Molecular genetic analysis of the catalytic site of Streptococcus mutans glucosyltransferases.

In the present communication molecular genetic approaches have been utilized to confirm the nature of the catalytic site of Streptococcus mutans glucosyltransferases (GTF)s. Site-directed mutagenesis was used to convert the putative sucrose binding Asp-451 of the GTF-I enzyme from S. mutans GS5 to Glu, Asn, and Thr. All three of the resulting mutated enzymes displayed no detectable sucrase or GTF activities. By contrast, mutation of nearby Asp residues did not markedly reduce enzymatic activity. The inactive enzymes also appear to bind acceptor dextrans as well as the parental enzyme. These results confirm the essential role of Asp-451 of the GTF-I from strain GS5 and analogous Asp residues in other related GTFs in enzymatic activity.     

Identification of essential amino acids in the Streptococcus mutans glucosyltransferases.

A comparison of the amino acid sequences of the glucosyltransferases (GTFs) of mutans streptococci with those from the alpha-amylase family of enzymes revealed a number of conserved amino acid positions which have been implicated as essential in catalysis. Utilizing a site-directed mutagenesis approach with the GTF-I enzyme of Streptococcus mutans GS-5, we identified three of these conserved amino acid positions, Asp413, Trp491, and His561, as being important in enzymatic activity. Mutagenesis of Asp413 to Thr resulted in a GTF which expressed only about 12% of the wild-type activity. In contrast, mutagenesis of Asp411 did not inhibit enzyme activity. In addition, the D413T mutant was less stable than was the parental enzyme when expressed in Escherichia coli. Moreover, conversion of Trp491 or His561 to either Gly or Ala resulted in enzymes devoid of GTF activity, indicating the essential nature of these two amino acids for activity. Furthermore, mutagenesis of the four Tyr residues present at positions 169 to 172 which are part of a subdomain with homology to the direct repeating sequences present in the glucan-binding domain of the GTFs had little overall effect on enzymatic activity, although the glucan products appeared to be less adhesive. These results are discussed relative to the mechanisms of catalysis proposed for the GTFs and related enzymes.     

Regulation (alteration) of activity and conformation of sucrase by coaggregation with cellobiase in culture medium of Termitomyces clypeatus

Extracellular sucrase (S) of Termitomyces clypeatus was aggregated with cellobiase (C) in culture filtrate and coaggregates of sucrase to cellobiase with different activity ratios (S/C) were obtained during purification. Specific activity of the enzyme decreased significantly, after purification of sucrase free from cellobiase. Purified sucrase was characterized as a glycoprotein of molar mass around 55kDa as indicated by SDS-PAGE and HPGPLC. K(m) and V(max) of the purified enzyme were determined as 34.48 mM and 13.3 U/mg, respectively, at optimum temperature (45 degrees C) and pH (5.0). Substrate affinity and reaction velocity of the purified enzyme, free from cellobiase, was lowered by approximately 3.5 and 55 times, respectively, than that of the enzyme obtained from culture filtrate. The instant regain of sucrase activity up to the extent of 41% was obtained on in vitro addition of cellobiase (free from sucrase) to the enzyme in incubation mixture. Conformation of the enzyme free from cellobiase appeared to be significantly different from that of the coaggregate, as analyzed by circular dichroic and light scattering spectroscopy. It was concluded that activity and conformation of sucrase is regulated (altered) by heteroaggregation with cellobiase in the fungus.     

普鲁兰多糖高产菌株Y68葡萄糖基转移酶的纯化及特性研究

运用离子交换层析法和凝胶过滤层析法分离纯化普鲁兰多糖高产菌株Y68胞内葡萄糖基转移酶(简称GTF),并以SDS-PAGE、Native-PAGE对蛋白质进行量化、比较及特性鉴定,研究其酶学特性。结果表明短梗霉Y68中GTF被分离纯化,纯化酶在SDS-PAGE凝胶电泳上显示分子量50.8 ku单一条带,而在Native-PAGE凝胶电泳上显示分子量350 ku单一条带。纯化酶的最适酶反应pH和最适酶反应温度分别为6.0和40℃,酶对pH十分敏感,稳定性较差,对温度的稳定性则稍好。GTF为金属酶,Na+和K+对酶有激活作用,Ca2+、Mn2+、Mg2+、Ba2+、Cu2+、Fe2+、Hg2+、Co2+对酶活性有抑制作用,Hg2+对酶活性的抑制作用说明酶活性中心含有二锍键。EDTA、PMSF、SDS和碘乙酸对GTF活性有很大的抑制作用,而二硫苏糖醇(DTT)对酶活性有保护作用。     

