Mitsuru Momma

Interdomain Disulfide Bridge in the Rice Granule Bound Starch Synthase I Catalytic Domain as Elucidated by X-Ray Structure Analysis

The catalytic domain of rice (Oryza sativa japonica) granule bound starch synthase I (OsGBSSI-CD) was overexpressed and the three-dimensional structures of the ligand-free and ADP-bound forms were determined. The structures were similar to those reported for bacterial and archaeal glycogen synthases, which belong to glycosyltransferase family 5. They had Rossmann fold N- and C-domains connected by canonical two-hinge peptides, and an interdomain disulfide bond that appears to be conserved in the Poaceae plant family. The presence of three covalent linkages might explain why both OsGBSSI-CD structures adopted only the closed domain arrangement.

The Structure of a Streptomyces avermitilis α-l-Rhamnosidase Reveals a Novel Carbohydrate-binding Module CBM67 within the Six-domain Arrangement

Background: α-l-Rhamnosidase hydrolyzes α-linked l-rhamnose from rhamnoglycosides or polysaccharides. Results: The crystal structure of Streptomyces avermitilis α-l-rhamnosidase belonging to glycoside hydrolase family 78 was determined. Conclusion: The l-rhamnose complexed structure revealed the catalytic mechanism of the enzyme and a calcium-dependent carbohydrate-binding module. Significance: Efficient catalysis of an exo-rhamnosidase requires a novel carbohydrate-binding module that binds terminal l-rhamnose sugars. α-l-Rhamnosidases hydrolyze α-linked l-rhamnosides from oligosaccharides or polysaccharides. We determined the crystal structure of the glycoside hydrolase family 78 Streptomyces avermitilis α-l-rhamnosidase (SaRha78A) in its free and l-rhamnose complexed forms, which revealed the presence of six domains N, D, E, F, A, and C. In the ligand complex, l-rhamnose was bound in the proposed active site of the catalytic module, revealing the likely catalytic mechanism of SaRha78A. Glu636 is predicted to donate protons to the glycosidic oxygen, and Glu895 is the likely catalytic general base, activating the nucleophilic water, indicating that the enzyme operates through an inverting mechanism. Replacement of Glu636 and Glu895 resulted in significant loss of α-rhamnosidase activity. Domain D also bound l-rhamnose in a calcium-dependent manner, with a KD of 135 μm. Domain D is thus a non-catalytic carbohydrate binding module (designated SaCBM67). Mutagenesis and structural data identified the amino acids in SaCBM67 that target the features of l-rhamnose that distinguishes it from the other major sugars present in plant cell walls. Inactivation of SaCBM67 caused a substantial reduction in the activity of SaRha78A against the polysaccharide composite gum arabic, but not against aryl rhamnosides, indicating that SaCBM67 contributes to enzyme function against insoluble substrates.

Crystal Structure of Bacillus subtilis α-Amylase in Complex with Acarbose

The crystal structure of Bacillus subtilis α-amylase, in complex with the pseudotetrasaccharide inhibitor acarbose, revealed an hexasaccharide in the active site as a result of transglycosylation. After comparison with the known structure of the catalytic-site mutant complexed with the native substrate maltopentaose, it is suggested that the present structure represents a mimic intermediate in the initial stage of the catalytic process.

Structural mechanism of JH delivery in hemolymph by JHBP of silkworm, Bombyx mori

Juvenile hormone (JH) plays crucial roles in many aspects of the insect life. All the JH actions are initiated by transport of JH in the hemolymph as a complex with JH-binding protein (JHBP) to target tissues. Here, we report structural mechanism of JH delivery by JHBP based upon the crystal and solution structures of apo and JH-bound JHBP. In solution, apo-JHBP exists in equilibrium of multiple conformations with different orientations of the gate helix for the hormone-binding pocket ranging from closed to open forms. JH-binding to the gate-open form results in the fully closed JHBP-JH complex structure where the bound JH is completely buried inside the protein. JH-bound JHBP opens the gate helix to release the bound hormone likely by sensing the less polar environment at the membrane surface of target cells. This is the first report that provides structural insight into JH signaling.

