Screening and Application of Microbial Esterases for the Enantioselective Synthesis of Chiral Glycerol Derivatives

Glucansucrases from family 70 of glycoside-hydrolases catalyse the synthesis of α-glucans with various types of osidic linkages from sucrose. Among these enzymes, alternansucrase (ASR) and dextransucrase E (DSR-E) catalyse the formation of unusual α-glucans. ASR catalyses the synthesis of linear glucan with α-1,3 and α-1,6 alternating linkages and DSR-E synthesizes a glucan containing α-1,6 linkages in the linear chain and α-1,2 branches. The sequence analysis of these enzymes enabled the identification of structural elements suspected to be involved in the enzyme specificities. Biochemical characterization of ASR and DSR-E variants obtained from gene truncations or site-directed mutagenesis experiments showed that the specificity of these enzymes to form different types of osidic linkage is controlled by two different approaches. For ASR, the double specificity is controlled by only one catalytic domain where important amino acids involved in the enzyme specificity have been identified. In the case of DSR-E, the double specificity is controlled by two different catalytic domains both belonging to family 70, each domain being specific of one type of linkage.     

Isolation of a gene from Leuconostoc citreum B/110-1-2 encoding a novel dextransucrase enzyme

The amplicon encoding dextransucrase DSR-F from Leuconostoc citreum B/110-1-2, a novel sucrose glucosyltransferase (GTF)-specific for α-1,6 and α-1,3 glucosidic bond synthesis, with α-1,4 branching was cloned, sequenced, and expressed into Escherichia coli JM109. Recombinant enzyme catalyzed oligosaccharides synthesis from sucrose as donor and maltose acceptor. The dsrF gene encodes for a protein (DSR-F) of 1,528 amino acids, with a theoretical molecular mass of 170447.72 Da (~170 kDa). From amino acid sequence comparison, it appears that DSR-F possesses the same domains as those described for GTFs. However, the variable region is longer than in other GTFs (by 100 amino acids) and two APY repeats (a 79 residue long motif with a high number of conserved glycine and aromatic residues, characterized by the presence of the three consecutive residues Ala, Pro, and Tyr) were identified in the glucan binding domain. The DSR-F catalytic domain possesses the catalytic triad involved in the glucosyl enzyme formation. The amino acid sequence of this domain shares a 56% identity with catalytic domain of the alternansucrase ASR from L. citreum NRRL B-1355 and with the catalytic domain of a putative alternansucrase sequence found in the genome of L. citreum KM20. A truncated active variant DSR-F-∆SP-∆GBD of 1,251 amino acids, with a molecular mass of 145 544 Da (~145 kDa), was obtained.     

Molecular characterization of DSR-E,an alpha-1,2 linkage-synthesizing dextransucrase with two catalytic domains

A novel Leuconostoc mesenteroides NRRL B-1299 dextransucrase gene, dsrE, was isolated, sequenced, and cloned in Escherichia coli, and the recombinant enzyme was shown to be an original glucansucrase which catalyses the synthesis of alpha-1,6 and alpha-1,2 linkages. The nucleotide sequence of the dsrE gene consists of an open reading frame of 8,508 bp coding for a 2,835-amino-acid protein with a molecular mass of 313,267 Da. This is twice the average mass of the glucosyltransferases (GTFs) known so far, which is consistent with the presence of an additional catalytic domain located at the carboxy terminus of the protein and of a central glucan-binding domain, which is also significantly longer than in other glucansucrases. From sequence comparison with family 70 and alpha-amylase enzymes, crucial amino acids involved in the catalytic mechanism were identified, and several original sequences located at some highly conserved regions in GTFs were observed in the second catalytic domain.     

