Theoretical Studies for the Discovery of Potential Sucrase-Isomaltase Inhibitors from Maize Silk Phytochemicals: An Approach to Treatment of Type 2 Diabetes.

A theoretical analysis of the potential inhibition of human sucrase-isomaltase (SI) by flavonoids was carried out with the aim of identifying potential candidates for an alternative treatment of type 2 diabetes. Two compounds from maize silks, maysin and luteolin, were selected to be studied with the structure-based density functional theory (DFT), molecular docking (MDock), and molecular dynamics (MD) approaches. The docking score and MD simulations suggested that the compounds maysin and luteolin presented higher binding affinities in N-terminal sucrase-isomaltase (NtSI) than in C-terminal sucrase-isomaltase (CtSI). The reactivity parameters, such as chemical hardness (η) and chemical potential (µ), of the ligands, as well as of the active site amino acids of the NtSI, were calculated by the meta-GGA M06 functional in combination with the 6-31G(d) basis set. The lower value of chemical hardness calculated for the maysin molecule indicated that this might interact more easily with the active site of NtSI, in comparison with the values of the acarbose and luteolin structures. Additionally, a possible oxidative process was proposed through the quantum chemical calculations of the electronic charge transfer values (∆N) between the active site amino acids of the NtSI and the ligands. In addition, maysin displayed a higher ability to generate more oxidative damage in the NtSI active site. Our results suggest that maysin and luteolin can be used to develop novel α-glucosidase inhibitors via NtSI inhibition.

Engineering of Thermal Stability in a Cold-Active Oligo-1,6-Glucosidase from Exiguobacterium sibiricum with Unusual Amino Acid Content.

A gene coding for a novel putative amylase, oligo-1,6-glucosidase from a psychrotrophic bacterium Exiguobacterium sibiricum from Siberian permafrost soil was cloned and expressed in Escherichia coli. The amino acid sequence of the predicted protein EsOgl and its 3D model displayed several features characteristic for the cold-active enzymes while possessing an unusually high number of proline residues in the loops-a typical feature of thermophilic enzymes. The activity of the purified recombinant protein was tested with p-nitrophenyl α-D-glucopyranoside as a substrate. The enzyme displayed a plateau-shaped temperature-activity profile with the optimum at 25 °C and a pronounced activity at low temperatures (50% of maximum activity at 5 °C). To improve the thermal stability at temperatures above 40 °C, we have introduced proline residues into four positions of EsOgl by site-directed mutagenesis according to "the proline rule". Two of the mutants, S130P and A109P demonstrated a three- and two-fold increased half-life at 45 °C. Moreover, S130P mutation led to a 60% increase in the catalytic rate constant. Combining the mutations resulted in a further increase in stability transforming the temperature-activity profile to a typical mesophilic pattern. In the most thermostable variant A109P/S130P/E176P, the half-life at 45 °C was increased from 11 min (wild-type) to 129 min.

Investigation of α-Glucosidase Inhibitory Metabolites from Tetracera scandens Leaves by GC-MS Metabolite Profiling and Docking Studies.

Stone leaf (Tetracera scandens) is a Southeast Asian medicinal plant that has been traditionally used for the management of diabetes mellitus. The underlying mechanisms of the antidiabetic activity have not been fully explored yet. Hence, this study aimed to evaluate the α-glucosidase inhibitory potential of the hydromethanolic extracts of T. scandens leaves and to characterize the metabolites responsible for such activity through gas chromatography-mass spectrometry (GC-MS) metabolomics. Crude hydromethanolic extracts of different strengths were prepared and in vitro assayed for α-glucosidase inhibition. GC-MS analysis was further carried out and the mass spectral data were correlated to the corresponding α-glucosidase inhibitory IC50 values via an orthogonal partial least squares (OPLS) model. The 100%, 80%, 60% and 40% methanol extracts displayed potent α-glucosidase inhibitory potentials. Moreover, the established model identified 16 metabolites to be responsible for the α-glucosidase inhibitory activity of T. scandens. The putative α-glucosidase inhibitory metabolites showed moderate to high affinities (binding energies of -5.9 to -9.8 kcal/mol) upon docking into the active site of Saccharomyces cerevisiae isomaltase. To sum up, an OPLS model was developed as a rapid method to characterize the α-glucosidase inhibitory metabolites existing in the hydromethanolic extracts of T. scandens leaves based on GC-MS metabolite profiling.