Specific and non-specific affinities of the extracellular glucosyltransferase complex of Streptococcus mutans 6715

Glucosyltransferases (GTF) from different strains of streptococci exhibited different elution profiles when fractionated on insoluble-dextran affinity columns. The proportions of unadsorbed and adsorbed GTF were not related to their extent of stimulation by exogenous dextran, and GTF preparations exposed to, and freed from, clinical dextran prior to fractionation lost their ability to bind to the dextran columns. Different proportions of bound GTF were released by irrigation of columns with different concentrations of salt and clinical dextran, and the “specific” binding and release of GTF exhibited by a column possessing covalently linked, clinical dextran ligands was duplicated on a control column that did not possess the dextran ligands. These results, and the high affinity of GTF for hydrophobic alkyl (Shaltiel) ligands, demonstrate that ionic and hydrophobic properties of impure GTF aggregates may lead to erroneous characterization of the dextran affinity of some protein fractions. Fractionations on DEAE-Sepharose and on hydroxylapatite showed that the two dextran-dependant GTF activities (GTF-S and GTF-I) were present in the major enzyme fraction (Streptococcus mutans 6715) recovered from a Sephacryl S-200 affinity column. A minor, dextran-independent GTF was not adsorbed onto the Sephacryl column. The presence of SDS (0.005%) and Triton X100 (0.01%) stabilized GTF activity during gel filtration and improved the separation of GTF-S and GTF-I in hydroxylapatite fractionation of the highly aggregated enzyme. A comparable separation of the two enzyme forms on DEAE-Sepharose was achieved only if T10 dextran (10 mg/mL) was included with the detergent mixture in the column irrigant.     

Physical, chemical, and enzymatic studies on the major sucrase of honey bees (Apis mellifera).

A sucrase from honey bees (Apis mellifera) which precipitates between ammonium sulfate saturations of 50 and 70% (5 mg protein per millilitre) and which makes up the major portion of the sucrases of honey bees was purified to homogeneity as shown by several criteria. A large part of the sucrase was found in the head while most of the rest was in the abdomen (a small amount was in the thorax). The enzyme precipitated between the same values of ammonium sulfate saturation as did the sucrase in honey and honey sucrase exhibited kinetics very similar to those of this enzyme. The enzyme was found to be a relatively nonspecific alpha-glucosidase and was shown to have transglucosidase activity. The production of glucose from sucrose was rectilinear when plotted by the Hofstee method at low substrate concentrations but decreased at high sucrose concentrations. The production of fructose was rectilinear throughout the concentration range used. The production of both glucose and rho-nitrophenol when rho nitrophenyl alpha-D-glucoside was the substrate was linear by the Hofstee plot. These effects were found to be due to transglucolysis and a mechanism of action is proposed. Amino acid and amino sugar analyses indicated that the sucrase was a glycoprotein. The molecular weight was found to be between 51000 and 82000 by three different methods and an so20.w value of 4.0 S was obtained. There was no evidence for subunit structure. Tests of the enzyme under various denaturation conditions did not reveal any unusual stabilities. The sucrase bound very tightly to a hydrophobic column. Iodoacetic acid decreased the activity of the sucrase but a large concentration was needed to bring about a 50% activity loss. Reducing agents caused some activity declines. Diethyl pyrocarbonate activated the enzyme.     