Role of the two catalytic domains of DSR-E dextransucrase and their involvement in the formation of highly alpha-1,2 branched dextran

The dsrE gene from Leuconostoc mesenteroides NRRL B-1299 was shown to encode a very large protein with two potentially active catalytic domains (CD1 and CD2) separated by a glucan binding domain (GBD). From sequence analysis, DSR-E was classified in glucoside hydrolase family 70, where it is the only enzyme to have two catalytic domains. The recombinant protein DSR-E synthesizes both alpha-1,6 and alpha-1,2 glucosidic linkages in transglucosylation reactions using sucrose as the donor and maltose as the acceptor. To investigate the specific roles of CD1 and CD2 in the catalytic mechanism, truncated forms of dsrE were cloned and expressed in Escherichia coli. Gene products were then small-scale purified to isolate the various corresponding enzymes. Dextran and oligosaccharide syntheses were performed. Structural characterization by (13)C nuclear magnetic resonance and/or high-performance liquid chromatography showed that enzymes devoid of CD2 synthesized products containing only alpha-1,6 linkages. On the other hand, enzymes devoid of CD1 modified alpha-1,6 linear oligosaccharides and dextran acceptors through the formation of alpha-1,2 linkages. Therefore, each domain is highly regiospecific, CD1 being specific for the synthesis of alpha-1,6 glucosidic bonds and CD2 only catalyzing the formation of alpha-1,2 linkages. This finding permitted us to elucidate the mechanism of alpha-1,2 branching formation and to engineer a novel transglucosidase specific for the formation of alpha-1,2 linkages. This enzyme will be very useful to control the rate of alpha-1,2 linkage synthesis in dextran or oligosaccharide production.     

Conserved repeat motifs and glucan binding by glucansucrases of oral streptococci and Leuconostoc mesenteroides

Glucansucrases of oral streptococci and Leuconostoc mesenteroides have a common pattern of structural organization and characteristically contain a domain with a series of tandem amino acid repeats in which certain residues are highly conserved, particularly aromatic amino acids and glycine. In some glucosyltransferases (GTFs) the repeat region has been identified as a glucan binding domain (GBD). Such GBDs are also found in several glucan binding proteins (GBP) of oral streptococci that do not have glucansucrase activity. Alignment of the amino acid sequences of 20 glucansucrases and GBP showed the widespread conservation of the 33-residue A repeat first identified in GtfI of Streptococcus downei. Site-directed mutagenesis of individual highly conserved residues in recombinant GBD of GtfI demonstrated the importance of the first tryptophan and the tyrosine-phenylalanine pair in the binding of dextran, as well as the essential contribution of a basic residue (arginine or lysine). A microplate binding assay was developed to measure the binding affinity of recombinant GBDs. GBD of GtfI was shown to be capable of binding glucans with predominantly alpha-1,3 or alpha-1,6 links, as well as alternating alpha-1,3 and alpha-1,6 links (alternan). Western blot experiments using biotinylated dextran or alternan as probes demonstrated a difference between the binding of streptococcal GTF and GBP and that of Leuconostoc glucansucrases. Experimental data and bioinformatics analysis showed that the A repeat motif is distinct from the 20-residue CW motif, which also has conserved aromatic amino acids and glycine and which occurs in the choline-binding proteins of Streptococcus pneumoniae and other organisms.     

Gluco-oligosaccharides synthesized by glucosyltransferases from constitutive mutants of Leuconostoc mesenteroides strain Lm 28

Aims:  To find different types of glucosyltransferases (GTFs) produced by Leuconostoc mesenteroides strain Lm 28 and its mutant forms, and to check the effectiveness of gluco-oligosaccharide synthesis using maltose as the acceptor.
Methods and Results:  Constitutive mutants were obtained after chemical mutagenesis by ethyl methane sulfonate. Lm M281 produced more active GTFs than that obtained by the parental strain cultivated on sucrose. GTF from Lm M286 produced a resistant glucan, based on endo-dextranase and amyloglucosidase hydrolysis. The extracellular enzymes from Lm M286 catalyse acceptor reactions and transfer the glucose unit from sucrose to maltose to produce gluco-oligosaccharides (GOS). By increasing the sucrose/maltose ratio, it was possible to catalyse the synthesis of oligosaccharides of increasing degree of polymerization (DP).
Conclusions:  Different types of GTFs (dextransucrase, alternansucrase and levansucrase) were produced from new constitutive mutants of Leuc. mesenteroides . GTFs from Lm M286 can catalyse the acceptor reaction in the presence of maltose, leading to the synthesis of branched oligosaccharides.
Significance and Impact of the Study:  Conditions were optimized to synthesize GOS by using GTFs from Lm M286, with the aim of producing maximum quantities of branched-chain oligosaccharides with DP 3–5. This would allow the use of the latter as prebiotics.     