Characterization of α-Glucosidase Inhibitors from Clinacanthus nutans Lindau Leaves by Gas Chromatography-Mass Spectrometry-Based Metabolomics and Molecular Docking Simulation.

BACKGROUND: Clinacanthus nutans (C. nutans) is an Acanthaceae herbal shrub traditionally consumed to treat various diseases including diabetes in Malaysia. This study was designed to evaluate the α-glucosidase inhibitory activity of C. nutans leaves extracts, and to identify the metabolites responsible for the bioactivity. METHODS: Crude extract obtained from the dried leaves using 80% methanolic solution was further partitioned using different polarity solvents. The resultant extracts were investigated for their α-glucosidase inhibitory potential followed by metabolites profiling using the gas chromatography tandem with mass spectrometry (GC-MS). RESULTS: Multivariate data analysis was developed by correlating the bioactivity, and GC-MS data generated a suitable partial least square (PLS) model resulting in 11 bioactive compounds, namely, palmitic acid, phytol, hexadecanoic acid (methyl ester), 1-monopalmitin, stigmast-5-ene, pentadecanoic acid, heptadecanoic acid, 1-linolenoylglycerol, glycerol monostearate, alpha-tocospiro B, and stigmasterol. In-silico study via molecular docking was carried out using the crystal structure Saccharomyces cerevisiae isomaltase (PDB code: 3A4A). Interactions between the inhibitors and the protein were predicted involving residues, namely LYS156, THR310, PRO312, LEU313, GLU411, and ASN415 with hydrogen bond, while PHE314 and ARG315 with hydrophobic bonding. CONCLUSION: The study provides informative data on the potential α-glucosidase inhibitors identified in C. nutans leaves, indicating the plant's therapeutic effect to manage hyperglycemia.

Characterization of divergent pseudo-sucrose isomerase from Azotobacter vinelandii: Deciphering the absence of sucrose isomerase activity.

Among members of the glycoside hydrolase (GH) family, sucrose isomerase (SIase) and oligo-1,6-glucosidase (O16G) are evolutionarily closely related even though their activities show different specificities. A gene (Avin_08330) encoding a putative SIase (AZOG: Azotobacterglucocosidase) from the nitrogen-fixing bacterium Azotobacter vinelandii is a type of pseudo-SIase harboring the "RLDRD" motif, a SIase-specific region in 329-333. However, neither sucrose isomerization nor hydrolysis activities were observed in recombinant AZOG (rAZOG). The rAZOG showed similar substrate specificity to Bacillus O16G as it catalyzes the hydrolysis of isomaltulose and isomaltose, which contain α-1,6-glycosidic linkages. Interestingly, rAZOG could generate isomaltose from the small substrate methyl-α-glucoside (MαG) via intermolecular transglycosylation. In addition, sucrose isomers isomaltulose and trehalulose were produced when 250 mM fructose was added to the MαG reaction mixture. The conserved regions I and II of AZOG are shared with many O16Gs, while regions III and IV are very similar to those of SIases. Strikingly, a shuffled AZOG, in which the N-terminal region of SIase containing conserved regions I and II was exchanged with the original enzyme, exhibited a production of sucrose isomers. This study demonstrates an evolutionary relationship between SIase and O16G and suggests some of the main regions that determine the specificity of SIase and O16G.

Biochemical properties of Glu-SH3 as a family 13 glycoside hydrolase with remarkable substrate specificity for trehalose: Implications to sequence-based classification of CAZymes.

A novel glycoside hydrolase from Exiguobacterium sp. SH3 was characterized. The enzyme, designated as Glu-SH3, was predicted by in silico analysis to have structural similarity with members of oligo-1,6-glucosidase and trehalose-6-phosphate hydrolase subfamilies in the GH-13 family of glycoside hydrolases. The gene was expressed in Escherichia coli and the recombinant enzyme was purified as a His-tagged protein of about 60 kDa. The enzyme was shown to have remarkable substrate specificity for trehalose. The characteristic ability of Glu-SH3 to hydrolyze trehalose was ascertained by zymography, thin layer chromatography, and NMR spectroscopy. The maximum activity of Glu-SH3 was obtained at 35 °C and pH 7, but it was able to exhibit more than 90% of the activity within the pH range of 5-8. The Vmax and Km values were estimated to be 170 U and 4.5 mg ml(-1), respectively. By comparison with trehalases, Glu-SH3 with Kcat and Kcat/Km values of 1552 s(-1) and 119.4 mM(-1) s(-1) can be recognized as a very efficient trehalose-hydrolyzing glycosidase. Given the phylogeny and the substrate specificity of Glu-SH3, it may be assumed that the enzyme shares a common ancestor with oligo-1,6-glucosidases but have evolved distinctly to serve a physiological function in trehalose metabolism.

