title: Crystal structure of mannanase 26A from Pseudomonas cellulosa and analysis of residues involved in substrate binding.
abstract: The crystal structure of Pseudomonas cellulosa mannanase 26A has been solved by multiple isomorphous replacement and refined at 1.85 A resolution to an R-factor of 0.182 (R-free = 0.211). The enzyme comprises (beta/alpha)(8)-barrel architecture with two catalytic glutamates at the ends of beta-strands 4 and 7 in precisely the same location as the corresponding glutamates in other 4/7-superfamily glycoside hydrolase enzymes (clan GH-A glycoside hydrolases). The family 26 glycoside hydrolases are therefore members of clan GH-A. Functional analyses of mannanase 26A, informed by the crystal structure of the enzyme, provided important insights into the role of residues close to the catalytic glutamates. These data showed that Trp-360 played a critical role in binding substrate at the -1 subsite, whereas Tyr-285 was important to the function of the nucleophile catalyst. His-211 in mannanase 26A does not have the same function as the equivalent asparagine in the other GH-A enzymes. The data also suggest that Trp-217 and Trp-162 are important for the activity of mannanase 26A against mannooligosaccharides but are less important for activity against polysaccharides.
authors.affiliation: Department of Biological and Nutritional Sciences, University of Newcastle, Newcastle upon Tyne NE1 7RU, United Kingdom.
authors.forename: D
authors.initials: D
authors.lastname: Hogg
authors.forename: E J
authors.initials: EJ
authors.lastname: Woo
authors.forename: D N
authors.initials: DN
authors.lastname: Bolam
authors.forename: V A
authors.initials: VA
authors.lastname: McKie
authors.forename: H J
authors.initials: HJ
authors.lastname: Gilbert
authors.forename: R W
authors.initials: RW
authors.lastname: Pickersgill
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D003461:Crystallography; D008351:Mannans / Q000378:metabolism; D008361:Mannosidases / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D011549:Pseudomonas / Q000201:enzymology*; D013329:Structure-Activity Relationship; D044902:beta-Mannosidase
chemical_list: D008351:Mannans; D008361:Mannosidases; D044902:beta-Mannosidase