Molecular Cloning, Expression and Biochemical Characterization of a Family 5 Glycoside Hydrolase First Endo-Mannanase (RfGH5_7) from Ruminococcus flavefaciens FD-1 v3.

The cellulosomal enzyme, RfGH51/2, of Ruminococcus flavefaciens contains an N-terminal module, a family 5 glycoside hydrolase GH5_4 with a putative endoglucanase activity, while C-terminal domain is a putative endo-mannanase (GH5_7). The two putative catalytic modules are separated by family 80 carbohydrate binding module (CBM80) having wide ligand specificity. The putative endo-mannanase module, GH5_7 (RfGH5_7), was cloned, expressed in Escherichia coli BL-21(DE3) cells and purified. SDS-PAGE analysis of purified RfGH5_7 showed molecular size ~ 35 kDa. Substrate specificity analysis of RfGH5_7 showed maximum activity against locust bean galactomannan (298.5 U/mg) followed by konjac glucomannan (256.2 U/mg) and carob galactomannan (177.2 U/mg). RfGH5_7 showed maximum activity at optimum pH 6.0 and temperature 60 °C. RfGH5_7 displayed stability in between pH 6.0 and 9.0 and thermostability till 50 °C. 10 mM Ca2+ ions increased the enzyme activity by 33%. The melting temperature of RfGH5_7 was 84 °C that was not affected by Ca2+ ions or chelating agents. RfGH5_7 showed, Vmax, 389 U/mg and Km, 0.92 mg/mL for locust bean galactomannan. TLC analysis revealed that RfGH5_7 hydrolysed locust bean galactomannan predominantly to mannose, mannobiose, mannotriose and higher degree of polymerization of manno-oligosaccharides indicating an endo-acting catalytic mechanism. This study revealed a highly active and thermostable endo-mannanase with considerable biotechnological potential.

Role of pectinolytic enzymes identified in Clostridium thermocellum cellulosome.

The cloning, expression and characterization of three cellulosomal pectinolytic enzymes viz., two variants of PL1 (PL1A and PL1B) and PL9 from Clostridium thermocellum was carried out. The comparison of the primary sequences of PL1A, PL1B and PL9 revealed that these proteins displayed considerable sequence similarities with family 1 and 9 polysaccharide lyases, respectively. PL1A, PL1B and PL9 are the putative catalytic domains of protein sequence ABN54148.1 and ABN53381.1 respectively. These two protein sequences also contain putative carbohydrate binding module (CBM) and type-I dockerin. The associated putative CBM of PL1A showed strong homology with family 6 CBMs while those of PL1B and PL9 showed homology with family 35 CBMs. Recombinant derivatives of these three enzymes showed molecular masses of approximately 34 kDa, 40 kDa and 32 kDa for PL1A, PL1B and PL9, respectively. PL1A, PL1B and PL9 displayed high activity toward polygalacturonic acid and pectin (up to 55% methyl-esterified) from citrus fruits. However, PL1B showed relatively higher activity towards 55% and 85% methyl-esterified pectin (citrus). PL1A and PL9 showed higher activity on rhamnogalacturonan than PL1B. Both PL1A and PL9 displayed maximum activity at pH 8.5 with optimum temperature of 50°C and 60°C respectively. PL1B achieved highest activity at pH 9.8, under an optimum temperature of 50°C. PL1A, PL1B and PL9 all produced two or more unsaturated galacturonates from pectic substrates as displayed by TLC analysis confirming that they are endo-pectate lyase belonging to family 1 and 9, respectively. This report reveals that pectinolytic activity displayed by Clostridium thermocellum cellulosome is coordinated by a sub-set of at least three multi-modular enzymes.

Expression, purification and crystallization of a novel carbohydrate-binding module from the Ruminococcus flavefaciens cellulosome.

Anaerobic bacteria organize carbohydrate-active enzymes into a multi-component complex, the cellulosome, which degrades cellulose and hemicellulose highly efficiently. Genome sequencing of Ruminococcus flavefaciens FD-1 offers extensive information on the range and diversity of the enzymatic and structural components of the cellulosome. The R. flavefaciens FD-1 genome encodes over 200 dockerin-containing proteins, most of which are of unknown function. One of these modular proteins comprises a glycoside hydrolase family 5 catalytic module (GH5) linked to an unclassified carbohydrate-binding module (CBM-Rf1) and a dockerin. The novel CBM-Rf1 has been purified and crystallized. The crystals belonged to the trigonal space group R32:H. The CBM-Rf1 structure was determined by a multiple-wavelength anomalous dispersion experiment using AutoSol from the PHENIX suite using both selenomethionyl-derivative and native data to resolutions of 2.28 and 2.0 Å, respectively.

Novel modular enzymes encoded by a cellulase gene cluster in Cellvibrio mixtus.