Maltase protein of Ogataea (Hansenula) polymorpha is a counterpart to the resurrected ancestor protein ancMALS of yeast maltases and isomaltases.

Saccharomyces cerevisiae maltases use maltose, maltulose, turanose and maltotriose as substrates, isomaltases use isomaltose, α-methylglucoside and palatinose and both use sucrose. These enzymes are hypothesized to have evolved from a promiscuous α-glucosidase ancMALS through duplication and mutation of the genes. We studied substrate specificity of the maltase protein MAL1 from an earlier diverged yeast, Ogataea polymorpha (Op), in the light of this hypothesis. MAL1 has extended substrate specificity and its properties are strikingly similar to those of resurrected ancMALS. Moreover, amino acids considered to determine selective substrate binding are highly conserved between Op MAL1 and ancMALS. Op MAL1 represents an α-glucosidase in which both maltase and isomaltase activities are well optimized in a single enzyme. Substitution of Thr200 (corresponds to Val216 in S. cerevisiae isomaltase IMA1) with Val in MAL1 drastically reduced the hydrolysis of maltose-like substrates (α-1,4-glucosides), confirming the requirement of Thr at the respective position for this function. Differential scanning fluorimetry (DSF) of the catalytically inactive mutant Asp199Ala of MAL1 in the presence of its substrates and selected monosaccharides suggested that the substrate-binding pocket of MAL1 has three subsites (-1, +1 and +2) and that binding is strongest at the -1 subsite. The DSF assay results were in good accordance with affinity (Km ) and inhibition (Ki ) data of the enzyme for tested substrates, indicating the power of the method to predict substrate binding. Deletion of either the maltase (MAL1) or α-glucoside permease (MAL2) gene in Op abolished the growth of yeast on MAL1 substrates, confirming the requirement of both proteins for usage of these sugars. © 2016 The Authors. Yeast published by John Wiley & Sons, Ltd.

Dietary phenolic compounds selectively inhibit the individual subunits of maltase-glucoamylase and sucrase-isomaltase with the potential of modulating glucose release.

In this study, it was hypothesized that dietary phenolic compounds selectively inhibit the individual C- and N-terminal (Ct, Nt) subunits of the two small intestinal α-glucosidases, maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI), for a modulated glycemic carbohydrate digestion. The inhibition by chlorogenic acid, caffeic acid, gallic acid, (+)-catechin, and (-)-epigallocatechin gallate (EGCG) on individual recombinant human Nt-MGAM and Nt-SI and on mouse Ct-MGAM and Ct-SI was assayed using maltose as the substrate. Inhibition constants, inhibition mechanisms, and IC50 values for each combination of phenolic compound and enzymatic subunit were determined. EGCG and chlorogenic acid were found to be more potent inhibitors for selectively inhibiting the two subunits with highest activity, Ct-MGAM and Ct-SI. All compounds displayed noncompetitive type inhibition. Inhibition of fast-digesting Ct-MGAM and Ct-SI by EGCG and chlorogenic acid could lead to a slow, but complete, digestion of starch for improved glycemic response of starchy foods with potential health benefit.

Steric hindrance by 2 amino acid residues determines the substrate specificity of isomaltase from Saccharomyces cerevisiae.

