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Structure of the GH76 α-mannanase homolog, BT2949, from the gut symbiont Bacteroides thetaiotaomicron.

Thompson AJ, Cuskin F, Spears RJ, Dabin J, Turkenburg JP, Gilbert HJ, Davies GJ - Acta Crystallogr. D Biol. Crystallogr. (2015)

Bottom Line: A significant member of this community, Bacteroides thetaiotaomicron, has evolved a complex system for sensing and processing a wide variety of natural glycoproducts in such a way as to provide maximum benefit to itself, the wider microbial community and the host.BT2949 presents a classical (α/α)6-barrel structure comprising a large extended surface cleft common to other GH76 family members.Analysis of the structure in conjunction with sequence alignments reveals the likely location of the catalytic active site of this noncanonical GH76.

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Affiliation: Department of Chemistry, University of York, Heslington, York YO10 5DD, England.

ABSTRACT
The large bowel microbiota, a complex ecosystem resident within the gastrointestinal tract of all human beings and large mammals, functions as an essential, nonsomatic metabolic organ, hydrolysing complex dietary polysaccharides and modulating the host immune system to adequately tolerate ingested antigens. A significant member of this community, Bacteroides thetaiotaomicron, has evolved a complex system for sensing and processing a wide variety of natural glycoproducts in such a way as to provide maximum benefit to itself, the wider microbial community and the host. The immense ability of B. thetaiotaomicron as a `glycan specialist' resides in its enormous array of carbohydrate-active enzymes, many of which are arranged into polysaccharide-utilization loci (PULs) that are able to degrade sugar polymers that are often inaccessible to other gut residents, notably α-mannan. The B. thetaiotaomicron genome encodes ten putative α-mannanases spread across various PULs; however, little is known about the activity of these enzymes or the wider implications of α-mannan metabolism for the health of both the microbiota and the host. In this study, SAD phasing of a selenomethionine derivative has been used to investigate the structure of one such B. thetaiotaomicron enzyme, BT2949, which belongs to the GH76 family of α-mannanases. BT2949 presents a classical (α/α)6-barrel structure comprising a large extended surface cleft common to other GH76 family members. Analysis of the structure in conjunction with sequence alignments reveals the likely location of the catalytic active site of this noncanonical GH76.

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(a) BT2949 native Patterson map, v = 0.5 section, showing a large non-origin peak at w = 0.43 (peak heights are depicted relative to the origin). (b) Arrangement of molecules along the crystallographic b axis within the structure of BT2949. The two molecules that make up the asymmetric unit are labelled A and B (red and blue). Molecule A is related to molecule B* (yellow) by a translation of (½x, ½y, 0.43z). (a) was prepared using FFT/MapSlicer within the CCP4 suite (Winn et al., 2011 ▶). (b) was assembled using PyMOL v.1.6 (Schrödinger).
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fig2: (a) BT2949 native Patterson map, v = 0.5 section, showing a large non-origin peak at w = 0.43 (peak heights are depicted relative to the origin). (b) Arrangement of molecules along the crystallographic b axis within the structure of BT2949. The two molecules that make up the asymmetric unit are labelled A and B (red and blue). Molecule A is related to molecule B* (yellow) by a translation of (½x, ½y, 0.43z). (a) was prepared using FFT/MapSlicer within the CCP4 suite (Winn et al., 2011 ▶). (b) was assembled using PyMOL v.1.6 (Schrödinger).

Mentions: The crystals of BT2949 belonged to the orthorhombic space group P22121 (unit-cell parameters a = 81, b = 121, c = 126 Å, α = β = γ = 90°) with two protein molecules present in each asymmetric unit (molecules A and B appear identical, with an average r.m.s.d of 0.07 Å across matching Cα positions). The final atomic model for each monomer spans a continuous peptide chain comprising residues Pro37–Asn395 (residues 1–22 form a signal peptide and were removed in this construct to allow soluble gene expression). Initial merging of the data with AIMLESS in space group P212121 appeared satisfactory (systematic absence probability of ≥0.98 for three 21 screw axes); however, the structure could not be solved after several attempts at SAD phasing, with poor contrast and mapCC (correlation coefficient) between the two possible hands for the structure. Closer inspection of diffraction intensities and the native Patterson (Fig. 2 ▶a) revealed likely translational noncrystallographic symmetry (tNCS). Subsequent reprocessing of the data in P22121 allowed facile phasing of the selenium substructure and model building of two complete BT2949 molecules. Analysis of the refined structure revealed the observed tNCS to be owing to two identically orientated molecules in neighbouring asymmetric units related to the origin by a translation of (½x, ½y, 0.43z) (see Fig. 2 ▶b).


