Purification and properties of a 1,3-1,4-beta-D-glucanase (lichenase, 1,3-1,4-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.73) from Bacteroides succinogenes cloned in Escherichia coli.

A 1,3-1,4-beta-D-glucanase (lichenase, 1,3-1,4-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.73) from Bacteroides succinogenes cloned in Escherichia coli was purified 600-fold by chromatography on Q-Sepharose and hydroxyapatite. The cloned enzyme hydrolysed lichenin and oat beta-D-glucan but not starch, CM(carboxymethyl)-cellulose, CM-pachyman, laminarin or xylan. The enzyme had a broad pH optimum with maximum activity at approx. pH 6.0 and a temperature optimum of 50 degrees C. The pH of elution from a chromatofocusing column for the cloned enzyme was 4.7 (purified) and 4.9 (crude) compared with 4.8 for the mixed-linkage beta-D-glucanase activity in B. succinogenes. The Mr of the cloned enzyme was estimated to be 37,200 by gel filtration and 35,200 by electrophoresis. The Km values estimated for lichenin and oat beta-D-glucan were 0.35 and 0.71 mg/ml respectively. The major hydrolytic products with lichenin as substrate were a trisaccharide (82%) and a pentasaccharide (9.5%). Hydrolysis of oat beta-D-glucan yielded a trisaccharide (63.5%) and a tetrasaccharide (29.6%) as the major products. The chromatographic patterns of the products from the cloned enzyme appear to be similar to those reported for the mixed-linkage beta-D-glucanase isolated from Bacillus subtilis. The data presented illustrate the similarity in properties of the cloned mixed-linkage enzyme and the 1,3-1,4-beta-D-glucanase from B. subtilis and the similarity with the 1,4-beta-glucanase in B. succinogenes.     

Prediction and Fourier transform infrared spectroscopy estimation of the secondary structure of a Bacillus licheniformis endo-beta-1,3-1,4-D-glucanase.

The secondary structure of a recombinant Bacillus licheniformis endo-beta-1,3-1,4-D-glucanase (EC.3.2.1.73) has been estimated by Fourier Transform Infrared Spectroscopy and also predicted by the algorithm of Chou and Fasman. From the curve fitting of the deconvolved IR spectrum, the most probable distribution of the secondary structural classes appears to be about 40% beta-sheet, 25% reverse turn, 24% non-ordered and 11% alpha-helix. From theoretical prediction of secondary structure the protein would present 37% beta-sheet, 31% reverse turn, 22% non-ordered and 10% alpha-helix.     

A catalytic role for threonine-12 of E. coli asparaginase II as established by site-directed mutagenesis

A threonine-12 to alanine mutant of E. coli asparaginase II (EC 3.5.1.1) has less than 0.01% of the activity of wild-type enzyme. Both tertiary and quaternary structure of the enzyme are essentially unaffected by the mutation; thus the activity loss seems to be the result of a direct impairment of catalytic function. As aspartate is still bound by the mutant enzyme, Thr-12 appears not be involved in substrate binding.     

DNA sequence of a Fibrobacter succinogenes mixed-linkage beta-glucanase (1,3-1,4-beta-D-glucan 4-glucanohydrolase) gene.

The DNA sequence of a mixed-linkage beta-glucanase (1,3-1,4-beta-D-glucan 4-glucanohydrolase [EC 3.2.1.73]) gene from Fibrobacter succinogenes cloned in Escherichia coli was determined. The general features of this gene are very similar to the consensus features for other gram-negative bacterial genes. The gene product was processed for export in E. coli. There is a high level of sequence homology between the structure of this glucanase and the structure of a mixed-linkage beta-glucanase from Bacillus subtilis. The nonhomologous region of the amino acid sequence includes a serine-rich region containing five repeats of the sequence Pro-Xxx-Ser-Ser-Ser-Ser-(Ala or Val) which may be functionally related to the serine-rich region observed in Pseudomonas fluorescens cellulase and the serine- and/or threonine-rich regions observed in Cellulomonas fimi endoglucanase and exoglucanase, in Clostridium thermocellum endoglucanases A and B, and in Trichoderma reesei cellobiohydrolase I, cellobiohydrolase II, and endoglucanase I.     