The structures of the E277A isomaltase mutant from Saccharomyces cerevisiae in complex with isomaltose or maltose were determined at resolutions of 1.80 and 1.40Å, respectively. The root mean square deviations between the corresponding main-chain atoms of free isomaltase and the E277Α-isomaltose complex structures and those of free isomaltase and the E277A-maltose complex structures were found to be 0.131Å and 0.083Å, respectively. Thus, the amino acid substitution and ligand binding do not affect the overall structure of isomaltase. In the E277A-isomaltose structure, the bound isomaltose was readily identified by electron densities in the active site pocket; however, the reducing end of maltose was not observed in the E277A-maltose structure. The superposition of maltose onto the E277A-maltose structure revealed that the reducing end of maltose cannot bind to the subsite +1 due to the steric hindrance from Val216 and Gln279. The amino acid sequence comparisons with α-glucosidases showed that a bulky hydrophobic amino acid residue is conserved at the position of Val216 in α-1,6-glucosidic linkage hydrolyzing enzymes. Similarly, a bulky amino acid residue is conserved at the position of Gln279 in α-1,6-glucosidic linkage-only hydrolyzing α-glucosidases. Ala, Gly, or Asn residues were located at the position of α-1,4-glucosidic linkage hydrolyzing α-glucosidases. Two isomaltase mutant enzymes - V216T and Q279A - hydrolyzed maltose. Thus, the amino acid residues at these positions may be largely responsible for determining the substrate specificity of α-glucosidases.

Crystal structures of isomaltase from Saccharomyces cerevisiae and in complex with its competitive inhibitor maltose.

The structures of isomaltase from Saccharomyces cerevisiae and in complex with maltose were determined at resolutions of 1.30 and 1.60 Å, respectively. Isomaltase contains three domains, namely, A, B, and C. Domain A consists of the (ß/α)(8) -barrel common to glycoside hydrolase family 13. However, the folding of domain C is rarely seen in other glycoside hydrolase family 13 enzymes. An electron density corresponding to a nonreducing end glucose residue was observed in the active site of isomaltase in complex with maltose; however, only incomplete density was observed for the reducing end. The active site pocket contains two water chains. One water chain is a water path from the bottom of the pocket to the surface of the protein, and may act as a water drain during substrate binding. The other water chain, which consists of six water molecules, is located near the catalytic residues Glu277 and Asp352. These water molecules may act as a reservoir that provides water for subsequent hydrolytic events. The best substrate for oligo-1,6-glucosidase is isomaltotriose; other, longer-chain, oligosaccharides are also good substrates. However, isomaltase shows the highest activity towards isomaltose and very little activity towards longer oligosaccharides. This is because the entrance to the active site pocket of isomaltose is severely narrowed by Tyr158, His280, and loop 310-315, and because the isomaltase pocket is shallower than that of other oligo-1,6-glucosidases. These features of the isomaltase active site pocket prevent isomalto-oligosaccharides from binding to the active site effectively.

Structural basis for substrate selectivity in human maltase-glucoamylase and sucrase-isomaltase N-terminal domains.

Human maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) are small intestinal enzymes that work concurrently to hydrolyze the mixture of linear alpha-1,4- and branched alpha-1,6-oligosaccharide substrates that typically make up terminal starch digestion products. MGAM and SI are each composed of duplicated catalytic domains, N- and C-terminal, which display overlapping substrate specificities. The N-terminal catalytic domain of human MGAM (ntMGAM) has a preference for short linear alpha-1,4-oligosaccharides, whereas N-terminal SI (ntSI) has a broader specificity for both alpha-1,4- and alpha-1,6-oligosaccharides. Here we present the crystal structure of the human ntSI, in apo form to 3.2 A and in complex with the inhibitor kotalanol to 2.15 A resolution. Structural comparison with the previously solved structure of ntMGAM reveals key active site differences in ntSI, including a narrow hydrophobic +1 subsite, which may account for its additional substrate specificity for alpha-1,6 substrates.

Crystallization and preliminary X-ray analysis of isomaltase from Saccharomyces cerevisiae.

Isomaltase from Saccharomyces cerevisiae is an oligo-1,6-glucosidase that preferentially hydrolyzes isomaltose, with little activity towards isomaltotriose or longer oligosaccharides. An amino-acid sequence analysis of the isomaltase revealed that it belongs to glucoside hydrolase family 13. Recombinant isomaltase was purified and crystallized by the hanging-drop vapour-diffusion method with PEG 3350 as the precipitant. The crystals belonged to space group C2, with unit-cell parameters a = 95.67, b = 115.42, c = 61.77 A, beta = 91.17 degrees . X-ray diffraction data were collected to 1.35 A resolution from a single crystal on a synchrotron-radiation source.

A novel type of detergent-resistant membranes may contribute to an early protein sorting event in epithelial cells.