Structural and Biochemical Characterization of Glycoside Hydrolase Family 79 β-Glucuronidase from Acidobacterium capsulatum

Background: The three-dimensional structures of β-glucuronidase have been solved only for the GH2 enzymes. Results: AcGlcA79A is composed of a (β/α)8-barrel domain and a β-domain. Conclusion: The substrate binding site of AcGlcA79A is adapted for recognition of GlcA as a substrate. Significance: This is the first report describing the crystal structure, mechanism, and catalytic residues of a GH79 enzyme. We present the first structure of a glycoside hydrolase family 79 β-glucuronidase from Acidobacterium capsulatum, both as a product complex with β-d-glucuronic acid (GlcA) and as its trapped covalent 2-fluoroglucuronyl intermediate. This enzyme consists of a catalytic (β/α)8-barrel domain and a β-domain with irregular Greek key motifs that is of unknown function. The enzyme showed β-glucuronidase activity and trace levels of β-glucosidase and β-xylosidase activities. In conjunction with mutagenesis studies, these structures identify the catalytic residues as Glu173 (acid base) and Glu287 (nucleophile), consistent with the retaining mechanism demonstrated by 1H NMR analysis. Glu45, Tyr243, Tyr292–Gly294, and Tyr334 form the catalytic pocket and provide substrate discrimination. Consistent with this, the Y292A mutation, which affects the interaction between the main chains of Gln293 and Gly294 and the GlcA carboxyl group, resulted in significant loss of β-glucuronidase activity while retaining the side activities at wild-type levels. Likewise, although the β-glucuronidase activity of the Y334F mutant is ∼200-fold lower (kcat/Km) than that of the wild-type enzyme, the β-glucosidase activity is actually 3 times higher and the β-xylosidase activity is only 2.5-fold lower than the equivalent parameters for wild type, consistent with a role for Tyr334 in recognition of the C6 position of GlcA. The involvement of Glu45 in discriminating against binding of the O-methyl group at the C4 position of GlcA is revealed in the fact that the E45D mutant hydrolyzes PNP-β-GlcA approximately 300-fold slower (kcat/Km) than does the wild-type enzyme, whereas 4-O-methyl-GlcA-containing oligosaccharides are hydrolyzed only 7-fold slower.

Structural elucidation of dextran degradation mechanism by streptococcus mutans dextranase belonging to glycoside hydrolase family 66

Background: Dextranase hydrolyzes α-1,6-linkages of dextran, producing isomaltooligosaccharides. Results: Crystal structure of Streptococcus mutans dextranase belonging to the glycoside hydrolase family 66 was determined. Conclusion: The enzyme structures complexed with isomaltotriose and suicide substrate revealed the enzymes catalytically important residues. Significance: This is the first structural report for a GH-66 enzyme elucidating the enzymes catalytic machinery. Dextranase is an enzyme that hydrolyzes dextran α-1,6 linkages. Streptococcus mutans dextranase belongs to glycoside hydrolase family 66, producing isomaltooligosaccharides of various sizes and consisting of at least five amino acid sequence regions. The crystal structure of the conserved fragment from Gln100 to Ile732 of S. mutans dextranase, devoid of its N- and C-terminal variable regions, was determined at 1.6 Å resolution and found to contain three structural domains. Domain N possessed an immunoglobulin-like β-sandwich fold; domain A contained the enzymes catalytic module, comprising a (β/α)8-barrel; and domain C formed a β-sandwich structure containing two Greek key motifs. Two ligand complex structures were also determined, and, in the enzyme-isomaltotriose complex structure, the bound isomaltooligosaccharide with four glucose moieties was observed in the catalytic glycone cleft and considered to be the transglycosylation product of the enzyme, indicating the presence of four subsites, −4 to −1, in the catalytic cleft. The complexed structure with 4′,5′-epoxypentyl-α-d-glucopyranoside, a suicide substrate of the enzyme, revealed that the epoxide ring reacted to form a covalent bond with the Asp385 side chain. These structures collectively indicated that Asp385 was the catalytic nucleophile and that Glu453 was the acid/base of the double displacement mechanism, in which the enzyme showed a retaining catalytic character. This is the first structural report for the enzyme belonging to glycoside hydrolase family 66, elucidating the enzymes catalytic machinery.

