Pseudomonas cellulosa expresses a single membrane-bound glycoside hydrolase family 51 arabinofuranosidase

BEYLOT, Marie-Helene; Emami, Kaveh; McKie, Vincent A.; Gilbert, Harry J.; Pell, G.

doi:10.1042/0264-6021:3580599

Cited by 23 publications

(35 citation statements)

References 24 publications

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“…In C. japonicus, CjAbf43A converts the double substitutions into single O3-linked decorations, which are then removed by the extracellular, membrane-associated, GH51 arabinofuranosidase (43). The linear arabinan generated is then hydrolyzed, exclusively, to arabinotriose by an endo-processive arabinanase (9).…”

Section: Discussionmentioning

confidence: 99%

The Structure and Function of an Arabinan-specific α-1,2-Arabinofuranosidase Identified from Screening the Activities of Bacterial GH43 Glycoside Hydrolases

Cartmell

McKee

Peña

et al. 2011

Journal of Biological Chemistry

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Reflecting the diverse chemistry of plant cell walls, microorganisms that degrade these composite structures synthesize an array of glycoside hydrolases. These enzymes are organized into sequence-, mechanism-, and structure-based families. Genomic data have shown that several organisms that degrade the plant cell wall contain a large number of genes encoding family 43 (GH43) glycoside hydrolases. Here we report the biochemical properties of the GH43 enzymes of a saprophytic soil bacterium, Cellvibrio japonicus, and a human colonic symbiont, Bacteroides thetaiotaomicron. The data show that C. japonicus uses predominantly exo-acting enzymes to degrade arabinan into arabinose, whereas B. thetaiotaomicron deploys a combination of endo-and side chain-cleaving glycoside hydrolases. Both organisms, however, utilize an arabinan-specific ␣-1,2-arabinofuranosidase in the degradative process, an activity that has not previously been reported. The enzyme can cleave ␣-1,2-arabinofuranose decorations in single or double substitutions, the latter being recalcitrant to the action of other arabinofuranosidases. The crystal structure of the C. japonicus arabinan-specific ␣-1,2-arabinofuranosidase, CjAbf43A, displays a fivebladed ␤-propeller fold. The specificity of the enzyme for arabinan is conferred by a surface cleft that is complementary to the helical backbone of the polysaccharide. The specificity of CjAbf43A for ␣-1,2-L-arabinofuranose side chains is conferred by a polar residue that orientates the arabinan backbone such that O2 arabinose decorations are directed into the active site pocket. A shelflike structure adjacent to the active site pocket accommodates O3 arabinose side chains, explaining how the enzyme can target O2 linkages that are components of single or double substitutions.

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Section: Discussionmentioning

confidence: 99%

The Structure and Function of an Arabinan-specific α-1,2-Arabinofuranosidase Identified from Screening the Activities of Bacterial GH43 Glycoside Hydrolases

Cartmell

McKee

Peña

et al. 2011

Journal of Biological Chemistry

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“…It is likely, however, that the loss of the esterase activity of Xyn11A in the P. cellulosa mutant would limit its capacity to utilize "natural" highly acetylated xylans. Indeed, the importance of arabinose removal has already been shown to be a limiting factor in the utilization of arabinoxylans as a carbon source by the bacterium (3).…”

Section: Discussionmentioning

confidence: 99%

“…RNA quantification. RNA was isolated from cultures of P. cellulosa grown at 30°C at different stages of the growth phase as described previously (3). Aliquots of RNA (5 g) were electrophoresed on 1.2% (wt/vol) agarose-formaldehyde denaturing gels (27) and transferred to Hybond N membranes (AmershamPharmacia) by using 300 mM sodium citrate buffer (pH 7.2) containing 3 M NaCl, and the nucleic acid was immobilized by UV cross-linking.…”

Section: Methodsmentioning

confidence: 99%

Evidence for Temporal Regulation of the Two Pseudomonas cellulosa Xylanases Belonging to Glycoside Hydrolase Family 11