Probing suggested catalytic domains of glycosyltransferases by site-directed mutagenesis.

The plant enzyme arbutin synthase isolated from cell suspension cultures of Rauvolfia serpentina and heterologously expressed in Escherichia coli is a member of the NRD1beta family of glycosyltransferases. This enzyme was used to prove, by site-directed mutagenesis, suggested catalytic domains and reaction mechanisms proposed for enzyme-catalyzed glycosylation. Replacement of amino acids far from the NRD domain do not significantly affect arbutin synthase activity. Exchange of amino acids at the NRD site leads to a decrease of enzymatic activity, e.g. substitution of Glu368 by Asp. Glu368, which is a conserved amino acid in glycosyltransferases located at position 2 and is important for enzyme activity, does not serve as the nucleophile in the catalytic centre as proposed. When it is replaced by Ala, the resulting mutant enzyme E368A exhibits comparable activity as found for E368D in respect to vanillin. Enzyme activities of wild-type and E368A towards several substrates were not affected at the same level. His360 at position 1 of NRD1beta glycosyltransferases occupies a more crucial role as expected. When it is exchanged against other basic amino acids such as Lys or Arg the enzyme activity decreases approximately 1000-fold. Replacement of His360 by Glu leads to a mutant enzyme (H360E) with an approximately 4000-fold lower activity compared with the wild-type. This mutein still produces a beta-glucoside, not an alpha-glucoside and therefore indicates that generation of the typical E-E motif of NRD1alpha glycosyltransferases does not convert a NRD1beta enzyme into a NRD1alpha enzyme. The presented data do not support several suggestions made in the literature about catalytic amino acids involved in the glycosyltransfer reaction.     

Identification of catalytic residues of pepstatin-insensitive carboxyl proteinases from prokaryotes by site-directed mutagenesis.

Pepstatin-insensitive carboxyl proteinases from Pseudomonas sp. (PCP) and Xanthomonas sp. (XCP) have no conserved catalytic residue sequences, -Asp*-Thr-Gly- (Asp is the catalytic residue) for aspartic proteinases. To identify the catalytic residues of PCP and XCP, we selected presumed catalytic residues based on their high sequence similarity, assuming that such significant sites as catalytic residues will be generally conserved. Several Ala mutants of Asp or Glu residues were constructed and analyzed. The D170A, E222A, and D328A mutants for PCP and XD79A, XD169A, and XD348A mutants for XCP were not converted to mature protein after activation, and no catalytic activity could be detected in these mutants. The specificity constants toward chromogenic substrate of the other PCP and XCP mutants, except for the D84A mutant of PCP, were similar to that of wild-type PCP or XCP. Coupled with the result of chemical modification (Ito, M., Narutaki, S., Uchida, K., and Oda, K. (1999) J. Biochem. (Tokyo) 125, 210-216), a pair of Asp residues (170 and 328) for PCP and a pair of Asp residues (169 and 348) for XCP were elucidated to be their catalytic residues, respectively. The Glu(222) residue in PCP or Asp(79) residue in XCP was excluded from the candidates as catalytic residues, since the corresponding mutant retained its original activity.     

The structure of endo-beta-1,4-galactanase from Bacillus licheniformis in complex with two oligosaccharide products

The beta-1,4-galactanase from Bacillus licheniformis (BLGAL) is a plant cell-wall-degrading enzyme involved in the hydrolysis of beta-1,4-galactan in the hairy regions of pectin. The crystal structure of BLGAL was determined by molecular replacement both alone and in complex with the products galactobiose and galactotriose, catching a first crystallographic glimpse of fragments of beta-1,4-galactan. As expected for an enzyme belonging to GH-53, the BLGAL structure reveals a (betaalpha)(8)-barrel architecture. However, BLGAL betaalpha-loops 2, 7 and 8 are long in contrast to the corresponding loops in structures of fungal galactanases determined previously. The structure of BLGAL additionally shows a calcium ion linking the long betaalpha-loops 7 and 8, which replaces a disulphide bridge in the fungal galactanases. Compared to the substrate-binding subsites predicted for Aspergillus aculeatus galactanase (AAGAL), two additional subsites for substrate binding are found in BLGAL, -3 and -4. A comparison of the pattern of galactan and galactooligosaccharides degradation by AAGAL and BLGAL shows that, although both are most active on substrates with a high degree of polymerization, AAGAL can degrade galactotriose and galactotetraose efficiently, whereas BLGAL prefers longer oligosaccharides and cannot hydrolyze galactotriose to any appreciable extent. This difference in substrate preference can be explained structurally by the presence of the extra subsites -3 and -4 in BLGAL.     