Leuconostoc mesenteroides B-1355 Mutants Producing Alternansucrases Exhibiting Decreases in Apparent Molecular Mass

Mutants of Leuconostoc mesenteroides B-1355 exhibiting decreases in the apparent molecular mass of alternansucrase on sodium dodecyl sulfate (SDS)-polyacrylamide gels stained for enzyme activity were isolated after mutagenizing strain R15 with N-methyl-N(prm1)-nitro-N-nitrosoguanidine. Strain R15 was a UV mutant of strain B-1355 which was enriched for production of alternansucrase. All strains produced principal and minor alternansucrase bands on SDS gels when cultures were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The patterns of the principal and minor activity bands on our SDS gels did not result from dextran-enzyme complexes, because mutants constitutive for synthesis of glucosyltransferases (GTFs) on sugars other than sucrose produced activity bands after growth in glucose medium that were the same as those produced after growth in sucrose medium. Dextransucrase, which had been inactivated by heating at 45(deg)C, was reactivated when subjected to SDS-PAGE, showing that our SDS-PAGE conditions were reversibly denaturing. Thermal denaturation at 45(deg)C did not involve a dispersal of GTFs into subunits. Densitometry measurements showed a roughly linear relationship between enzyme activity and band intensity over a loading range of 0.2 to 0.8 mU per sample well. We concluded that SDS-PAGE followed by activity staining was a reliable method for estimating numbers and ratios of GTFs produced by Leuconostoc sp. in media containing sucrose.     

Understanding the polymerization mechanism of glycoside-hydrolase family 70 glucansucrases

Glucan formation catalyzed by two GH-family 70 enzymes, Leuconostoc mesenteroides NRRL B-512F dextransucrase and L. mesenteroides NRRL B-1355 alternansucrase, was investigated by combining biochemical and kinetic characterization of the recombinant enzymes and their respective products. Using HPAEC analysis, we showed that two molecules act as initiator of polymerization: sucrose itself and glucose produced by hydrolysis, the latter being preferred when produced in sufficient amounts. Then, elongation occurs by transfer of the glucosyl residue coming from sucrose to the non-reducing end of initially formed products. Dextransucrase preferentially produces an isomaltooligosaccharide series, whose concentration is always low because of the high ability of these products to be elongated and form high molecular weight dextran. Compared with dextransucrase, alternansucrase has a broader specificity. It produces a myriad of oligosaccharides with various alpha-1,3 and/or alpha-1,6 links in early reaction stages. Only some of them are further elongated. Overall alternan polymer is smaller in size than dextran. In dextransucrase, the A repeats often found in C-terminal domain of GH family 70 were found to play a major role in efficient dextran elongation. Their truncation result in an enzyme much less efficient to catalyze high molecular weight polymer formation. It is thus proposed that, in dextransucrase, the A repeats define anchoring zones for the growing chains, favoring their elongation. Based on these results, a semi-processive mechanism involving only one active site and an elongation by the non-reducing end is proposed for the GH-family 70 glucansucrases.     