Structure of the GH76 α-mannanase homolog, BT2949, from the gut symbiont Bacteroides thetaiotaomicron.

Thompson AJ, Cuskin F, Spears RJ, Dabin J, Turkenburg JP, Gilbert HJ, Davies GJ - Acta Crystallogr. D Biol. Crystallogr. (2015)

(a) BT2949 native Patterson map, v = 0.5 section, showing a large non-origin peak at w = 0.43 (peak heights are depicted relative to the origin). (b) Arrangement of molecules along the crystallographic b axis within the structure of BT2949. The two molecules that make up the asymmetric unit are labelled A and B (red and blue). Molecule A is related to molecule B* (yellow) by a translation of (½x, ½y, 0.43z). (a) was prepared using FFT/MapSlicer within the CCP4 suite (Winn et al., 2011 ▶). (b) was assembled using PyMOL v.1.6 (Schrödinger).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4321491&req=5

fig2: (a) BT2949 native Patterson map, v = 0.5 section, showing a large non-origin peak at w = 0.43 (peak heights are depicted relative to the origin). (b) Arrangement of molecules along the crystallographic b axis within the structure of BT2949. The two molecules that make up the asymmetric unit are labelled A and B (red and blue). Molecule A is related to molecule B* (yellow) by a translation of (½x, ½y, 0.43z). (a) was prepared using FFT/MapSlicer within the CCP4 suite (Winn et al., 2011 ▶). (b) was assembled using PyMOL v.1.6 (Schrödinger).
Mentions: The crystals of BT2949 belonged to the orthorhombic space group P22121 (unit-cell parameters a = 81, b = 121, c = 126 Å, α = β = γ = 90°) with two protein molecules present in each asymmetric unit (molecules A and B appear identical, with an average r.m.s.d of 0.07 Å across matching Cα positions). The final atomic model for each monomer spans a continuous peptide chain comprising residues Pro37–Asn395 (residues 1–22 form a signal peptide and were removed in this construct to allow soluble gene expression). Initial merging of the data with AIMLESS in space group P212121 appeared satisfactory (systematic absence probability of ≥0.98 for three 21 screw axes); however, the structure could not be solved after several attempts at SAD phasing, with poor contrast and mapCC (correlation coefficient) between the two possible hands for the structure. Closer inspection of diffraction intensities and the native Patterson (Fig. 2 ▶a) revealed likely translational noncrystallographic symmetry (tNCS). Subsequent reprocessing of the data in P22121 allowed facile phasing of the selenium substructure and model building of two complete BT2949 molecules. Analysis of the refined structure revealed the observed tNCS to be owing to two identically orientated molecules in neighbouring asymmetric units related to the origin by a translation of (½x, ½y, 0.43z) (see Fig. 2 ▶b).

Bottom Line: A significant member of this community, Bacteroides thetaiotaomicron, has evolved a complex system for sensing and processing a wide variety of natural glycoproducts in such a way as to provide maximum benefit to itself, the wider microbial community and the host.BT2949 presents a classical (α/α)6-barrel structure comprising a large extended surface cleft common to other GH76 family members.Analysis of the structure in conjunction with sequence alignments reveals the likely location of the catalytic active site of this noncanonical GH76.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry, University of York, Heslington, York YO10 5DD, England.

ABSTRACT
The large bowel microbiota, a complex ecosystem resident within the gastrointestinal tract of all human beings and large mammals, functions as an essential, nonsomatic metabolic organ, hydrolysing complex dietary polysaccharides and modulating the host immune system to adequately tolerate ingested antigens. A significant member of this community, Bacteroides thetaiotaomicron, has evolved a complex system for sensing and processing a wide variety of natural glycoproducts in such a way as to provide maximum benefit to itself, the wider microbial community and the host. The immense ability of B. thetaiotaomicron as a `glycan specialist' resides in its enormous array of carbohydrate-active enzymes, many of which are arranged into polysaccharide-utilization loci (PULs) that are able to degrade sugar polymers that are often inaccessible to other gut residents, notably α-mannan. The B. thetaiotaomicron genome encodes ten putative α-mannanases spread across various PULs; however, little is known about the activity of these enzymes or the wider implications of α-mannan metabolism for the health of both the microbiota and the host. In this study, SAD phasing of a selenomethionine derivative has been used to investigate the structure of one such B. thetaiotaomicron enzyme, BT2949, which belongs to the GH76 family of α-mannanases. BT2949 presents a classical (α/α)6-barrel structure comprising a large extended surface cleft common to other GH76 family members. Analysis of the structure in conjunction with sequence alignments reveals the likely location of the catalytic active site of this noncanonical GH76.

Show MeSH
Related in: MedlinePlus