One sorting mechanism of apical and basolateral proteins in epithelial cells is based on their solubility profiles with Triton X-100. Nevertheless, apical proteins themselves are also segregated beyond the trans-Golgi network by virtue of their association or nonassociation with cholesterol/sphingolipid-rich microdomains (Jacob, R., and Naim, H. Y. (2001) Curr. Biol. 11, 1444-1450). Therefore, extractability with Triton X-100 does not constitute an absolute criterion of protein sorting. Here, we investigate the solubility patterns of apical and basolateral proteins with other detergents and demonstrate that the mild detergent Tween 20 is adequate to discriminate between apical and basolateral proteins during early stages in their biosynthesis. Although the mannose-rich forms of the apical proteins sucrase-isomaltase, lactase-phlorizin hydrolase, aminopeptidase N, and dipeptidylpeptidase IV reveal similar solubility profiles comprising soluble and nonsoluble fractions, the basolateral proteins, vesicular stomatitis virus G protein, major histocompatibility complex class I, and CD46 are entirely soluble with this detergent. The insoluble Tween 20 membranes are enriched in phosphatidylinositol and phosphatidylglycerol compatible with their synthesis in the endoplasmic reticulum and the existence of a novel class of detergent-resistant membranes. The association of the mannose-rich biosynthetic forms of the apical proteins, sucraseisomaltase, lactase-phlorizin hydrolase, aminopeptidase N, and dipeptidylpeptidase IV with the Tween 20-resistant membranes suggests an early polarized sorting mechanism prior to maturation in the Golgi apparatus.

Crystal structure of Thermotoga maritima 4-alpha-glucanotransferase and its acarbose complex: implications for substrate specificity and catalysis.

4-alpha-Glucanotransferase (GTase) is an essential enzyme in alpha-1,4-glucan metabolism in bacteria and plants. It catalyses the transfer of maltooligosaccharides from an 1,4-alpha-D-glucan molecule to the 4-hydroxyl group of an acceptor sugar molecule. The crystal structures of Thermotoga maritima GTase and its complex with the inhibitor acarbose have been determined at 2.6A and 2.5A resolution, respectively. The GTase structure consists of three domains, an N-terminal domain with the (beta/alpha)(8) barrel topology (domain A), a 65 residue domain, domain B, inserted between strand beta3 and helix alpha6 of the barrel, and a C-terminal domain, domain C, which forms an antiparallel beta-structure. Analysis of the complex of GTase with acarbose has revealed the locations of five sugar-binding subsites (-2 to +3) in the active-site cleft lying between domain B and the C-terminal end of the (beta/alpha)(8) barrel. The structure of GTase closely resembles the family 13 glycoside hydrolases and conservation of key catalytic residues previously identified for this family is consistent with a double-displacement catalytic mechanism for this enzyme. A distinguishing feature of GTase is a pair of tryptophan residues, W131 and W218, which, upon the carbohydrate inhibitor binding, form a remarkable aromatic "clamp" that captures the sugar rings at the acceptor-binding sites +1 and +2. Analysis of the structure of the complex shows that sugar residues occupying subsites from -2 to +2 engage in extensive interactions with the protein, whereas the +3 glucosyl residue makes relatively few contacts with the enzyme. Thus, the structure suggests that four subsites, from -2 to +2, play the dominant role in enzyme-substrate recognition, consistent with the observation that the smallest donor for T.maritima GTase is maltotetraose, the smallest chain transferred is a maltosyl unit and that the smallest residual fragment after transfer is maltose. A close similarity between the structures of GTase and oligo-1,6-glucosidase has allowed the structural features that determine differences in substrate specificity of these two enzymes to be analysed.

Oligo-1,6-glucosidase from a thermophile, Bacillus thermoglucosidasius KP1006, was efficiently produced by combinatorial expression of GroEL in Escherichia coli.