Purification and characterization of Acremonium implicatum alpha-glucosidase having regioselectivity for alpha-1,3-glucosidic linkage

alpha-Glucosidase with a high regioselectivity for alpha-1,3-glucosidic linkages for hydrolysis and transglucosylation was purified from culture broth of Acremonium implicatum. The enzyme was a tetrameric protein (M.W. 440,000), of which the monomer (M.W. 103,000; monomeric structure was expected from cDNA sequence) was composed of two polypeptides (M.W. 51,000 and 60,000) formed possibly by posttranslational proteolysis. Nigerose and maltose were hydrolyzed by the enzyme rapidly, but slowly for kojibiose. The k(0)/K(m) value for nigerose was 2.5-fold higher than that of maltose. Isomaltose was cleaved slightly, and sucrose was not. Maltotriose, maltotetraose, p-nitrophenyl alpha-maltoside and soluble starch were good substrates. The enzyme showed high affinity for maltooligosaccharides and p-nitrophenyl alpha-maltoside. The enzyme had the alpha-1,3- and alpha-1,4-glucosyl transfer activities to synthesize oligosaccharides, but no ability to form alpha-1,2- and alpha-1,6-glucosidic linkages. Ability for the formation of alpha-1,3-glucosidic linkage was two to three times higher than that for alpha-1,4-glucosidic linkage. Eight kinds of transglucosylation products were synthesized from maltose, in which 3(2)-O-alpha-nigerosyl-maltose and 3(2)-O-alpha-maltosyl-maltose were novel saccharides.     

Production of glucosyltransferases by wild-type Leuconostoc mesenteroides in media containing sugars other than sucrose

Leuconostoc mesenteroides produces glucosyltransferases (GTFs) and fructosyltransferases (FTFs) which are inducible enzymes which respectively synthesize dextrans and levans from sucrose. Except for a few mutant strains which produce high activities in glucose medium, L. mesenteroides is thought not to produce GTFs and FTFs unless sucrose is present. We show here that cultures of eight strains produced low, but detectable GTF activity when glucose, maltose or melibiose replaced sucrose as the growth substrate. Four strains also produced FTFs of approximately 130 kDa in medium with or without sucrose. The GTFs and FTFs produced on sugars other than sucrose could be detected as bands on SDS gels even when not detected by other methods. Except for strain B-523, the number, sizes and relative intensities of the bands were independent of the sugar used for growing the cultures. Alternansucrase from strains B-1355 and B-1501 in glucose or maltose medium was almost entirely associated with the cell fraction, ruling out binding to glucans as the cause of the association. Received 06 October 1998/ Accepted in revised form 05 February 1999     

The influence of mutanase and dextranase on the production and structure of glucans synthesized by streptococcal glucosyltransferases

Glucanohydrolases, especially mutanase [alpha-(1-->3) glucanase; EC 3.2.1.59] and dextranase [alpha-(1-->6) glucanase; EC 3.2.1.11], which are present in the biofilm known as dental plaque, may affect the synthesis and structure of glucans formed by glucosyltransferases (GTFs) from sucrose within dental plaque. We examined the production and the structure of glucans synthesized by GTFs B (synthesis of alpha-(1-->3)-linked glucans) or C [synthesis of alpha-(1-->6)- and alpha-(1-->3)-linked glucans] in the presence of mutanase and dextranase, alone or in combination, in solution phase and on saliva-coated hydroxyapatite beads (surface phase). The ability of Streptococcus sobrinus 6715 to adhere to the glucan, which was formed in the presence of the glucanohydrolases was also explored. The presence of mutanase and/or dextranase during the synthesis of glucans by GTF B and C altered the proportions of soluble to insoluble glucan. The presence of either dextranase or mutanase alone had a modest effect on total amount of glucan formed, especially in the surface phase; the glucanohydrolases in combination reduced the total amount of glucan. The amount of (1-->6)-linked glucan was reduced in presence of dextranase. In contrast, mutanase enhanced the formation of soluble glucan, and reduced the percentage of 3-linked glucose of GTF B and C glucans whereas dextranase was mostly without effect. Glucan formed in the presence of dextranase provided fewer binding sites for S. sobrinus; mutanase was devoid of any effect. We also noted that the GTFs bind to dextranase and mutanase. Glucanohydrolases, even in the presence of GTFs, influence glucan synthesis, linkage remodeling, and branching, which may have an impact on the formation, maturation, physical properties, and bacterial binding sites of the polysaccharide matrix in dental plaque. Our data have relevance for the formation of polysaccharide matrix of other biofilms.     

