase (HN) protein. In contrast, the F protein of SV5 strain WR induces cell fusion only when coexpressed with the HN protein, the same as do other paramyxovirus F proteins. When Leu-22 in the subunit F2 of the WR F protein is replaced with the counterpart (Pro) in the W3A F protein, the resulting mutant L22P induces extensive cell fusion by itself. In the present study, we obtained anti-L22P monoclonal antibodies (MAbs) 21-1 and 6-7, whose epitopes were located in the middle (amino acids [aa] 227 to 320) of subunit F1. The amino-terminal region (aa 20 to 47) of subunit F2 was also involved in the formation of MAb 21-1 epitope. Flow cytometric analysis revealed that both the MAbs reacted very faintly with native WR F protein that was expressed on the cell surface whereas they reacted efficiently with native L22P irrespective of whether it is cleaved into F1 and F2. However, by heating the cells at 47 degrees C after mild formaldehyde fixation, the epitopes for MAb 6-7 and mAb 21-1 in the WR F protein were exposed and the reactivity of the MAbs with the WR F protein became comparable to their reactivity with L22P. Thus, the two MAbs seem to distinguish the difference in native conformation between fusogenic mutant L22P and its parental nonfusogenic WR F protein. The native conformation of L22P may represent an intermediate between native and postfusion conformations of a typical paramyxovirus F protein.