Dataset Information

Structural and biochemical basis for mannan utilization by Caldanaerobius polysaccharolyticus strain ATCC BAA-17.

ABSTRACT: Hemicelluloses, the polysaccharide component of plant cell walls, represent one of the most abundant biopolymers in nature. The most common hemicellulosic constituents of softwoods, such as conifers and cycads, are mannans consisting of a 1,4-linked ?-mannopyranosyl main chain with branch decorations. Efforts toward the utilization of hemicellulose for bioconversion into cellulosic biofuels have resulted in the identification of several families of glycoside hydrolases that can degrade mannan. However, effective biofermentation of manno-oligosaccharides is limited by a lack of appropriate uptake route in ethanologenic organisms. Here, we used transcriptome sequencing to gain insights into mannan degradation by the thermophilic anaerobic bacterium Caldanaerobius polysaccharolyticus. The most highly up-regulated genes during mannan fermentation occur in a cluster containing several genes encoding enzymes for efficient mannan hydrolysis as well as a solute-binding protein (CpMnBP1) that exhibits specificity for short mannose polymers but exhibited the flexibility to accommodate branched polysaccharide decorations. Co-crystal structures of CpMnBP1 in complex with mannobiose (1.4-Å resolution) and mannotriose (2.2-Å resolution) revealed the molecular rationale for chain length and oligosaccharide specificity. Calorimetric analysis of several active site variants confirmed the roles of residues critical to the function of CpMnBP1. This work represents the first biochemical characterization of a mannose-specific solute-binding protein and provides a framework for engineering mannan utilization capabilities for microbial fermentation.

SUBMITTER: Chekan JR

PROVIDER: S-EPMC4263893 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Structural and biochemical basis for mannan utilization by Caldanaerobius polysaccharolyticus strain ATCC BAA-17.

Chekan Jonathan R JR Kwon In Hyuk IH Agarwal Vinayak V Dodd Dylan D Revindran Vanessa V Mackie Roderick I RI Cann Isaac I Nair Satish K SK

The Journal of biological chemistry 20141023 50

Hemicelluloses, the polysaccharide component of plant cell walls, represent one of the most abundant biopolymers in nature. The most common hemicellulosic constituents of softwoods, such as conifers and cycads, are mannans consisting of a 1,4-linked β-mannopyranosyl main chain with branch decorations. Efforts toward the utilization of hemicellulose for bioconversion into cellulosic biofuels have resulted in the identification of several families of glycoside hydrolases that can degrade mannan. H ...[more]

PMID: 25342756

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Project description:The plant cell wall polysaccharide arabinan provides an important supply of arabinose, and unraveling arabinan-degrading strategies by microbes is important for understanding its use as a source of energy. Here, we explored the arabinan-degrading enzymes in the thermophilic bacterium Caldanaerobius polysaccharolyticus and identified a gene cluster encoding two glycoside hydrolase (GH) family 51 α-l-arabinofuranosidases (CpAbf51A, CpAbf51B), a GH43 endoarabinanase (CpAbn43A), a GH27 β-l-arabinopyranosidase (CpAbp27A), and two GH127 β-l-arabinofuranosidases (CpAbf127A, CpAbf127B). The genes were expressed as recombinant proteins, and the functions of the purified proteins were determined with para-nitrophenyl (pNP)-linked sugars and naturally occurring pectin structural elements as the substrates. The results demonstrated that CpAbn43A is an endoarabinanase while CpAbf51A and CpAbf51B are α-l-arabinofuranosidases that exhibit diverse substrate specificities, cleaving α-1,2, α-1,3, and α-1,5 linkages of purified arabinan-oligosaccharides. Furthermore, both CpAbf127A and CpAbf127B cleaved β-arabinofuranose residues in complex arabinan side chains, thus providing evidence of the function of this family of enzymes on such polysaccharides. The optimal temperatures of the enzymes ranged between 60°C and 75°C, and CpAbf43A and CpAbf51A worked synergistically to release arabinose from branched and debranched arabinan. Furthermore, the hydrolytic activity on branched arabinan oligosaccharides and degradation of pectic substrates by the endoarabinanase and l-arabinofuranosidases suggested a microbe equipped with diverse activities to degrade complex arabinan in the environment. Based on our functional analyses of the genes in the arabinan degradation cluster and the substrate-binding studies on a component of the cognate transporter system, we propose a model for arabinan degradation and transport by C. polysaccharolyticusIMPORTANCE Genomic DNA sequencing and bioinformatic analysis allowed the identification of a gene cluster encoding several proteins predicted to function in arabinan degradation and transport in C. polysaccharolyticus The analysis of the recombinant proteins yielded detailed insights into the putative arabinan metabolism of this thermophilic bacterium. The use of various branched arabinan oligosaccharides provided a detailed understanding of the substrate specificities of the enzymes and allowed assignment of two new GH127 polypeptides as β-l-arabinofuranosidases able to degrade pectic substrates, thus expanding our knowledge of this rare group of glycoside hydrolases. In addition, the enzymes showed synergistic effects for the degradation of arabinans at elevated temperatures. The enzymes characterized from the gene cluster are, therefore, of utility for arabinose production in both the biofuel and food industries.

Dataset Information

Structural and biochemical basis for mannan utilization by Caldanaerobius polysaccharolyticus strain ATCC BAA-17.

Publications

Structural and biochemical basis for mannan utilization by Caldanaerobius polysaccharolyticus strain ATCC BAA-17.

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