Stimulation of various functions in murine peritoneal macrophages by glucans produced by glucosyltransferases from Streptococcus mutans

The glucan that was produced by glucosyltransferases (GTFs) from Streptococcus mutans was examined for its stimulating functions toward murine peritoneal macrophages. Soluble glucan was obtained by the reaction with cell-free crude GTFs and sucrose, followed by ethanol precipitation, dispersion in water and re-precipitation by ethanol. Soluble glucan, those average molecular weight was about 3 x 10(5), was composed of mixture of alpha-1,6 and alpha-1,3 linkages in a 3:1 ratio. When 30 and 60 microg/ml of the glucan was incubated with peritoneal macrophages, the lysosomal phosphatase activity was increased in a dose-dependent manner, indicating that soluble glucan may activate macrophages. To examine its effects on the various functions of macrophages, soluble glucan was orally administered daily at a level of 100 mg/kg of body weight to C57BL/6 mice. Significant stimulation of the production of H2O2 by the macrophages was observed without any increase in NO production. The production of tumor necrosis factor-alpha (TNF-alpha) by the macrophages was also stimulated from 538.73-555.06 pg/ml to 585.73-596.40 pg/ml during 15 days of oral administration of soluble glucan. The cytotoxicity of peritoneal macrophages against B16 tumor cells was significantly enhanced by 25-38% during 15 days of oral administration. These results may indicate that soluble glucan stimulates the immune functions of macrophages.     

Production and catabolite repression of Penicillium italicum beta-glucanases.

The filamentous fungus Penicillium italicum, grown in a defined liquid medium, produced beta-1,3-glucanase, which remained essentially bound to the cells, and beta-1,6-glucanase, an essentially extracellular enzyme. When glucose was depleted from the medium, when a limited concentration of glucose (0.2%) was maintained, or when the carbon source was galactose (3%) or lactose (3%), a significant increase in the specific activity of beta-1,3-glucanase, in cell extracts, took place. This was paralleled by a very slow rate of growth, and under glucose limitation, the appearance of beta-1,3-glucanase in the medium was also observed. On the other hand, when an excess of glucose, fructose, or sucrose was present, the specific activity remained constant and active growth was promoted. Laminarin, cellobiose, gentiobiose, and isolated Penicillium italicum walls were not capable of significantly inducing beta-1,3-glucanase synthesis to a level beyond that attained by glucose limitation. A similar behavior was observed for beta-1,6-glucanase. beta-1,3-Glucanase and beta-1,6-glucanase are therefore constitutive enzymes subjected to catabolite repression. The results are discussed in the context of the possible functions that have been suggested for glucanases and related enzymes.     

Activation and inhibition of dextransucrase by calcium

Initial rate kinetics of dextran synthesis by dextransucrase (sucrose:1,6-alpha-D-glucan-6-alpha-D-glucosyltransferase, EC 2.4.1.5) from Leuconostoc mesenteroides NRRL B-512F showed that below 1 mM, Ca2+ activated the enzyme by increasing Vmax and decreasing the Km for sucrose. Above 1 mM, Ca2+ was a weak competitive inhibitor (Ki = 59 mM). Although it was an activator at low concentration, Ca2+ was not required for dextran synthesis, either of main chain or branch linkages. Neither was it required for sucrose hydrolysis, acceptor reactions, or enzyme renaturation after SDS-polyacrylamide gel electrophoresis. A model for dextran synthesis is proposed in which dextransucrase has two Ca2+ sites, one activating and one inhibitory. Ca2+ at the inhibitory site prevents the binding of sucrose.     