Effect of harmaline on rat intestinal brush border sucrase activity

The effect of harmaline, a plant alkaloid has been studied on rat intestinal brush border sucrase activity. Stimulation of sucrase activity by Na+ was found to be pH-dependent. At neutral pH, 20 mM Na+ stimulated sucrase activity by reducing K(m) by 30%, while at acidic pH (5.2), the activity increased 4-fold compared to Na+-free enzyme. At 1.0 mM, harmaline markedly inhibited (67%) the enzyme activity at pH 5.2 in the absence of Na+. However, inhibition was reduced in presence of 20 mM sodium, whereas 4.0 mM harmaline was required to inhibit the enzyme activity by 65%. In the absence of Na+ ions, harmaline inhibition of sucrase activity was of competitive type, but it changed to non-competitive type in presence of 20 mM Na+ at pH 5.2. Sucrase-harmaline interactions as a function of pH, both in presence and absence of Na+ revealed a shift in pH optima of the enzyme towards a higher pH in presence of 4 mM and 1 mM harmaline respectively. The observed inhibition was reversible in nature and was only partially overcome by sodium, lithium, potassium, cesium, rubidium and ammonium ions. These findings suggest that harmaline also inhibits rat brush border sucrase and that the presence of Na+ site is not a pre-requisite for the inhibition.     

Cortisone and thyroxine modulate intestinal lactase and sucrase mRNA levels and activities in the suckling rat.

Glucocorticoids and thyroxine modulate postnatal intestinal sucrase and lactase activities. Whether changes in enzyme activity are accompanied by changes in enzyme mRNA levels were determined in day 6 rats given thyroxine, cortisone, or thyroxine plus cortisone and killed 3 days later. Cortisone induced precocious expression of jejunal sucrase activity which was enhanced when cortisone plus thyroxine was administered; sucrase mRNA changed in parallel. Jejunal lactase activity was unaffected by thyroxine and was increased after cortisone, but not after thyroxine plus cortisone. Jejunal lactase mRNA levels increased equally after cortisone or after cortisone plus thyroxine. Thus, cortisone induces coordinated increases in sucrase and lactase activities and in corresponding mRNA levels. Thyroxine only enhances cortisone induced sucrase expression and antagonizes cortisone by depressing lactase activity post-translationally.     

Blood group activity of human sucrase from intestinal metaplasia.

Sucrases were purified from human small intestine and from areas of intestinal metaplasia of the stomach mucosa surrounding stomach cancers. The kinetic constants and pH activity profiles of enzyme preparations from the two sources were similar. No blood group activity of sucrase was detectable in preparations from three cases of intestinal metaplasia, but preparations from two other cases showed activity like that of the small intestine. These results indicate that sucrase from areas of intestinal metaplasia has similar enzymatic properties to those of enzyme from the small intestine, but that the antigenic sugar moiety of the enzyme associated with blood group activity varies.     

Castanospermine: a potent inhibitor of sucrase from the human enterocyte-like cell line Caco-2

The addition of castanospermine (5-50 microM) to a culture medium of Caco-2 cells results in a specific suppression of sucrase activity without modification of the biosynthesis of the enzyme. This effect is due to a direct inhibiting effect of castanospermine on Caco-2 sucrase activity. This inhibition is time-dependent (half-maximum efficiency at 10 min for 100 nM), enhanced by preincubation (suggesting a strong interaction with the enzyme), dose-dependent (ED50 at 4 nM after 1 h preincubation period) and of the fully non-competitive type. The calculated Ki (2.6 nM) suggests that castanospermine is the most potent inhibitor of sucrase so far reported.     

Dextran synthesis by immobilized dextran sucrase

Dextran sucrase has been produced by fermentation of Leuconostoc mesenteroides NRRL B-512, with and without continuous sucrose addition to improve enzyme production. The enzyme preparation has been concentrated from the fermentation broth by ultrafiltration and purified by gel permeation chromatography on Ultrogel. The specific activity of the dextran sucrase was greatly enhanced by calcium chloride addition to the purified enzyme. This enzyme preparation has been immobilized by covalent coupling onto an amino porous silica support (Spherosil) activated with glutaraldehyde. Immobilized dextran sucrase derivatives with an activity up to 830 dextran sucrase units per g. support could thus be obtained. The effect of the support specific area on coupling efficiency and reaction kinetics has been investigated, and the effect of intraparticular diffusion underlined. The molecular weight distribution of the dextran has been determined when varying several parameters.     