Yeast phosphoglycerate kinase: investigation of catalytic function by site-directed mutagenesis.

A salt link buried in the domain interface of phosphoglycerate kinase has been implicated as being important in controlling the conformational transition from the open, or substrate-binding, to the closed, or catalytically competent, form of the enzyme. The residues contributing to the salt link are remote from the active site, but are connected to the substrate-binding sites through strands of beta-sheet. It has been suggested that these residues may also mediate sulphate and anion activation. These assumptions have been tested by examining the properties of a site-directed mutant (histidine-388----glutamine-388). The expression and overall structural integrity of the mutant, produced in yeast from a multicopy plasmid, remains essentially unaltered from the wild-type enzyme. However, the mutant enzyme has a kcat. reduced by 5-fold. The Km for ATP is lowered by 3-fold, and the Km for 3-phosphoglycerate is unaffected. The effects of sulphate on activity over a wide range of substrate concentrations appear to be the same for both the mutant and wild-type enzymes. These results lead to a reappraisal of the mechanistic role of the inter-domain histidine-glutamate interaction, as well as a refinement of the kinetic model of the enzyme.     

Identification of glutamic acid 105 at the active site of Bacillus amyloliquefaciens 1,3-1,4-beta-D-glucan 4-glucanohydrolase using epoxide-based inhibitors.

Bacillus amyloliquefaciens 1,3-1,4-beta-D-glucan 4-glucanohydrolase (EC 3.2.1.73) was modified by the mechanism-based, affinity-labeling reagent [14C](3,4)-epoxybutyl beta-D-cellobioside. Following partial inactivation a completely inactivated enzyme preparation containing 1.1 mol of covalently bound inhibitor/mol of protein was obtained by chromatography on a cellulosic matrix. The inactivated enzyme was digested with endoproteinase Glu-C and radioactive peptides purified by reversed-phase high performance liquid chromatography (HPLC). The affinity label was esterified exclusively to the gamma-carboxylate of Glu105 in the sequence Gly-Thr-Pro-Trp-Asp-Glu-Ile-Asp-Ile-Glu109. The sequence motif Glu-(Ile/Leu)-Asp-Ile is found in many glucanases and xylanases and may therefore serve to identify the catalytic nucleophile in beta-glycanases, which otherwise exhibit a low degree of sequence identity. The esterification of Glu105 by the affinity label abolished endoproteinase Glu-C-mediated hydrolysis of the Glu-Ile106 peptide bond. Identification of phenylthiohydantoin-Glu105 during automated sequence analysis was not possible unless the affinity label was liberated by prior base hydrolysis. These observations formed the basis for the development of a highly sensitive approach for the identification of catalytic carboxylates in polysaccharide hydrolases employing non-radioactive inhibitors, comparative HPLC mapping, electrospray mass spectrometry, and Edman degradation.     

Partial identification of carbohydrate-binding sites of a Galalpha1,3Galbeta1,4GlcNAc-specific lectin from the mushroom Marasmius oreades by site-directed mutagenesis