Factors affecting alpha,-1,2 glucooligosaccharide synthesis by Leuconostoc mesenteroides NRRL B-1299 dextransucrase

The optimization of alpha-1,2 glucooligosaccharide (GOS) synthesis from maltose and sucrose by Leuconostoc mesenteroides NRRL B-1299 dextransucrase was achieved using experimental design and consecutive analysis of the key parameters. An increase of the pH of the reaction from 5.4 to 6.7 and of the temperature from 25 to 40 degrees C significantly favored alpha-1,2 GOS synthesis, thanks to a significant decrease of the side reactions, i.e., dextran and leucrose synthesis. These positive effects were not sufficient to compensate for the decrease of enzyme stability caused by the use of high pH and temperature. However, the critical parameters were the sucrose to maltose concentration ratio (S/M) and the total sugar concentration (TSC). Alpha1,2 GOS synthesis was favored at high S/M ratios. But using these conditions also led to an increase of side reactions which could be modulated by choosing the appropriate TSC. Finally, with S/M = 4 and TSC = 45% w/v, dextran and leucrose productions were limited and the final alpha-1,2 GOS yield reached 56.7%, the total GOS yield being 88%.     

Purification and properties of extracellular glucosyltransferase synthesizing 1,6-, 1,3-alpha-D-glucan from Streptococcus mutans serotype a

An extracellular glucosyltransferase (sucrose: 1,6-, 1,3-alpha-D-glucan 3-alpha- and 6-alpha-D-glucosyltransferase, EC 2.4.1.-) of Streptococcus mutans HS6 (serotype a) was purified from culture supernatant by DEAE-Sepharose chromatography and preparative isoelectric focusing. The molecular weight measured by SDS-PAGE was 159 000 and the isoelectric point was pH 4.9. The specific activity was 89.7 i.u. (mg protein)-1 and the optimum pH was 6.0. The Km value for sucrose was 4.9 mM and the enzyme activity was not stimulated by exogenous dextran T10. Glucan was synthesized de novo from sucrose by the purified enzyme and consisted of 49.1 mol% 1,6-alpha-linked glucose and 33.9 mol% 1,3-alpha-linked glucose, with 13.6 mol% terminal glucose and 3.3 mol% 1,3,6-alpha-branched glucose.     

Structure and enzymatic properties of genetically truncated forms of the water-insoluble glucan-synthesizing glucosyltransferase from Streptococcus sobrinus.

Glucosyltransferase-I (GTF-I: 175 kDa) of a cariogenic bacterium, Streptococcus sobrinus 6715, mediates the conversion of water-soluble dextran (alpha-1,6-glucan) into a water-insoluble form by making numerous alpha-1,3-glucan branches along the dextran chains with sucrose as the glucosyl donor. The structures and catalytic properties were compared for two GTF-I fragments, GTF-I' (138 kDa) and GS (110 kDa). Both lack the N-terminal 84 residues of GTF-I. While GTF-I' still contains four of the six C-terminal repeats characteristic of streptococcal glucosyltransferases, GS lacks all of them. Electron microscopy of negatively stained samples indicated a double-domain structure for GTF-I', consisting of a spherical head with a smaller spherical tail, which was occasionally seen as a long extension. GS was seen just as the head portion of GTF-I'. In the absence of dextran, both fragments simply hydrolyzed sucrose with similar K(m) and k(cat) values at low concentrations (<5 mM). At higher sucrose concentrations (>10 mM), however, GTF-I' exhibited glucosyl transfer activity to form insoluble alpha-1, 3-glucans. So did GS, but less efficiently. Dextran increased the rate and efficiency of the glucosyl transfer by GTF-I'. On removal of the C-terminal repeats of GTF-I' by mild trypsin treatment, this dextran-stimulated transfer was completely lost and the dextran-independent transfer became less efficient. These results indicate that the N-terminal two-thirds of the GTF-I sequence are organized as a structurally and functionally independent domain to catalyze not only sucrose hydrolysis but also glucosyl transfer to form alpha-1,3-glucan chains, although not efficiently; the C-terminal repeat increases the efficiency of the intrinsic glucosyl transfer by the N-terminal domain as well as rendering the whole molecule primer-dependent for far more efficient insoluble glucan synthesis.     

