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Crystal structure of a homotrimeric verrucomicrobial exo-β-1,4-mannosidase active in the hindgut of the wood-feeding termite Reticulitermes flavipes.

ABSTRACT: The termite Reticulitermes flavipes causes extensive damage due to the high efficiency and broad specificity of the ligno- and hemicellulolytic enzyme systems produced by its symbionts. Thus, the R. flavipes gut microbiome is expected to constitute an excellent source of enzymes that can be used for the degradation and valorization of plant biomass. The symbiont Opitutaceae bacterium strain TAV5 belongs to the phylum Verrucomicrobia and thrives in the hindgut of R. flavipes. The sequence of the gene with the locus tag opit5_10225 in the Opitutaceae bacterium strain TAV5 genome has been classified as a member of glycoside hydrolase family 5 (GH5), and provisionally annotated as an endo-β-mannanase. We characterized biochemically and structurally the opit5_10225 gene product, and show that the enzyme, Op5Man5, is an exo-β-1,4-mannosidase [EC 3.2.1.25] that is highly specific for β-1,4-mannosidic bonds in mannooligosaccharides and ivory nut mannan. The structure of Op5Man5 was phased using electron cryo-microscopy and further determined and refined at 2.2 Å resolution using X-ray crystallography. Op5Man5 features a 200-kDa large homotrimer composed of three modular monomers. Despite insignificant sequence similarity, the structure of the monomer, and homotrimeric assembly are similar to that of the GH42-family β-galactosidases and the GH164-family exo-β-1,4-mannosidase Bs164 from Bacteroides salyersiae. To the best of our knowledge Op5Man5 is the first structure of a glycoside hydrolase from a bacterial symbiont isolated from the R. flavipes digestive tract, as well as the first example of a GH5 glycoside hydrolase with a GH42 β-galactosidase-type homotrimeric structure.

SUBMITTER: Kalyani DC

PROVIDER: S-EPMC8233224 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Two morphologically distinct, H2- and CO2-utilizing methanogens were isolated from gut homogenates of the subterranean termite, Reticulitermes-flavipes (Kollar) (Rhinotermitidae). Strain RFM-1 was a short straight rod (0.4 by 1.2 micron), whereas strain RFM-2 was a slightly curved rod (0.34 by 1.6 microns) that possessed polar fibers. Their morphology, gram-positive staining reaction, resistance to cell lysis by chemical agents, and narrow range of utilizable substracts were typical of species belonging to the family Methanobacteriaceae. Analysis of the nearly complete sequences of the small-subunit rRNA-encoding genes confirmed this affiliation and supported their recognition as new species of Methanobrevibacter: M. cuticularis (RFM-1) and M. curvatus (RFM-2). The per cell rates of methanogenesis by strains RFM-1 and RFM-2 in vitro, taken together with their in situ population densities (ca. 10(6) cells.gut-1; equivalent to 10(9) cells . ml of gut fluid-1), could fully account for the rate of methane emission by the live termites. UV epifluorescence and electron microscopy confirmed that RFM-1- and RFM-2-type cells were the dominant methanogens in R.flavipes collected in Michigan (but were not the only methanogens associated with this species) and that they colonized the peripheral, microoxic region of the hindgut, i.e., residing on or near the hindgut epithelium and also attached to filamentous prokaryotes associated with the gut wall. An examination of their oxygen tolerance revealed that both strains possessed catalase-like activity. Moreover, when dispersed in tubes or agar medium under H2-CO2-O2 (75: 18.8:6.2, vol/vol/vol), both strains grew to form a thin plate about 6 mm below the meniscus, just beneath the oxic-anoxic interface. Such growth plates were capable of mediating a net consumption of O2 that otherwise penetrated much deeper into uninoculated control tubes. Similar results were obtained with an authentic strain of Methanobrevibacter arboriphilicus. This is the first detailed description of an important and often cited but poorly understood component of the termite gut microbiota.

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Crystal structure of a homotrimeric verrucomicrobial exo-β-1,4-mannosidase active in the hindgut of the wood-feeding termite Reticulitermes flavipes.

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