Sugar-complex structures of the C-half domain of the galactose-binding lectin EW29 from the earthworm Lumbricus terrestris

R-type lectins are one of the most prominent types of lectin; they exist ubiquitously in nature and mainly bind to the galactose unit of sugar chains. The galactose-binding lectin EW29 from the earthworm Lumbricus terrestris belongs to the R-type lectin family as represented by the plant lectin ricin. It shows haemagglutination activity and is composed of a single peptide chain that includes two homologous domains: N-terminal and C-terminal domains. A truncated mutant of EW29 comprising the C-terminal domain (rC-half) has haemagglutination activity by itself. In order to clarify how rC-half recognizes ligands and shows haemagglutination activity, X-ray crystal structures of rC-half in complex with D-lactose and N-acetyl-D-galactosamine have been determined. The structure of rC-half is similar to that of the ricin B chain and consists of a beta-trefoil fold; the fold is further divided into three similar subdomains referred to as subdomains alpha, beta and gamma, which are gathered around the pseudo-threefold axis. The structures of sugar complexes demonstrated that subdomains alpha and gamma of rC-half bind terminal galactosyl and N-acetylgalactosaminyl glycans. The sugar-binding properties are common to both ligands in both subdomains and are quite similar to those of ricin B chain-lactose complexes. These results indicate that the C-terminal domain of EW29 uses these two galactose-binding sites for its function as a single-domain-type haemagglutinin.

Biochemical characterization of a novel cycloisomaltooligosaccharide glucanotransferase from Paenibacillus sp. 598K

Cycloisomaltooligosaccharide glucanotransferase (CITase; EC 2.4.1.248), a member of the glycoside hydrolase family 66 (GH66), catalyzes the intramolecular transglucosylation of dextran to produce cycloisomaltooligosaccharides (CIs; cyclodextrans) of varying lengths. Eight CI-producing bacteria have been found; however, CITase from Bacillus circulans T-3040 (CITase-T3040) is the only CI-producing enzyme that has been characterized to date. In this study, we report the gene cloning, enzyme characterization, and analysis of essential Asp and Glu residues of a novel CITase from Paenibacillus sp. 598K (CITase-598K). The cit genes from T-3040 and 598K strains were expressed recombinantly, and the properties of Escherichia coli recombinant enzymes were compared. The two CITases exhibited high primary amino acid sequence identity (67%). The major product of CITase-598K was cycloisomaltoheptaose (CI-7), whereas that of CITase-T3040 was cycloisomaltooctaose (CI-8). Some of the properties of CITase-598K are more favorable for practical use compared with CITase-T3040, i.e., the thermal stability for CITase-598K (≤50°C) was 10°C higher than that for CITase-T3040 (≤40°C); the k(cat)/K(M) value of CITase-598K was approximately two times higher (32.2s(-1)mM(-1)) than that of CITase-T3040 (17.8s(-1)mM(-1)). Isomaltotetraose was the smallest substrate for both CITases. When isomaltoheptaose or smaller substrates were used, a lag time was observed before the intramolecular transglucosylation reaction began. As substrate length increased, the lag time shortened. Catalytically important residues of CITase-598K were predicted to be Asp144, Asp269, and Glu341. These findings will serve as a basis for understanding the reaction mechanism and substrate recognition of GH66 enzymes.

Structural Elucidation of the Cyclization Mechanism of α-1,6-Glucan by Bacillus circulans T-3040 Cycloisomaltooligosaccharide Glucanotransferase