Characterization of a novel low molecular weight sucrase from filamentous fungus Termitomyces clypeatus

An extracellular sucrase from the culture filtrate of filamentous basidiomycota Termitomyces clypeatus grown on high sucrose (5%, w/v) was purified by gel filtration chromatography, ion exchange chromatography and HPGPLC. The biochemical properties, molecular weight and conformation of sucrase produced were significantly different from the sucrase earlier purified from sucrose (1%, w/v) medium in the fungus. Purified sucrase was characterized as a low molecular weight protein of 13.5 kDa as approximated by SDS-PAGE and HPGPLC and exhibited predominantly random coil conformation in far-UV CD spectra. The enzyme was optimally active at 47 °C and pH 5.0. K_m and catalytic activity of the enzyme for sucrose were found to be 3.5 mM and 1.06 U/mg/mM, respectively. The enzyme was maximally active towards sucrose than to raffinose and sucrase activity was significantly inhibited by bivalent metal ions and reducing group agents. The results indicated that due to changes in aggregation pattern, molecular organization of purified sucrase, produced in high sucrose medium, was altered and was different from the previously reported enzyme. This is the first report of a sucrase of such low size showing activity.     

Structural analysis of the alpha-D-glucan (EPS180) produced by the Lactobacillus reuteri strain 180 glucansucrase GTF180 enzyme

The neutral exopolysaccharide EPS180 produced from sucrose by the glucansucrase GTF180 enzyme from Lactobacillus reuteri 180 was found to be a (1-->3,1-->6)-alpha-D-glucan, with no repeating units present. Based on linkage analysis, periodate oxidation, and 1D/2D 1H and 13C NMR spectroscopy of the intact EPS180, as well as MS and NMR analysis of oligosaccharides obtained by partial acid hydrolysis of EPS180, a composite model, that includes all identified structural features, was formulated as follows: [Formula: see text].     

Decrease in adenylate cyclase activity in spleen T- and B-lymphocytes and thymus T-lymphocytes in CBA mice treated with levamisole

The influence of various concentrations of levamisole on the activity of the membrane adenylate cyclase complex of lymphocytes is analyzed. It is ascertained that levamisole decreases the activity of adenylate cyclase at a concentration of 10(-9)-10(-7) M in thymic lymphocytes, and that the activity of the enzyme in spleen T- and B-lymphocytes remains constant. The influence of levamisole on the activity of adenylate cyclase thymocytes is studied under conditions of its stimulation by NaF, GTF and adrenaline. It is ascertained that levamisole at a concentration of 10(-9)-10(-7) M decreases the activity of the enzyme stimulated by NaF, GTF and adrenaline.     

Deletions in the carboxyl-terminal region of Streptococcus gordonii glucosyltransferase affect cell-associated enzyme activity and sucrose-associated accumulation of growing cells.

The single glucosyltransferase (GTF) of Streptococcus gordonii Challis CH1 makes alpha 1,3- and alpha 1,6-linked glucans from sucrose. The GTF carboxyl-terminal region has six direct repeats thought to be involved in glucan binding. Strains with defined mutations in this region have been described recently (M. M. Vickerman, M. C. Sulavik, P. E. Minick, and D. B. Clewell, Infect. Immun. 64:5117-5128, 1996). Strain CH107 GTF has three internal direct repeats deleted; the 59 carboxyl-terminal amino acids are identical to those of the parental strain. This deletion resulted in decreased enzyme activity but did not affect the amount of cell-associated GTF protein. The GTFs of strains CH2RPE and CH4RPE have six and eight direct repeats, respectively, but are both missing the 14 carboxyl-terminal amino acids. Strain CH2RPE had significantly decreased levels of cell-associated GTF; this decrease was not obviated by the increased number of direct repeats in strain CH4RPE. Thus, the carboxyl-terminal amino acids appeared to influence the amount of cell-associated GTF more than the direct repeats. The qualitative and quantitative differences in the GTFs did not affect the abilities of these strains to accumulate on hydroxyapatite beads in the absence of sucrose. However, when sucrose was added as a substrate for GTF, the mutant strains were unable to accumulate on these surfaces to the same extent as the parent. These differences in sucrose-associated accumulation may be due to changes in the nature of the glucans produced by the different enzymes and/or cohesive interactions between these glucans and the GTF on the surfaces of the growing streptococci.     