To improve the production of oligo-1,6-glucosidase from the obligately thermophilic Bacillus thermoglucosidasius KP1006 in Escherichia coli, the combined expression of oligo-1,6-glucosidase with various chaperone proteins of Hsp (heat-shock protein) 60 team proteins (GroES and GroEL) or Hsp70 team proteins (GrpE, DnaK and DnaJ) from the same thermophile was examined. This attempt was based on the facts that, (i) among glycosyl hydrolases of Family 13, bacillary oligo-1,6-glucosidases share highest homology with yeast alpha-glucosidase, and (ii) this yeast enzyme interacts with GroEL. In B. thermoglucosidasius Hsp60 team proteins, in particular, GroEL brought about a remarkable rise in expression of B. thermoglucosidasius oligo-1,6-glucosidase, while Hsp70 team proteins had no significant effect. The effect of B. thermoglucosidasius GroEL on oligo-1,6-glucosidase expression was supported by the finding that thermally inactivated B. thermoglucosidasius oligo-1,6-glucosidase was revived by B. thermoglucosidasius GroEL. Although the molecular mass of B. thermoglucosidasius oligo-1,6-glucosidase (66 kDa) exceeds the major range of substrates for GroEL proteins, the GroEL molecules probably recognized the alpha/beta motifs contained in the N-terminal domain and the subdomain of the oligo-1,6-glucosidase. Here we show that (i) the production of B. thermoglucosidasius oligo-1,6-glucosidase in E. coli was improved 3.8-fold by Hsp60 team proteins, (ii) the system can function for the expression of other glycosyl hydrolases of Family 13 that have defects in expression and (iii) the combinatorial expression of thermostable proteins with GroEL from the same thermophile in E. coli can increase the production of thermostable enzymes, preventing problems derived from differences in protein biogenesis.

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.

Comparison of sucrase-free isomaltase with sucrase-isomaltase purified from the house musk shrew Suncus murinus.

We purified sucrase-isomaltase and sucrase-free isomaltase from a normal and a sucrase-deficient line, respectively, of the house musk shrew Suncus murinus and examined the effects of mutation on enzyme structure and activities. Recent cDNA cloning studies have predicted that sucrase-free mutant isomaltase lacks the C-terminal 69 amino acids of normal isomaltase, as well as the entire sucrase. On SDS-polyacrylamide gel electrophoresis purified sucrase-free isomaltase gave a single protein band of 103 kDa, while sucrase-isomaltase gave two major protein bands of 106 and 115 kDa. The 115, but not 106, kDa band was quite similar to the 103 kDa band on Western blotting with Aleuria aurantia lectin and antibody against shrew sucrase-isomaltase, suggesting that the 115 and 103 kDa bands are due to normal and mutant isomaltases, respectively, in accordance with the above prediction. Purified isomaltase and sucrase-isomaltase were similar in Km and Vmax (based on isomaltase mass) values for isomaltose hydrolysis and in inhibition of isomaltase activity by antibody against rabbit sucrase-isomaltase, suggesting that the enzymatic properties of isomaltase are mostly unaffected by mutation.

Clustered proline residues around the active-site cleft in thermostable oligo-1,6-glucosidase of Bacillus flavocaldarius KP1228.

The gene that coded for a cellular oligo-1,6-glucosidase (dextrin 6-alpha-D-glucanohydrolase, EC 3.2.1.10) in Bacillus flavocaldarius KP1228 (FERM-P9542) cells growing at 51-82 degrees C was expressed in Escherichia coli JM109. The enzyme had a half-life of 10 min at 89.2 degrees C. Purification of the enzyme and its characterization showed that the enzyme was identical with the native one. Its primary structure of 529 residues with a molecular weight of 61,469 deduced from the gene was 40-42% identical to the sequences of less thermostable oligo-1,6-glucosidases from Bacillus cereus ATCC 7064, Bacillus coagulans ATCC 7050, and Bacillus thermoglucosidasius KP1006. Sequence analysis showed that the B. flavocaldarius enzyme shared 14 proline residues at the same positions as in the three other enzymes, and that the B. flavocaldarius enzyme had 22 of 33 additional proline residues (cf. 1/5, 5/10, and 9/18 in the respective counterparts) in three long polypeptides constituting the active-site cleft, which connected the third, fourth, and eighth beta-strands to the corresponding third, fourth, and eighth alpha-helices in the (beta/alpha)8-barrel.

The refined crystal structure of Bacillus cereus oligo-1,6-glucosidase at 2.0 A resolution: structural characterization of proline-substitution sites for protein thermostabilization.