Background: Cycloisomaltooligosaccharide glucanotransferase catalyzes an intramolecular transglucosylation reaction and produces cycloisomaltooligosaccharides from dextran. Results: The crystal structure of Bacillus circulans T-3040 cycloisomaltooligosaccharide glucanotransferase was determined. Conclusion: The enzyme structures complexed with isomaltooligosaccharides and cycloisomaltooctaose revealed the molecular mechanism of action. Significance: CBM35 functions in the product size determination and substrate recruitment. Bacillus circulans T-3040 cycloisomaltooligosaccharide glucanotransferase belongs to the glycoside hydrolase family 66 and catalyzes an intramolecular transglucosylation reaction that produces cycloisomaltooligosaccharides from dextran. The crystal structure of the core fragment from Ser-39 to Met-738 of B. circulans T-3040 cycloisomaltooligosaccharide glucanotransferase, devoid of its N-terminal signal peptide and C-terminal nonconserved regions, was determined. The structural model contained one catalytic (β/α)8-barrel domain and three β-domains. Domain N with an immunoglobulin-like β-sandwich fold was attached to the N terminus; domain C with a Greek key β-sandwich fold was located at the C terminus, and a carbohydrate-binding module family 35 (CBM35) β-jellyroll domain B was inserted between the 7th β-strand and the 7th α-helix of the catalytic domain A. The structures of the inactive catalytic nucleophile mutant enzyme complexed with isomaltohexaose, isomaltoheptaose, isomaltooctaose, and cycloisomaltooctaose revealed that the ligands bound in the catalytic cleft and the sugar-binding site of CBM35. Of these, isomaltooctaose bound in the catalytic site extended to the second sugar-binding site of CBM35, which acted as subsite −8, representing the enzyme·substrate complex when the enzyme produces cycloisomaltooctaose. The isomaltoheptaose and cycloisomaltooctaose bound in the catalytic cleft with a circular structure around Met-310, representing the enzyme·product complex. These structures collectively indicated that CBM35 functions in determining the size of the product, causing the predominant production of cycloisomaltooctaose by the enzyme. The canonical sugar-binding site of CBM35 bound the mid-part of isomaltooligosaccharides, indicating that the original function involved substrate binding required for efficient catalysis.

Novel dextranase catalyzing cycloisomaltooligosaccharide-formation and identification of catalytic amino acids and their functions using chemical rescue approach

Background: Catalytic residues and molecular mechanism of GH-66 enzymes were hitherto unknown. Results: Novel dextranase produced isomaltotetraose and cyclo-isomaltosaccharides. Its nucleophile (Asp340) and acid/base catalyst (Glu412) were identified by a chemical rescue approach. Conclusion: Three GH-66 enzyme types were newly classified for the first time. Significance: This work elucidates production of isomaltotetraose and cycloisomaltosaccharides, classification of GH-66, identification of catalytic residues, and novel dextran-forming type chemical rescue. A novel endodextranase from Paenibacillus sp. (Paenibacillus sp. dextranase; PsDex) was found to mainly produce isomaltotetraose and small amounts of cycloisomaltooligosaccharides (CIs) with a degree of polymerization of 7–14 from dextran. The 1,696-amino acid sequence belonging to the glycosyl hydrolase family 66 (GH-66) has a long insertion (632 residues; Thr451–Val1082), a portion of which shares identity (35% at Ala39–Ser1304 of PsDex) with Pro32–Ala755 of CI glucanotransferase (CITase), a GH-66 enzyme that catalyzes the formation of CIs from dextran. This homologous sequence (Val837–Met932 for PsDex and Tyr404–Tyr492 for CITase), similar to carbohydrate-binding module 35, was not found in other endodextranases (Dexs) devoid of CITase activity. These results support the classification of GH-66 enzymes into three types: (i) Dex showing only dextranolytic activity, (ii) Dex catalyzing hydrolysis with low cyclization activity, and (iii) CITase showing CI-forming activity with low dextranolytic activity. The fact that a C-terminal truncated enzyme (having Ala39–Ser1304) has 50% wild-type PsDex activity indicates that the C-terminal 392 residues are not involved in hydrolysis. GH-66 enzymes possess four conserved acidic residues (Asp189, Asp340, Glu412, and Asp1254 of PsDex) of catalytic candidates. Their amide mutants decreased activity (11,500 to 140,000 times), and D1254N had 36% activity. A chemical rescue approach was applied to D189A, D340G, and E412Q using α-isomaltotetraosyl fluoride with NaN3. D340G or E412Q formed a β- or α-isomaltotetraosyl azide, respectively, strongly indicating Asp340 and Glu412 as a nucleophile and acid/base catalyst, respectively. Interestingly, D189A synthesized small sized dextran from α-isomaltotetraosyl fluoride in the presence of NaN3.