Functional, genetic, and bioinformatic characterization of dextransucrase (DSRBCB4) gene in Leuconostoc mesenteroides B-1299CB4

A gene encoding a dextransucrase (dsrBCB4) that synthesizes only alpha-1,6-linked dextran was cloned from Leuconostoc mesenteroides B-1299CB4. The coding region consisted of an open reading frame (ORF) of 4,395 bp that coded a 1,465-amino-acids protein with a molecular mass 163,581 Da. The expressed recombinant DSRBCB4 (rDSRBCB4) synthesized oligosaccharides in the presence maltose or isomaltose as an acceptor, plus the products included alpha-1,6-linked glucosyl residues in addition to the maltosyl or isomaltosyl residue. Alignments of the amino acid sequence of DSRBCB4 with glucansucrases from Streptococcus and Leuconostoc identified conserved amino acid residues in the catalytic core that are critical for enzyme activity. The mutants D530N, E568Q, and D641N displayed a 98- to 10,000-fold reduction of total enzyme activity.     

Identification of catalytic and substrate-binding site residues in Bacillus cereus ATCC7064 oligo-1,6-glucosidase.

Three active site residues (Asp199, Glu255, Asp329) and two substrate-binding site residues (His103, His328) of oligo-1,6-glucosidase (EC 3.2.1.10) from Bacillus cereus ATCC7064 were identified by site-directed mutagenesis. These residues were deduced from the X-ray crystallographic analysis and the comparison of the primary structure of the oligo-1,6-glucosidase with those of Saccharomyces carlsbergensis alpha-glucosidase, Aspergillus oryzae alpha-amylase and pig pancreatic alpha-amylase which act on alpha-1,4-glucosidic linkages. The distances between these putative residues of B. cereus oligo-1,6-glucosidase calculated from the X-ray analysis data closely resemble those of A. oryzae alpha-amylase and pig pancreatic alpha-amylase. A single mutation of Asp199-->Asn, Glu255-->Gln, or Asp329-->Asn resulted in drastic reduction in activity, confirming that three residues are crucial for the reaction process of alpha-1,6-glucosidic bond cleavage. Thus, it is identified that the basic mechanism of oligo-1,6-glucosidase for the hydrolysis of alpha-1,6-glucosidic linkage is essentially the same as those of other amylolytic enzymes belonging to Family 13 (alpha-amylase family). On the other hand, mutations of histidine residues His103 and His328 resulted in pronounced dissimilarity in catalytic function. The mutation His328-->Asn caused the essential loss in activity, while the mutation His103-->Asn yielded a mutant enzyme that retained 59% of the k0/Km of that for the wild-type enzyme. Since mutants of other alpha-amylases acting on alpha-1,4-glucosidic bond linkage lost most of their activity by the site-directed mutagenesis at their equivalent residues to His103 and His328, the retaining of activity by His103-->Asn mutation in B. cereus oligo-1,6-glucosidase revealed the distinguished role of His103 for the hydrolysis of alpha-1,6-glucosidic bond linkage.     

Three-dimensional structure and substrate binding of Bacillus stearothermophilus neopullulanase

Crystal structures of Bacillus stearothermophilus TRS40 neopullulanase and its complexes with panose, maltotetraose and isopanose were determined at resolutions of 1.9, 2.4, 2.8 and 3.2A, respectively. Since the latter two carbohydrates are substrates of this enzyme, a deactivated mutant at the catalytic residue Glu357-->Gln was used for complex crystallization. The structures were refined at accuracies with r.m.s. deviations of bond lengths and bond angles ranging from 0.005A to 0.008A and 1.3 degrees to 1.4 degrees, respectively. The active enzyme forms a dimer in the crystalline state and in solution. The monomer enzyme is composed of four domains, N, A, B and C, and has a (beta/alpha)(8)-barrel in domain A. The active site lies between domain A and domain N from the other monomer. The results show that dimer formation makes the active-site cleft narrower than those of ordinary alpha-amylases, which may contribute to the unique substrate specificity of this enzyme toward both alpha-1,4 and alpha-1,6-glucosidic linkages. This specificity may be influenced by the subsite structure. Only subsites -1 and -2 are commonly occupied by the product and substrates, suggesting that equivocal recognition occurs at the other subsites, which contributes to the wide substrate specificity of this enzyme.