et al. 2002

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Pseudomonas cellulosa is a highly efficient xylan-degrading bacterium. Genes encoding five xylanases, and several accessory enzymes, which remove the various side chains that decorate the xylan backbone, have been isolated from the pseudomonad and characterized. The xylanase genes consist of xyn10A, xyn10B, xyn10C, xyn10D, and xyn11A, which encode Xyn10A, Xyn10B, Xyn10C, Xyn10D, and Xyn11A, respectively. In this study a sixth xylanase gene, xyn11B, was isolated which encodes a 357-residue modular enzyme, designated Xyn11B, comprising a glycoside hydrolase family 11 catalytic domain appended to a C-terminal X-14 module, a homologue of which binds to xylan. Localization studies showed that the two xylanases with glycoside hydrolase family (GH) 11 catalytic modules, Xyn11A and Xyn11B, are secreted into the culture medium, whereas Xyn10C is membrane bound. xyn10C, xyn10D, xyn11A, and xyn11B were all abundantly expressed when the bacterium was cultured on xylan or ␤-glucan but not on medium containing mannan, whereas glucose repressed transcription of these genes. Although all of the xylanase genes were induced by the same polysaccharides, temporal regulation of xyn11A and xyn11B was apparent on xylan-containing media. Transcription of xyn11A occurred earlier than transcription of xyn11B, which is consistent with the predicted mode of action of the encoded enzymes. Xyn11A, but not Xyn11B, exhibits xylan esterase activity, and the removal of acetate side chains is required for xylanases to hydrolyze the xylan backbone. A transposon mutant of P. cellulosa in which xyn11A and xyn11B were inactive displayed greatly reduced extracellular but normal cell-associated xylanase activity, and its growth rate on medium containing xylan was indistinguishable from wild-type P. cellulosa. Based on the data presented here, we propose a model for xylan degradation by P. cellulosa in which the GH11 enzymes convert decorated xylans into substituted xylooligosaccharides, which are then hydrolyzed to their constituent sugars by the combined action of cell-associated GH10 xylanases and side chain-cleaving enzymes.

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“…Although the majority of microbial AFs are secreted into the culture medium, some are retained within the cytoplasm or are membrane-associated (Beylot et al, 2001a;. AbfA and Abf2 activity was detected in whole cells and the natural substrates of these enzymes are most probably degradation products or arabinose-containing polysaccharides (see above), suggesting that the two AFs are retained in the cytoplasm.…”

Section: Localization Of Abfa and Abf2 Activitiesmentioning

confidence: 99%

“…However, Bacillus species and other aerobic bacteria secrete a moderate number of enzymes that attack the polysaccharide backbone, releasing quite large oligosaccharides (Numan & Bhosle, 2006;. The complete hydrolysis of these products is accomplished by intracellular or membrane-associated enzymes (Beylot et al, 2001a;Shulami et al, 2007). Thus, the cytoplasmic nature of AbfA and Abf2 in B. subtilis might represent an adaptive advantage for the bacterium since competing non-hemicellulolytic micro-organisms are unable to utilize the relatively large arabino-oligomers and arabino-oligosaccharides products released by the extracellular enzymes of B. subtilis .…”

Section: Enzymic Characterization Of Purified Abfa and Abf2mentioning

confidence: 99%

Two distinct arabinofuranosidases contribute to arabino-oligosaccharide degradation in Bacillus subtilis

2008

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Bacillus subtilis produces a-L-arabinofuranosidases (EC 3.2.1.55; AFs) capable of releasing arabinosyl oligomers and L-arabinose from plant cell walls. Here, we show by insertion-deletion mutational analysis that genes abfA and xsa(asd), herein renamed abf2, encode AFs responsible for the majority of the intracellular AF activity in B. subtilis. Both enzyme activities were shown to be cytosolic and functional studies indicated that arabino-oligomers are natural substrates for the AFs. The products of the two genes were overproduced in Escherichia coli, purified and characterized. The molecular mass of the purified AbfA and Abf2 was about 58 kDa and 57 kDa, respectively. However, native PAGE gradient gel analysis and cross-linking assays detected higher-order structures (.250 kDa), suggesting a multimeric organization of both enzymes. Kinetic experiments at 37 6C, with p-nitrophenyl-a-L-arabinofuranoside as substrate, gave an apparent K m of 0.498 mM and 0.421 mM, and V max of 317 U mg "1 and 311 U mg "1 for AbfA and Abf2, respectively. The two enzymes displayed maximum activity at 50 6C and 60 6C, respectively, and both proteins were most active at pH 8.0. AbfA and Abf2 both belong to family 51 of the glycoside hydrolases but have different substrate specificity. AbfA acts preferentially on (1A5) linkages of linear a-1,5-L-arabinan and a-1,5-linked arabino-oligomers, and is much less effective on branched sugar beet arabinan and arabinoxylan and arabinogalactan. In contrast, Abf2 is most active on (1A2) and (1A3) linkages of branched arabinan and arabinoxylan, suggesting a concerted contribution of these enzymes to optimal utilization of arabinosecontaining polysaccharides by B. subtilis.

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Pseudomonas cellulosa expresses a single membrane-bound glycoside hydrolase family 51 arabinofuranosidase

Cited by 23 publications

References 24 publications

The Structure and Function of an Arabinan-specific α-1,2-Arabinofuranosidase Identified from Screening the Activities of Bacterial GH43 Glycoside Hydrolases

The Structure and Function of an Arabinan-specific α-1,2-Arabinofuranosidase Identified from Screening the Activities of Bacterial GH43 Glycoside Hydrolases

Evidence for Temporal Regulation of the Two Pseudomonas cellulosa Xylanases Belonging to Glycoside Hydrolase Family 11

Two distinct arabinofuranosidases contribute to arabino-oligosaccharide degradation in Bacillus subtilis

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