Hydrolysis of plant cell wall polysaccharides, a process which is of intrinsic biological and biotechnological importance, requires the concerted action of an extensive repertoire of microbial cellulases and hemicellulases. Here, we report the identification of the gene cluster unk16A, regA and cel5B in the aerobic soil bacterium Cellvibrio mixtus, encoding a family 16 (CmUnk16A) glycoside hydrolase (GH), an AraC/XylS transcription activator (CmRegA) and a family 5 (CmCel5B) endo-glucanase, respectively. CmUnk16A is a modular enzyme comprising, in addition to the catalytic domain, two family 32 carbohydrate-binding modules (CBMs), termed CBM32-1 and CBM32-2, a CBM4 and a domain of unknown function. We show that CBM32-2 binds weakly to laminarin and pustulan. CmRegA is also a modular protein containing a highly hydrophobic N-terminal domain and a C-terminal DNA-binding domain of the AraC/XylS family. The role of the identified enzymes in the hydrolysis of cell wall polysaccharides by aerobic bacteria is discussed.

Galactomannan hydrolysis and mannose metabolism in Cellvibrio mixtus.

Galactomannan hydrolysis results from the concerted action of microbial endo-mannanases, manosidases and alpha-galactosidases and is a mechanism of intrinsic biological importance. Here we report the identification of a gene cluster in the aerobic soil bacterium Cellvibrio mixtus encoding enzymes involved in the degradation of this polymeric substrate. The family 27 alpha-galactosidase, termed CmAga27A, preferentially hydrolyse galactose containing polysaccharides. In addition, we have characterized an enzyme with epimerase activity, which might be responsible for the conversion of mannose into glucose. The role of the identified enzymes in the hydrolysis of galactomannan by aerobic bacteria is discussed.

Structure and activity of two metal ion-dependent acetylxylan esterases involved in plant cell wall degradation reveals a close similarity to peptidoglycan deacetylases.

The enzymatic degradation of plant cell wall xylan requires the concerted action of a diverse enzymatic syndicate. Among these enzymes are xylan esterases, which hydrolyze the O-acetyl substituents, primarily at the O-2 position of the xylan backbone. All acetylxylan esterase structures described previously display a alpha/beta hydrolase fold with a "Ser-His-Asp" catalytic triad. Here we report the structures of two distinct acetylxylan esterases, those from Streptomyces lividans and Clostridium thermocellum, in native and complex forms, with x-ray data to between 1.6 and 1.0 A resolution. We show, using a novel linked assay system with PNP-2-O-acetylxyloside and a beta-xylosidase, that the enzymes are sugar-specific and metal ion-dependent and possess a single metal center with a chemical preference for Co2+. Asp and His side chains complete the catalytic machinery. Different metal ion preferences for the two enzymes may reflect the surprising diversity with which the metal ion coordinates residues and ligands in the active center environment of the S. lividans and C. thermocellum enzymes. These "CE4" esterases involved in plant cell wall degradation are shown to be closely related to the de-N-acetylases involved in chitin and peptidoglycan degradation (Blair, D. E., Schuettelkopf, A. W., MacRae, J. I., and Aalten, D. M. (2005) Proc. Natl. Acad. Sci. U. S. A., 102, 15429-15434), which form the NodB deacetylase "superfamily."

Insights into the molecular determinants of substrate specificity in glycoside hydrolase family 5 revealed by the crystal structure and kinetics of Cellvibrio mixtus mannosidase 5A.

The enzymatic hydrolysis of the glycosidic bond is central to numerous biological processes. Glycoside hydrolases, which catalyze these reactions, are grouped into families based on primary sequence similarities. One of the largest glycoside hydrolase families is glycoside hydrolase family 5 (GH5), which contains primarily endo-acting enzymes that hydrolyze beta-mannans and beta-glucans. Here we report the cloning, characterization, and three-dimensional structure of the Cellvibrio mixtus GH5 beta-mannosidase (CmMan5A). This enzyme releases mannose from the nonreducing end of mannooligosaccharides and polysaccharides, an activity not previously observed in this enzyme family. CmMan5A contains a single glycone (-1) and two aglycone (+1 and +2) sugar-binding subsites. The -1 subsite displays absolute specificity for mannose, whereas the +1 subsite does not accommodate galactosyl side chains but will bind weakly to glucose. The +2 subsite is able to bind to decorated mannose residues. CmMan5A displays similar activity against crystalline and amorphous mannans, a property rarely attributed to glycoside hydrolases. The 1.5 A crystal structure reveals that CmMan5A adopts a (beta/alpha)(8) barrel fold, and superimposition with GH5 endo-mannanases shows that dramatic differences in the length of three loops modify the active center accessibility and thus modulate the specificity from endo to exo. The most striking and significant difference is the extended loop between strand beta8 and helix alpha8 comprising residues 378-412. This insertion forms a "double" steric barrier, formed by two short beta-strands that function to "block" the substrate binding cleft at the edge of the -1 subsite forming the "exo" active center topology of CmMan5A.