Identification of peptides from autolysates of Saccharomyces cerevisiae that exhibit glucose tolerance factor activity in a yeast assay

1. Cationic fractions were isolated from a low chromium (less than 0.2 ppm) commercial yeast extract in an attempt to purify the material responsible for glucose tolerance factor (GTF) activity observed in a standard yeast assay system. 2. Following previously described procedures a fraction with GTF activity but containing negligible chromium was isolated, which on further purification was found to be composed of many separate small basic peptides. 3. Much of the activity of the yeast GTF material in the yeast assay could be attributed to the presence of basic peptides and free amino acids acting as nitrogen sources for the yeast. 4. Additional activity was present in the yeast GTF sample, which was not due to a synergistic effect of the mixed amino acids and peptides although the component of the yeast extract responsible for this activity was not identified. 5. The results show that the GTF fractions isolated according to most previously published procedures are highly impure, and conclusions drawn about the nature of GTF based on these isolates must remain open to question. 6. The activity due to the presence of peptides and amino acids is a major cause of lack of specificity of the yeast systems as an assay for GTF.     

Presence of sucrase in the yolk sac of the chick

The presence of sucrase in the yolk sac of the chick was studied biochemically and immunologically. The sucrase was partially purified from the yolk sac of hatched chicks and was compared with the sucrase purified from the small intestine. Immunodiffusion with antiserum against intestinal sucrase and characterization of the activity revealed that the two enzymes were almost identical. However, the size of the yolk sac sucrase was found to be slightly smaller than that of the intestinal enzyme by chromatography on Sephadex G-200 and polyacrylamide gel electrophoresis. Immunocytochemical studies showed that the sucrase was located on the free surface of yolk sac endodermal cells, but the sucrase may also be present in the cytoplasm.     

Mutants of Streptococcus gordonii Challis over-producing glucosyltransferase

Two mutants of Streptococcus gordonii which over-produced extracellular polysaccharide when grown on sucrose-containing medium were isolated after mutagenesis of strain Challis with ethyl methanesulphonate. The mutants, designated strains OB20 and OB30, expressed 2.6-fold and 4.7-fold respectively more glucosyltransferase (GTF) activities than the wild-type strain. Transformation experiments suggested that the two mutants carried different mutations, denoted gtf-20 and gtf-30. A double mutant (gtf-20 gtf-30) was constructed and this strain produced 6.4-fold more GTF. Enzymes from wild-type and mutant strains were biochemically indistinguishable and they synthesized structurally identical glucans. Increasing the Na+ concentration of the bacterial growth medium reduced GTF production in all strains by about 60%. Tween 80 also inhibited enzyme production and more specifically reduced GTF synthesis by the mutants. The mutations gtf-20 and gtf-30 appear to define separate genetic loci involved in regulating expression of GTF activity in S. gordonii.     

Development of high-molar-mass cellobiase complex by spontaneous protein-protein interaction in the culture filtrate ofTermitomyces clypeatus

The 450 kDa cellobiase fromTermitomyces clypeatus which migrates as a single band on IEF, PAGE and SDS-PAGE, was found to possess appreciable sucrase activity. The fungus produced sucrase and cellobiase constitutively in different media but with different activity ratios. The kinetics of secretion of the two enzymes was similar underin vivo andin vitro conditions. HPGPLC analysis of the culture filtrates indicated the presence of both sucrase and cellobiase in the same protein fractions of different molar mass, even in the 30-kDa protein fraction. No free sucrase or cellobiase could be detected in the culture filtrates. It was also observed that fractionation of cellobiase by (NH₄)₂SO₄ precipitation was different with different amounts of associated sucrase activity present in the culture filtrate. The (NH₄)₂SO₄-precipitated cellobiase fraction also contained cellobiases in proteins of widely varied molar mass ranges. However, none of the low-molar mass proteins other than the 450-kDa enzyme could be purified, as all low-molar-mass fractions spontaneously aggregated to the 450-kDa enzyme. Hydrophobic chromatography of the (NH₄)₂SO₄-precipitated fractions followed by HPGPLC of the eluted active fraction yielded both cellobiase-free sucrase and a very low sucrase-containing cellobiase fraction. The cellobiase fraction, homogeneous in PAGE, was also a high-molar-mass protein complex dissociating into a number of protein bands on SDS-PAGE. It was suggested that the 450-kDa cellobiase was not liberated by the fungus as a preformed enzyme complex but that the complex developed through interaction of cellobiase with sucrase underin vitro conditions and the possibility of the involvement of other proteins in the aggregation cannot be excluded.