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Structural and biochemical analyses of glycoside hydrolase family 26 ?-mannanase from a symbiotic protist of the termite Reticulitermes speratus.

ABSTRACT: Termites and their symbiotic protists have established a prominent dual lignocellulolytic system, which can be applied to the biorefinery process. One of the major components of lignocellulose from conifers is glucomannan, which comprises a heterogeneous combination of ?-1,4-linked mannose and glucose. Mannanases are known to hydrolyze the internal linkage of the glucomannan backbone, but the specific mechanism by which they recognize and accommodate heteropolysaccharides is currently unclear. Here, we report biochemical and structural analyses of glycoside hydrolase family 26 mannanase C (RsMan26C) from a symbiotic protist of the termite Reticulitermes speratus. RsMan26C was characterized based on its catalytic efficiency toward glucomannan, compared with pure mannan. The crystal structure of RsMan26C complexed with gluco-manno-oligosaccharide(s) explained its specificities for glucose and mannose at subsites -5 and -2, respectively, in addition to accommodation of both glucose and mannose at subsites -3 and -4. RsMan26C has a long open cleft with a hydrophobic platform of Trp(94) at subsite -5, facilitating enzyme binding to polysaccharides. Notably, a unique oxidized Met(85) specifically interacts with the equatorial O-2 of glucose at subsite -3. Our results collectively indicate that specific recognition and accommodation of glucose at the distal negative subsites confers efficient degradation of the heteropolysaccharide by mannanase.

SUBMITTER: Tsukagoshi H

PROVIDER: S-EPMC4036197 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Structural and biochemical analyses of glycoside hydrolase family 26 β-mannanase from a symbiotic protist of the termite Reticulitermes speratus.

Tsukagoshi Hikaru H Nakamura Akihiko A Ishida Takuya T Touhara Kouki K KK Otagiri Masato M Moriya Shigeharu S Samejima Masahiro M Igarashi Kiyohiko K Fushinobu Shinya S Kitamoto Katsuhiko K Arioka Manabu M

The Journal of biological chemistry 20140225 15

Termites and their symbiotic protists have established a prominent dual lignocellulolytic system, which can be applied to the biorefinery process. One of the major components of lignocellulose from conifers is glucomannan, which comprises a heterogeneous combination of β-1,4-linked mannose and glucose. Mannanases are known to hydrolyze the internal linkage of the glucomannan backbone, but the specific mechanism by which they recognize and accommodate heteropolysaccharides is currently unclear. H ...[more]

PMID: 24570006

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Project description:BACKGROUND:The yeast Saccharomyces cerevisiae, a promising host for lignocellulosic bioethanol production, is unable to metabolize xylose. In attempts to confer xylose utilization ability in S. cerevisiae, a number of xylose isomerase (XI) genes have been expressed heterologously in this yeast. Although several of these XI encoding genes were functionally expressed in S. cerevisiae, the need still exists for a S. cerevisiae strain with improved xylose utilization ability for use in the commercial production of bioethanol. Although currently much effort has been devoted to achieve the objective, one of the solutions is to search for a new XI gene that would confer superior xylose utilization in S. cerevisiae. Here, we searched for novel XI genes from the protists residing in the hindgut of the termite Reticulitermes speratus. RESULTS:Eight novel XI genes were obtained from a cDNA library, prepared from the protists of the R. speratus hindgut, by PCR amplification using degenerated primers based on highly conserved regions of amino acid sequences of different XIs. Phylogenetic analysis classified these cloned XIs into two groups, one showed relatively high similarities to Bacteroidetes and the other was comparatively similar to Firmicutes. The growth rate and the xylose consumption rate of the S. cerevisiae strain expressing the novel XI, which exhibited highest XI activity among the eight XIs, were superior to those exhibited by the strain expressing the XI gene from Piromyces sp. E2. Substitution of the asparagine residue at position 337 of the novel XI with a cysteine further improved the xylose utilization ability of the yeast strain. Interestingly, introducing point mutations in the corresponding asparagine residues in XIs originated from other organisms, such as Piromyces sp. E2 or Clostridium phytofermentans, similarly improved xylose utilization in S. cerevisiae. CONCLUSIONS:A novel XI gene conferring superior xylose utilization in S. cerevisiae was successfully isolated from the protists in the termite hindgut. Isolation of this XI gene and identification of the point mutation described in this study might contribute to improving the productivity of industrial bioethanol.

Dataset Information

Structural and biochemical analyses of glycoside hydrolase family 26 ?-mannanase from a symbiotic protist of the termite Reticulitermes speratus.

Publications

Structural and biochemical analyses of glycoside hydrolase family 26 β-mannanase from a symbiotic protist of the termite Reticulitermes speratus.

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