The Galalpha1,3Galbeta1,4GlcNAc-specific lectin from the mushroom Marasmius oreades (MOA) contains a ricin B chain-like (QXW)(3) domain at its N-terminus that is composed of three identical subdomains (alpha, beta, and gamma) and a C-terminal domain of unknown function. Here, we investigate the structure-function relationship of MOA to define the number and location of its carbohydrate-binding sites. Based on the sequence alignment of MOA to the ricin B-chain lactose-binding sites, we systematically constructed mutants by site-directed mutagenesis. We have used precipitation and hemagglutination assay for the primary analyses, and surface plasmon resonance for the kinetic analysis. Among amino acid residues at the putative carbohydrate-binding sites, Gln(46) in the alpha subdomain and Trp(138) in the gamma subdomain have been identified to be important amino acid residues directly or indirectly involved in carbohydrate recognition. By surface plasmon resonance, Q46A and W138A were 2.4- and 4.3-fold less active than that of the wild-type MOA (K(a) = 2 x 10(7)), respectively. A double-site mutant (Q46A/W138A) had activity similar to W138A. The C-terminal deletion mutant MOADeltaC showed hemagglutination and precipitation activity, although its binding constant was 12.5-fold less active (K(a) = 1.6 x 10(6)) than that of the wild-type MOA. A C-terminal deletion mutant with mutations at both Gln(46) and Trp(138) (MOADeltaC-Q46A/W138A) was 12,500-fold less active (K(a) = 1.6 x 10(3)) than that of the wild-type MOA. On the basis of this observation, we conclude that both alpha and gamma subdomains are most probably involved in carbohydrate binding, but the beta subdomain appears to be inactive.     

利用乳酸乳球菌AcmA表面展示β-1,3-1,4-葡聚糖酶

采用PCR扩增乳酸乳球(Lactococcus lactis)MBl91菌株的全长肽聚糖水解酶基因acmA,通过C-末端融合构建了与绿色荧光基因gfp的融合基因acmA-gfp,再连接于表达载体pMG36k上后得到可组成型表达AcmA-GFP融合蛋白的重组质粒pMB137,然后将该质粒电转化导入到乳酸乳球菌AS1.2829中获得重组菌MB137.经SDS-PAGE检测.重组菌MB137可表达预期的分子量约74 kD的蛋白质.Western blotting、细胞分级分离组分的荧光活性测定和特异GFP 二抗标记的流式细胞仪检测证实GFP被成功锚定在重组茵细胞表面,被锚定蛋白约占总表达融合蛋白的35%.进一步通过从枯草芽胞杆菌BF7658基因组中扩增去信号肽序列的β-1,3-1,4葡聚糖酶基因gls,来取代pMB137中的gfp,得到携带融合基因acmA-gls的重组质粒pMB138,经导入到乳酸乳球茵AS1.2829后得到重组菌MB138,其全细胞β-1,3-1,4-葡聚糖水解酶的活性约为12 U/mL茵液,明显高于对照茵株.     

Essential role of domain 4 of pneumolysin from Streptococcus pneumoniae in cytolytic activity as determined by truncated proteins

Pneumolysin (PLY), an important virulence factor of Streptococcus pneumoniae, is one of the members of thiol-activated cytolysins (TACYs) consisting of four domains. TACYs commonly bind to membrane cholesterol and oligomerize to form transmembrane pore. We have constructed full-length and various truncated PLYs to study the role of domains of PLY in the cytolytic activity. Full-length PLY had binding ability to both cell membrane and immobilized cholesterol. A truncated PLY which comprised only domain 4 molecule, the C-terminal domain of PLY, sustained the binding ability to cell membrane and cholesterol, whereas domain 1-3 molecule had no binding ability to them. Furthermore, the domain 4 molecule inhibited both the membrane binding and the hemolytic activity of full-length PLY. Accordingly, the present results provided the direct evidence that domain 4 was essential for the initial binding to membrane cholesterol and the interaction led to the subsequent membrane damage process.     

Analysis of the catalytic mechanism of juvenile hormone esterase by site-directed mutagenesis.

1. Juvenile hormone esterase (JHE) is a serine hydrolase selective for hydrolysis of the conjugated methyl esters of insect juvenile hormones. 2. We have investigated the mechanism of catalytic action of this enzyme by site-directed mutagenesis of the cloned enzyme and expression of the mutants in a baculovirus system. 3. A series of individual mutations of JHE were made to residues possibly involved in catalysis of juvenile hormones, and which are highly conserved in both esterases and lipases. 4. Mutation of the serine residue at position 201 to glycine (S201G), or aspartate 173 to asparagine (D173N), or histidine 446 to lysine (H446K), removed all detectable activity and these mutagenized enzymes were determined to be at least 10(6)-fold less active than wild type JHE. 5. Mutation of arginine 47 to histidine (R47H) decreased but did not abolish activity, with Km essentially unchanged at 66 nM for R47H compared to 34 nM for wild type JHE. 6. The kcat for R47H was decreased from 103 min-1 for wild type JHE to 1.9 min-1. 7. In addition, glutamate residue 332 was altered to glutamine (E332Q) and expressed in an Escherichia coli system. 8. This mutation was also found to remove all detectable activity. 9. From the results presented in this study and by comparison of JHE to other serine esterases and lipases, we predict that JHE possesses a Ser201-His446-Glu332 catalytic triad. 10. In addition, aspartate 173 and arginine 47 are essential for the efficient functioning of JHE.     

