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Systems analysis of the glycoside hydrolase family 18 enzymes from Cellvibrio japonicus characterizes essential chitin degradation functions.

ABSTRACT: Understanding the strategies used by bacteria to degrade polysaccharides constitutes an invaluable tool for biotechnological applications. Bacteria are major mediators of polysaccharide degradation in nature; however, the complex mechanisms used to detect, degrade, and consume these substrates are not well-understood, especially for recalcitrant polysaccharides such as chitin. It has been previously shown that the model bacterial saprophyte Cellvibrio japonicus is able to catabolize chitin, but little is known about the enzymatic machinery underlying this capability. Previous analyses of the C. japonicus genome and proteome indicated the presence of four glycoside hydrolase family 18 (GH18) enzymes, and studies of the proteome indicated that all are involved in chitin utilization. Using a combination of in vitro and in vivo approaches, we have studied the roles of these four chitinases in chitin bioconversion. Genetic analyses showed that only the chi18D gene product is essential for the degradation of chitin substrates. Biochemical characterization of the four enzymes showed functional differences and synergistic effects during chitin degradation, indicating non-redundant roles in the cell. Transcriptomic studies revealed complex regulation of the chitin degradation machinery of C. japonicus and confirmed the importance of CjChi18D and CjLPMO10A, a previously characterized chitin-active enzyme. With this systems biology approach, we deciphered the physiological relevance of the glycoside hydrolase family 18 enzymes for chitin degradation in C. japonicus, and the combination of in vitro and in vivo approaches provided a comprehensive understanding of the initial stages of chitin degradation by this bacterium.

SUBMITTER: Monge EC

PROVIDER: S-EPMC5846145 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Systems analysis of the glycoside hydrolase family 18 enzymes from <i>Cellvibrio japonicus</i> characterizes essential chitin degradation functions.

Monge Estela C EC Tuveng Tina R TR Vaaje-Kolstad Gustav G Eijsink Vincent G H VGH Gardner Jeffrey G JG

The Journal of biological chemistry 20180124 10

Understanding the strategies used by bacteria to degrade polysaccharides constitutes an invaluable tool for biotechnological applications. Bacteria are major mediators of polysaccharide degradation in nature; however, the complex mechanisms used to detect, degrade, and consume these substrates are not well-understood, especially for recalcitrant polysaccharides such as chitin. It has been previously shown that the model bacterial saprophyte <i>Cellvibrio japonicus</i> is able to catabolize chiti ...[more]

PMID: 29367339

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Project description:BackgroundXyloglucan (XyG) is a ubiquitous and fundamental polysaccharide of plant cell walls. Due to its structural complexity, XyG requires a combination of backbone-cleaving and sidechain-debranching enzymes for complete deconstruction into its component monosaccharides. The soil saprophyte Cellvibrio japonicus has emerged as a genetically tractable model system to study biomass saccharification, in part due to its innate capacity to utilize a wide range of plant polysaccharides for growth. Whereas the downstream debranching enzymes of the xyloglucan utilization system of C. japonicus have been functionally characterized, the requisite backbone-cleaving endo-xyloglucanases were unresolved.ResultsCombined bioinformatic and transcriptomic analyses implicated three glycoside hydrolase family 5 subfamily 4 (GH5_4) members, with distinct modular organization, as potential keystone endo-xyloglucanases in C. japonicus. Detailed biochemical and enzymatic characterization of the GH5_4 modules of all three recombinant proteins confirmed particularly high specificities for the XyG polysaccharide versus a panel of other cell wall glycans, including mixed-linkage beta-glucan and cellulose. Moreover, product analysis demonstrated that all three enzymes generated XyG oligosaccharides required for subsequent saccharification by known exo-glycosidases. Crystallographic analysis of GH5D, which was the only GH5_4 member specifically and highly upregulated during growth on XyG, in free, product-complex, and active-site affinity-labelled forms revealed the molecular basis for the exquisite XyG specificity among these GH5_4 enzymes. Strikingly, exhaustive reverse-genetic analysis of all three GH5_4 members and a previously biochemically characterized GH74 member failed to reveal a growth defect, thereby indicating functional compensation in vivo, both among members of this cohort and by other, yet unidentified, xyloglucanases in C. japonicus. Our systems-based analysis indicates distinct substrate-sensing (GH74, GH5E, GH5F) and attack-mounting (GH5D) functions for the endo-xyloglucanases characterized here.ConclusionsThrough a multi-faceted, molecular systems-based approach, this study provides a new insight into the saccharification pathway of xyloglucan utilization system of C. japonicus. The detailed structural-functional characterization of three distinct GH5_4 endo-xyloglucanases will inform future bioinformatic predictions across species, and provides new CAZymes with defined specificity that may be harnessed in industrial and other biotechnological applications.

Project description:beta-1,4-Mannanases (mannanases), which hydrolyse mannans and glucomannans, are located in glycoside hydrolase families (GHs) 5 and 26. To investigate whether there are fundamental differences in the molecular architecture and biochemical properties of GH5 and GH26 mannanases, four genes encoding these enzymes were isolated from Cellvibrio japonicus and the encoded glycoside hydrolases were characterized. The four genes, man5A, man5B, man5C and man26B, encode the mannanases Man5A, Man5B, Man5C and Man26B, respectively. Man26B consists of an N-terminal signal peptide linked via an extended serine-rich region to a GH26 catalytic domain. Man5A, Man5B and Man5C contain GH5 catalytic domains and non-catalytic carbohydrate-binding modules (CBMs) belonging to families 2a, 5 and 10; Man5C in addition contains a module defined as X4 of unknown function. The family 10 and 2a CBMs bound to crystalline cellulose and ivory nut crystalline mannan, displaying very similar properties to the corresponding family 10 and 2a CBMs from Cellvibrio cellulases and xylanases. CBM5 bound weakly to these crystalline polysaccharides. The catalytic domains of Man5A, Man5B and Man26B hydrolysed galactomannan and glucomannan, but displayed no activity against crystalline mannan or cellulosic substrates. Although Man5C was less active against glucomannan and galactomannan than the other mannanases, it did attack crystalline ivory nut mannan. All the enzymes exhibited classic endo-activity producing a mixture of oligosaccharides during the initial phase of the reaction, although their mode of action against manno-oligosaccharides and glucomannan indicated differences in the topology of the respective substrate-binding sites. This report points to a different role for GH5 and GH26 mannanases from C. japonicus. We propose that as the GH5 enzymes contain CBMs that bind crystalline polysaccharides, these enzymes are likely to target mannans that are integral to the plant cell wall, while GH26 mannanases, which lack CBMs and rapidly release mannose from polysaccharides and oligosaccharides, target the storage polysaccharide galactomannan and manno-oligosaccharides.

Dataset Information

Systems analysis of the glycoside hydrolase family 18 enzymes from Cellvibrio japonicus characterizes essential chitin degradation functions.

Publications

Systems analysis of the glycoside hydrolase family 18 enzymes from <i>Cellvibrio japonicus</i> characterizes essential chitin degradation functions.

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