A novel Cellvibrio mixtus family 10 xylanase that is both intracellular and expressed under non-inducing conditions. (1/33)

Hydrolysis of the plant cell wall polysaccharides cellulose and xylan requires the synergistic interaction of a repertoire of extracellular enzymes. Recently, evidence has emerged that anaerobic bacteria can synthesize high levels of periplasmic xylanases which may be involved in the hydrolysis of small xylo-oligosaccharides absorbed by the micro-organism. Cellvibrio mixtus, a saprophytic aerobic soil bacterium that is highly active against plant cell wall polysaccharides, was shown to express internal xylanase activity when cultured on media containing xylan or glucose as sole carbon source. A genomic library of C. mixtus DNA, constructed in lambdaZAPII, was screened for xylanase activity. The nucleotide sequence of the genomic insert from a xylanase-positive clone that expressed intracellular xylanase activity in Escherichia coli revealed an ORF of 1137 bp (xynC), encoding a polypeptide with a deduced M(r) of 43413, defined as xylanase C (XylC). Probing a gene library of Pseudomonas fluorescens subsp. cellulosa with C. mixtus xynC identified a xynC homologue (designated xynG) encoding XylG; XylG and xynG were 67% and 63% identical to the corresponding C. mixtus sequences, respectively. Both XylC and XylG exhibit extensive sequence identity with family 10 xylanases, particularly with non-modular enzymes, and gene deletion studies on xynC supported the suggestion that they are single-domain xylanases. Purified recombinant XylC had an M(r) of 41000, and displayed biochemical properties typical of family 10 polysaccharidases. However, unlike previously characterized xylanases, XylC was particularly sensitive to proteolytic inactivation by pancreatic proteinases and was thermolabile. C. mixtus was grown to late-exponential phase in the presence of glucose or xylan and the cytoplasmic, periplasmic and cell envelope fractions were probed with anti-XylC antibodies. The results showed that XylC was absent from the culture media but was predominantly present in the periplasm of C. mixtus cells grown on glucose, xylan, CM-cellulose or Avicel. These data suggest that C. mixtus can express non-modular internal xylanases whose potential roles in the hydrolysis of plant cell wall components are discussed.  (+info)

The membrane-bound alpha-glucuronidase from Pseudomonas cellulosa hydrolyzes 4-O-methyl-D-glucuronoxylooligosaccharides but not 4-O-methyl-D-glucuronoxylan. (2/33)

The microbial degradation of xylan is a key biological process. Hardwood 4-O-methyl-D-glucuronoxylans are extensively decorated with 4-O-methyl-D-glucuronic acid, which is cleaved from the polysaccharides by alpha-glucuronidases. In this report we describe the primary structures of the alpha-glucuronidase from Cellvibrio mixtus (C. mixtus GlcA67A) and the alpha-glucuronidase from Pseudomonas cellulosa (P. cellulosa GlcA67A) and characterize P. cellulosa GlcA67A. The primary structures of C. mixtus GlcA67A and P. cellulosa GlcA67A, which are 76% identical, exhibit similarities with alpha-glucuronidases in glycoside hydrolase family 67. The membrane-associated pseudomonad alpha-glucuronidase released 4-O-methyl-D-glucuronic acid from 4-O-methyl-D-glucuronoxylooligosaccharides but not from 4-O-methyl-D-glucuronoxylan. We propose that the role of the glucuronidase, in combination with cell-associated xylanases, is to hydrolyze decorated xylooligosaccharides, generated by extracellular hemicellulases, to xylose and 4-O-methyl-D-glucuronic acid, enabling the pseudomonad to preferentially utilize the sugars derived from these polymers.  (+info)

Convergent evolution sheds light on the anti-beta -elimination mechanism common to family 1 and 10 polysaccharide lyases. (3/33)