Tertiary structure in 7.9 M guanidinium chloride--the role of Glu53 and Asp287 in Pyrococcus furiosus endo-beta-1,3-glucanase

The thermodynamic stability of family 16 endo-beta-1,3-glucanase(EC 3.2.1.39) from the hyperthermophilic archaeon Pyrococcus furiosus is decreased upon single (D287A, E53A) and double (E53A/D287A) mutation of Asp287 and Glu53. In accordance with the homology model prediction,both carboxylic acids are involved in the composition of a calcium binding site, as shown by titration of the wild-type and the variant proteins with a chromophoric chelator. The present study shows that, in P. furiosus, endo-beta-1,3-glucanase residues Glu53 and Asp287 also make up a calcium binding site in 7.9 M guanidinium chloride. The persistence of tertiary structure in 7.9 M guanidinium chloride, a feature of the wild-type enzyme,is observed also for the three variant proteins. The DeltaG(H2O) values relative to the guanidinium chloride-induced equilibrium unfolding of the three variants are approximately 50% lower than that of the wild-type. The destabilizing effect of the combined mutations of the double mutant is non-additive, with an energy of interaction of 24.2 kJ x mol(-1), suggesting a communication between the two mutated residues. The decrease in the thermodynamic stability of D287A, E53A and E53A/D287A is contained almost exclusively in the m-values, a parameter which reflects the solvent exposed surface area upon unfolding. The decrease in m-value suggests that the substitution with alanine of two evenly charged repulsive side chains induces a stabilization of the non-native state in 7.9 M guanidinium chloride comparable to that induced by the presence of calcium on the wildtype. These results suggest that the stabilization of a compact non-native state may be a strategy for P. furiosus endo-beta-1,3-glucanase to thrive under adverse environmental conditions.     

The mechanism of aubstrate eecognition of pyroglutamyl-peptidase I from Bacillus amyloliquefaciens as determined by X-ray crystallography and site-directed mutagenesis

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:O.H-N:Inh and Gln71:N-H.OE: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.     

Nonessentiality of histidine 291 of Rhodospirillum rubrum ribulose-bisphosphate carboxylase/oxygenase as determined by site-directed mutagenesis

Chemical modification of spinach ribulosebisphosphate carboxylase/oxygenase by diethyl pyrocarbonate led to the conclusion that His-298 is an essential active-site residue (Igarashi, Y., McFadden, B. A., and El-Gul, T. (1985) Biochemistry 24, 3957-3962). From the pH dependence of inactivation, the pKa of His-298 was observed to be approximately 6.8, and it was suggested that this histidine might be the essential base that initiates catalysis (Paech, C. (1985) Biochemistry 24, 3194-3199). To explore further the possible function of His-298, we have used site-directed mutagenesis to replace the corresponding residue of the Rhodospirillum rubrum carboxylase (His-291) with alanine. Assays of extracts of Escherichia coli JM107, harboring either the wild-type or mutant gene in an expression vector, revealed that the mutant protein is approximately 40% as active catalytically as the normal carboxylase. After purification to near homogeneity by immunoaffinity chromatography, the mutant protein was partially characterized with respect to subunit structure, kinetic parameters, and interaction with a transition-state analogue. The purified mutant carboxylase had a kcat of 1.5 s-1 and a kcat/Km of 1.7 X 10(4) M-1 s-1 in contrast to values of 3.6 s-1 and 6 X 10(5) M-1 s-1 for the normal enzyme. The high level of enzyme activity exhibited by the Ala-291 mutant excludes His-291 in the R. rubrum carboxylase (and by inference His-298 in the spinach carboxylase) as a catalytically essential residue.