The crystal structure of oligo-1,6-glucosidase (dextrin 6-alpha-glucanohydrolase, EC 3.2.1.10) from Bacillus cereus ATCC7064 has been refined to 2.0 A resolution with an R-factor of 19.6% for 43,328 reflections. The final model contains 4646 protein atoms and 221 ordered water molecules with respective root-mean-square deviations of 0.015 A for bond lengths and of 3.166 degrees for bond angles from the ideal values. The structure consists of three domains: the N-terminal domain (residues 1 to 104 and 175 to 480), the subdomain (residues 105 to 174) and the C-terminal domain (residues 481 to 558). The N-terminal domain takes a (beta/alpha)8-barrel structure with additions of an alpha-helix (N alpha6') between the sixth strand Nbeta6 and the sixth helix N alpha6, an alpha-helix (N alpha7') between the seventh strand Nbeta7 and the seventh helix N alpha7 and three alpha-helices (N alpha8', N alpha8" and N alpha8'" between the eighth strand Nbeta8 and the eighth helix N alpha8. The subdomain is composed of an alpha-helix, a three-stranded antiparallel beta-sheet, and long intervening loops. The C-terminal domain has a beta-barrel structure of eight antiparallel beta-strands folded in double Greek key motifs, which is distorted in the sixth strand Cbeta6. Three catalytic residues, Asp199, Glu255 and Asp329, are located at the bottom of a deep cleft formed by the subdomain and a cluster of the two additional alpha-helices N alpha8' and N alpha8" in the (beta/alpha)8-barrel. The refined structure reveals the locations of 21 proline-substitution sites that are expected to be critical to protein thermostabilization from a sequence comparison among three Bacillus oligo-1,6-glucosidases with different thermostability. These sites lie in loops, beta-turns and alpha-helices, in order of frequency, except for Cys515 in the fourth beta-strand Cbeta4 of the C-terminal domain. The residues in beta-turns (Lys121, Glu208, Pro257, Glu290, Pro443, Lys457 and Glu487) are all found at their second positions, and those in alpha-helices (Asn109, Glu175, Thr261 and Ile403) are present at their N1 positions of the first helical turns. Those residues in both secondary structures adopt phi and phi values favorable for proline substitution. Residues preceding the 21 sites are mostly conserved upon proline occurrence at these 21 sites in more thermostable Bacillus oligo-1,6-glucosidases. These structural features with respect to the 21 sites indicate that the sites in beta-turns and alpha-helices have more essential prerequisites for proline substitution to thermostabilize the protein than those in loops. This well supports the previous finding that the replacement at the appropriate positions in beta-turns or alpha-helices is the most effective for protein thermostabilization by proline substitution.

Analysis of the critical sites for protein thermostabilization by proline substitution in oligo-1,6-glucosidase from Bacillus coagulans ATCC 7050 and the evolutionary consideration of proline residues.

To identify the critical sites for protein thermostabilization by proline substitution, the gene for oligo-1,6- glucosidase from a thermophilic Bacillus coagulans strain, ATCC 7050, was cloned as a 2.4-kb DNA fragment and sequenced. In spite of a big difference in their thermostabilities, B. coagulans oligo-1,6-glucosidase had a large number of points in its primary structure identical to respective points in the same enzymes from a mesophilic Bacillus cereus strain, ATCC 7064 (57%), and an obligately thermophilic Bacillus thermoglucosidasius strain, KP1006 (59%). The number of prolines (19 for B. cereus oligo-1,6-glucosidase, 24 for B. coagulans enzyme, and 32 for B. thermoglucosidasius enzyme) was observed to increase with the rise in thermostabilities of the oligo-1,6-glucosidases. Classification of proline residues in light of the amino acid sequence alignment and the protein structure revealed by X-ray crystallographic analysis also supported this tendency. Judging from proline residues occurring in B. coagulans oligo-1,6-glucosidase and the structural requirement for proline substitution (second site of the beta turn and first turn of the alpha helix) (K. Watanabe, T. Masuda, H. Ohashi, H. Mihara, and Y. Suzuki, Eur. J. Biochem. 226:277-283, 1994), the critical sites for thermostabilization were found to be Lys-121, Glu-290, Lys-457, and Glu-487 in B. cereus oligo-1,6-glucosidase. With regard to protein evolution, the oligo-1,6-glucosidases very likely follow the neutral theory. The adaptive mutations of the oligo-1,6-glucosidases that appear to increase thermostability are consistent with the substitution of proline residues for neutrally occurring residues. It is concluded that proline substitution is an important factor for the selection of thermostability in oligo-1,6-glucosidases.