Enzyme-catalyzed beta-elimination of sugar uronic acids, exemplified by the degradation of plant cell wall pectins, plays an important role in a wide spectrum of biological processes ranging from the recycling of plant biomass through to pathogen virulence. The three-dimensional crystal structure of the catalytic module of a "family PL-10" polysaccharide lyase, Pel10Acm from Cellvibrio japonicus, solved at a resolution of 1.3 A, reveals a new polysaccharide lyase fold and is the first example of a polygalacturonic acid lyase that does not exhibit the "parallel beta-helix" topology. The "Michaelis" complex of an inactive mutant in association with the substrate trigalacturonate/Ca2+ reveals the catalytic machinery harnessed by this polygalacturonate lyase, which displays a stunning resemblance, presumably through convergent evolution, to the tetragalacturonic acid complex observed for a structurally unrelated polygalacturonate lyase from family PL-1. Common coordination of the -1 and +1 subsite saccharide carboxylate groups by a protein-liganded Ca2+ ion, the positioning of an arginine catalytic base in close proximity to the alpha-carbon hydrogen and numerous other conserved enzyme-substrate interactions, considered in light of mutagenesis data for both families, suggest a generic polysaccharide anti-beta-elimination mechanism.  (+info)

The alpha-glucuronidase, GlcA67A, of Cellvibrio japonicus utilizes the carboxylate and methyl groups of aldobiouronic acid as important substrate recognition determinants. (4/33)

alpha-Glucuronidases are key components of the ensemble of enzymes that degrade the plant cell wall. They hydrolyze the alpha1,2-glycosidic bond between 4-O-methyl-d-glucuronic acid (4-O-MeGlcA) and the xylan or xylooligosaccharide backbone. Here we report the crystal structure of an inactive mutant (E292A) of the alpha-glucuronidase, GlcA67A, from Cellvibrio japonicus in complex with its substrate. The data show that the 4-O-methyl group of the substrate is accommodated within a hydrophobic sheath flanked by Val-210 and Trp-160, whereas the carboxylate moiety is located within a positively charged region of the substrate-binding pocket. The carboxylate side chains of Glu-393 and Asp-365, on the "beta-face" of 4-O-MeGlcA, form hydrogen bonds with a water molecule that is perfectly positioned to mount a nucleophilic attack at the anomeric carbon of the target glycosidic bond, providing further support for the view that, singly or together, these amino acids function as the catalytic base. The capacity of reaction products and product analogues to inhibit GlcA67A shows that the 4-O-methyl group, the carboxylate, and the xylose sugar of aldobiouronic acid all play an important role in substrate binding. Site-directed mutagenesis informed by the crystal structure of enzyme-ligand complexes was used to probe the importance of highly conserved residues at the active site of GlcA67A. The biochemical properties of K288A, R325A, and K360A show that a constellation of three basic amino acids (Lys-288, Arg-325, and Lys-360) plays a critical role in binding the carboxylate moiety of 4-O-MeGlcA. Disruption of the apolar nature of the pocket created by Val-210 (V210N and V210S) has a detrimental effect on substrate binding, although the reduction in affinity is not reflected by an inability to accommodate the 4-O-methyl group. Replacing the two tryptophan residues that stack against the sugar rings of the substrate with alanine (W160A and W543A) greatly reduced activity.  (+info)

Reclassification of 'Pseudomonas fluorescens subsp. cellulosa' NCIMB 10462 (Ueda et al. 1952) as Cellvibrio japonicus sp. nov. and revival of Cellvibrio vulgaris sp. nov., nom. rev. and Cellvibrio fulvus sp. nov., nom. rev. (5/33)

'Pseudomonas fluorescens subsp. cellulosa' NCIMB 10462 has been demonstrated by a polyphasic taxonomic approach to be a member of the genus Cellvibrio. 16S rDNA sequence analysis suggests that this is the only genus that could accept this specimen. The sequence is 95.5% similar to that of Cellvibrio mixtus subsp. mixtus ACM 2601T (the type strain of the type species of the genus), which is its closest relation. The genomic DNA G + C content was determined to be 53.3 mol%, which is similar to the values obtained for the validly described Cellvibrio species. DNA-DNA hybridization experiments have shown that strain NCIMB 10462T (= NCDO 2697T) represents a novel species; therefore, it is proposed that it be designated as the type strain of the novel species Cellvibrio japonicus sp. nov. This study also used 16S rDNA analysis, DNA-DNA hybridization experiments and phenotypic testing to revive the species Cellvibrio vulgaris sp. nov., nom. rev. and Cellvibrio fulvus sp. nov., nom. rev. C. vulgaris NCIMB 8633T (=LMG 2848T) and C. fulvus NCIMB 8634T (=LMG 2847T) are the proposed type strains.  (+info)

Taxonomic study of Cellvibrio strains and description of Cellvibrio ostraviensis sp. nov., Cellvibrio fibrivorans sp. nov. and Cellvibrio gandavensis sp. nov. (6/33)

