Crystal structure of mannanase 26A from Pseudomonas cellulosa and analysisof residues involved in substrate binding

The crystal structure of Pseudomonas cellulosa mannanase 26A has been solve d by multiple isomorphous replacement and refined at 1.85 Angstrom resoluti on to an R-factor of 0.182 (R-free = 0.211). The enzyme comprises (beta/alp ha)(8)-barrel architecture with two catalytic glutamates at the ends of bet a -strands 4 and 7 in precisely the same location as the corresponding glut amates in other 4/7-superfamily glycoside hydrolase enzymes (clan GH-A glyc oside hydrolases). The family 26 glycoside hydrolases are therefore members of clan GH-A. Functional analyses of mannanase 26A, informed by the crysta l structure of the enzyme, provided important insights into the role of res idues close to the catalytic glutamates. These data showed that Trp-360 pla yed 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 mann anase 26A does not have the same function as the equivalent asparagine in t he other GH-A enzymes. The data also suggest that Trp-217 and Trp-162 are i mportant for the activity of mannanase 26RA against mannooligosaccharides b ut are less important for activity against polysaccharides.