Crystal structure and characterization of the glycoside hydrolase family 62 α-L-arabinofuranosidase from Streptomyces coelicolor.

α-L-arabinofuranosidase, which belongs to the glycoside hydrolase family 62 (GH62), hydrolyzes arabinoxylan but not arabinan or arabinogalactan. The crystal structures of several α-L-arabinofuranosidases have been determined, although the structures, catalytic mechanisms, and substrate specificities of GH62 enzymes remain unclear. To evaluate the substrate specificity of a GH62 enzyme, we determined the crystal structure of α-L-arabinofuranosidase, which comprises a carbohydrate-binding module family 13 domain at its N terminus and a catalytic domain at its C terminus, from Streptomyces coelicolor. The catalytic domain was a five-bladed ß-propeller consisting of five radially oriented anti-parallel ß-sheets. Sugar complex structures with l-arabinose, xylotriose, and xylohexaose revealed five subsites in the catalytic cleft and an l-arabinose-binding pocket at the bottom of the cleft. The entire structure of this GH62 family enzyme was very similar to that of glycoside hydrolase 43 family enzymes, and the catalytically important acidic residues found in family 43 enzymes were conserved in GH62. Mutagenesis studies revealed that Asp(202) and Glu(361) were catalytic residues, and Trp(270), Tyr(461), and Asn(462) were involved in the substrate-binding site for discriminating the substrate structures. In particular, hydrogen bonding between Asn(462) and xylose at the nonreducing end subsite +2 was important for the higher activity of substituted arabinofuranosyl residues than that for terminal arabinofuranoses.

The structure of a Streptomyces avermitilis α-L-rhamnosidase reveals a novel carbohydrate-binding module CBM67 within the six-domain arrangement.

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. Glu(636) is predicted to donate protons to the glycosidic oxygen, and Glu(895) is the likely catalytic general base, activating the nucleophilic water, indicating that the enzyme operates through an inverting mechanism. Replacement of Glu(636) and Glu(895) 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.

Structural and biochemical characterization of glycoside hydrolase family 79 ß-glucuronidase from Acidobacterium capsulatum.

We present the first structure of a glycoside hydrolase family 79 ß-glucuronidase from Acidobacterium capsulatum, both as a product complex with ß-D-glucuronic acid (GlcA) and as its trapped covalent 2-fluoroglucuronyl intermediate. This enzyme consists of a catalytic (ß/α)(8)-barrel domain and a ß-domain with irregular Greek key motifs that is of unknown function. The enzyme showed ß-glucuronidase activity and trace levels of ß-glucosidase and ß-xylosidase activities. In conjunction with mutagenesis studies, these structures identify the catalytic residues as Glu(173) (acid base) and Glu(287) (nucleophile), consistent with the retaining mechanism demonstrated by (1)H NMR analysis. Glu(45), Tyr(243), Tyr(292)-Gly(294), and Tyr(334) form the catalytic pocket and provide substrate discrimination. Consistent with this, the Y292A mutation, which affects the interaction between the main chains of Gln(293) and Gly(294) and the GlcA carboxyl group, resulted in significant loss of ß-glucuronidase activity while retaining the side activities at wild-type levels. Likewise, although the ß-glucuronidase activity of the Y334F mutant is ~200-fold lower (k(cat)/K(m)) than that of the wild-type enzyme, the ß-glucosidase activity is actually 3 times higher and the ß-xylosidase activity is only 2.5-fold lower than the equivalent parameters for wild type, consistent with a role for Tyr(334) in recognition of the C6 position of GlcA. The involvement of Glu(45) 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-ß-GlcA approximately 300-fold slower (k(cat)/K(m)) than does the wild-type enzyme, whereas 4-O-methyl-GlcA-containing oligosaccharides are hydrolyzed only 7-fold slower.

Characterization of an endo-processive-type xyloglucanase having a ß-1,4-glucan-binding module and an endo-type xyloglucanase from Streptomyces avermitilis.

We cloned two glycoside hydrolase family 74 genes, the sav_1856 gene and the sav_2574 gene, from Streptomyces avermitilis NBRC14893 and characterized the resultant recombinant proteins. The sav_1856 gene product (SaGH74A) consisted of a catalytic domain and a family 2 carbohydrate-binding module at the C terminus, while the sav_2574 gene product (SaGH74B) consisted of only a catalytic domain. SaGH74A and SaGH74B were expressed successfully and had molecular masses of 92 and 78 kDa, respectively. Both recombinant proteins were xyloglucanases. SaGH74A had optimal activity at 60°C and pH 5.5, while SaGH74B had optimal activity at 55°C and pH 6.0. SaGH74A was stable over a broad pH range (pH 4.5 to 9.0), whereas SaGH74B was stable over a relatively narrow pH range (pH 6.0 to 6.5). Analysis of the hydrolysis products of tamarind xyloglucan and xyloglucan-derived oligosaccharides indicated that SaGH74A was endo-processive, while SaGH74B was a typical endo-enzyme. The C terminus of SaGH74A, which was annotated as a carbohydrate-binding module, bound to ß-1,4-linked glucan-containing soluble polysaccharides such as hydroxyethyl cellulose, barley glucan, and xyloglucan.

Purification, Cloning, Functional Expression, Structure, and Characterization of a Thermostable ß-Mannanase from Talaromyces trachyspermus B168 and Its Efficiency in Production of Mannooligosaccharides from Coffee Wastes.

Highly thermostable ß-mannanase, belonging to glycoside hydrolase family 5 subfamily 7, was purified from the culture supernatant of Talaromyces trachyspermus B168 and the cDNA of its transcript was cloned. The recombinant enzyme showed maximal activity at pH 4.5 and 85 °C. It retained more than 90 % of its activity below 60 °C. Obtaining the crystal structure of the enzyme helped us to understand the mechanism of its thermostability. An antiparallel ß-sheet, salt-bridges, hydrophobic packing, proline residues in the loops, and loop shortening are considered to be related to the thermostability of the enzyme. The enzyme hydrolyzed mannans such as locust bean gum, carob galactomannan, guar gum, konjac glucomannan, and ivory nut mannan. It hydrolyzed 50.7 % of the total mannans from coffee waste, producing mannooligosaccharides. The enzyme has the highest optimum temperature among the known fungal ß-mannanases and has potential for use in industrial applications.