Thirty-one cellulolytic bacterial isolates from soils that were phenotypically very similar and phylogenetically highly related to Cellvibrio strains were further characterized using a polyphasic taxonomic approach. By using repetitive extragenic palindromic DNA-PCR fingerprinting, six different fingerprints could be recognized among the isolates. Representative strains and four reference strains of the genus Cellvibrio were used for DNA-DNA hybridization, which yielded eight DNA hybridization groups at a cut-off level of 70% DNA binding. One group was formed by three isolates and Cellvibrio vulgaris LMG 2848T and a second group consisted of Cellvibrio mixtus strains ACM 2601T and ACM 2603. Two isolates and Cellvibrio fulvus LMG 2847T constituted single-member groups. For the remaining groups, three novel species are proposed: Cellvibrio fibrivorans sp. nov. (six strains, type strain LMG 18561T =ACM 5172T), Cellvibrio ostraviensis sp. nov. (eight strains, type strain LMG 19434T =ACM 5173T) and Cellvibrio gandavensis sp. nov. (12 strains, type strain LMG 18551T =ACM 5174T). The novel Cellvibrio species could be differentiated from each other and from C. mixtus, C. vulgaris and C. fulvus on the basis of phenotypic features, their fatty acid compositions and the G + C content of their DNA.  (+info)

Parallel induction of D-arabitol and D-sorbitol dehydrogenases. (7/33)

Scolnick, Edward M. (Harvard Medical School, Boston, Mass.) and Edmund C. C. Lin. Parallel induction of d-arabitol and d-sorbitol dehydrogenases. J. Bacteriol. 84:631-637. 1962.-Two inducible diphosphopyridine nucleotide-linked dehydrogenases are described in a bacterium isolated from the soil, Cellvibrio polyoltrophicus ATCC 14774. The first enzyme catalyzes the dehydrogenation of d-arabitol to d-xylulose and d-mannitol to d-fructose. The data suggest that in vivo this enzyme has the dual function of the utilization of both of these polyhydric alcohols. The second enzyme was found to act only on d-sorbitol, converting it to d-fructose. Evidence for its physiological function as a d-sorbitol dehydrogenase is also given. Both of these enzymes were found to be induced in parallel by any of the three polyhydric alcohols, d-arabitol, d-mannitol, and d-sorbitol. A common stereoconfiguration of the inducers for these enzymes is suggested. The parallel evolution of substrate specificity and inducer specificity is discussed with respect to the functional advantage that such a selective process might offer.  (+info)

The mechanisms by which family 10 glycoside hydrolases bind decorated substrates. (8/33)

Endo-beta-1,4-xylanases (xylanases), which cleave beta-1,4 glycosidic bonds in the xylan backbone, are important components of the repertoire of enzymes that catalyze plant cell wall degradation. The mechanism by which these enzymes are able to hydrolyze a range of decorated xylans remains unclear. Here we reveal the three-dimensional structure, determined by x-ray crystallography, and the catalytic properties of the Cellvibrio mixtus enzyme Xyn10B (CmXyn10B), the most active GH10 xylanase described to date. The crystal structure of the enzyme in complex with xylopentaose reveals that at the +1 subsite the xylose moiety is sandwiched between hydrophobic residues, which is likely to mediate tighter binding than in other GH10 xylanases. The crystal structure of the xylanase in complex with a range of decorated xylooligosaccharides reveals how this enzyme is able to hydrolyze substituted xylan. Solvent exposure of the O-2 groups of xylose at the +4, +3, +1, and -3 subsites may allow accommodation of the alpha-1,2-linked 4-O-methyl-d-glucuronic acid side chain in glucuronoxylan at these locations. Furthermore, the uronic acid makes hydrogen bonds and hydrophobic interactions with the enzyme at the +1 subsite, indicating that the sugar decorations in glucuronoxylan are targeted to this proximal aglycone binding site. Accommodation of 3'-linked l-arabinofuranoside decorations is observed in the -2 subsite and could, most likely, be tolerated when bound to xylosides in -3 and +4. A notable feature of the binding mode of decorated substrates is the way in which the subsite specificities are tailored both to prevent the formation of "dead-end" reaction products and to facilitate synergy with the xylan degradation-accessory enzymes such as alpha-glucuronidase. The data described in this report and in the accompanying paper indicate that the complementarity in the binding of decorated substrates between the glycone and aglycone regions appears to be a conserved feature of GH10 xylanases.  (+info)

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