title: Galactomannan Catabolism Conferred by a Polysaccharide Utilization Locus of Bacteroides ovatus: ENZYME SYNERGY AND CRYSTAL STRUCTURE OF A β-MANNANASE.
abstract: A recently identified polysaccharide utilization locus (PUL) from Bacteroides ovatus ATCC 8483 is transcriptionally up-regulated during growth on galacto- and glucomannans. It encodes two glycoside hydrolase family 26 (GH26) β-mannanases, BoMan26A and BoMan26B, and a GH36 α-galactosidase, BoGal36A. The PUL also includes two glycan-binding proteins, confirmed by β-mannan affinity electrophoresis. When this PUL was deleted, B. ovatus was no longer able to grow on locust bean galactomannan. BoMan26A primarily formed mannobiose from mannan polysaccharides. BoMan26B had higher activity on galactomannan with a high degree of galactosyl substitution and was shown to be endo-acting generating a more diverse mixture of oligosaccharides, including mannobiose. Of the two β-mannanases, only BoMan26B hydrolyzed galactoglucomannan. A crystal structure of BoMan26A revealed a similar structure to the exo-mannobiohydrolase CjMan26C from Cellvibrio japonicus, with a conserved glycone region (-1 and -2 subsites), including a conserved loop closing the active site beyond subsite -2. Analysis of cellular location by immunolabeling and fluorescence microscopy suggests that BoMan26B is surface-exposed and associated with the outer membrane, although BoMan26A and BoGal36A are likely periplasmic. In light of the cellular location and the biochemical properties of the two characterized β-mannanases, we propose a scheme of sequential action by the glycoside hydrolases encoded by the β-mannan PUL and involved in the β-mannan utilization pathway in B. ovatus. The outer membrane-associated BoMan26B initially acts on the polysaccharide galactomannan, producing comparably large oligosaccharide fragments. Galactomanno-oligosaccharides are further processed in the periplasm, degalactosylated by BoGal36A, and subsequently hydrolyzed into mainly mannobiose by the β-mannanase BoMan26A.
authors.affiliation: From the Department of Biochemistry and Structural Biology, Lund University P. O. Box 124, S-221 00 Lund, Sweden and.
authors.forename: Viktoria
authors.initials: V
authors.lastname: Bågenholm
authors.affiliation: From the Department of Biochemistry and Structural Biology, Lund University P. O. Box 124, S-221 00 Lund, Sweden and.
authors.forename: Sumitha K
authors.initials: SK
authors.lastname: Reddy
authors.affiliation: From the Department of Biochemistry and Structural Biology, Lund University P. O. Box 124, S-221 00 Lund, Sweden and.
authors.forename: Hanene
authors.initials: H
authors.lastname: Bouraoui
authors.affiliation: From the Department of Biochemistry and Structural Biology, Lund University P. O. Box 124, S-221 00 Lund, Sweden and.
authors.forename: Johan
authors.initials: J
authors.lastname: Morrill
authors.affiliation: From the Department of Biochemistry and Structural Biology, Lund University P. O. Box 124, S-221 00 Lund, Sweden and.
authors.forename: Evelina
authors.initials: E
authors.lastname: Kulcinskaja
authors.affiliation: the Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan 48109.
authors.forename: Constance M
authors.initials: CM
authors.lastname: Bahr
authors.affiliation: From the Department of Biochemistry and Structural Biology, Lund University P. O. Box 124, S-221 00 Lund, Sweden and.
authors.forename: Oskar
authors.initials: O
authors.lastname: Aurelius
authors.affiliation: the Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan 48109.
authors.forename: Theresa
authors.initials: T
authors.lastname: Rogers
authors.affiliation: the Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan 48109.
authors.forename: Yao
authors.initials: Y
authors.lastname: Xiao
authors.affiliation: From the Department of Biochemistry and Structural Biology, Lund University P. O. Box 124, S-221 00 Lund, Sweden and.
authors.forename: Derek T
authors.initials: DT
authors.lastname: Logan
authors.affiliation: the Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan 48109.
authors.forename: Eric C
authors.initials: EC
authors.lastname: Martens
authors.affiliation: the Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan 48109.
authors.forename: Nicole M
authors.initials: NM
authors.lastname: Koropatkin
authors.affiliation: From the Department of Biochemistry and Structural Biology, Lund University P. O. Box 124, S-221 00 Lund, Sweden and henrik.stalbrand@biochemistry.lu.se.
authors.forename: Henrik
authors.initials: H
authors.lastname: Stålbrand
mesh_terms: D001439:Bacteroides / Q000201:enzymology*; D002384:Catalysis; D018360:Crystallography, X-Ray; D005690:Galactose / Q000031:analogs & derivatives; D006868:Hydrolysis; D008351:Mannans / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*; D011487:Protein Conformation; D013379:Substrate Specificity; D044902:beta-Mannosidase / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D008351:Mannans; D011134:Polysaccharides; C012990:galactomannan; D044902:beta-Mannosidase; D005690:Galactose
keywords: Bacteroides ovatus; Gram-negative bacteria; carbohydrate metabolism; enzyme kinetics; galactomannan; glycobiology; glycoside hydrolase; human gut bacteria; polysaccharide utilization loci; structure-function