Three-dimensional crystal structure and enzymic characterization of beta-mannanase Man5A from blue mussel Mytilus edulis.
Endo-beta-1,4-d-mannanase is the key depolymerizing enzyme for beta-1,4-mannan polymers present in the cell walls of plants and some algae, as well as in some types of plant seeds. Endo-1,4-beta-mannanase from blue mussel Mytilus edulis (MeMan5A) belongs to the glycoside hydrolase (GH) family 5 enzymes. The MeMan5A structure has been determined to 1.6A resolution using the multiple-wavelength anomalous dispersion method at the selenium K edge with selenomethionyl MeMan5A expressed in the yeast Pichia pastoris. As expected for GH 5 enzymes, the structure showed a (betaalpha)(8)-barrel fold. An unusually large number of histidine side-chains are exposed on the surface, which may relate to its location within the crystalline style of the digestive tract of the mussel. Kinetic analysis of MeMan5A revealed that the enzyme requires at least six subsites for efficient hydrolysis. Mannotetraose (M4) and mannopentaose (M5) were shown to interact with subsites -3 to +1, and -3 to +2, respectively. A clear kinetic threshold was observed when going from M4 to M5, indicating that the +2 subsite provides important interaction in the hydrolysis of short oligomeric mannose substrates. The catalytic centre motif at subsite -1 found in superfamily GH clan A is, as expected, conserved in MeMan5A, but the architecture of the catalytic cleft differs significantly from other GH 5 enzyme structures. We therefore suggest that MeMan5A represents a new subfamily in GH 5.
Department of Cell and Molecular Biology, Uppsala University, Biomedical Center, Box 596, SE-751 24 Uppsala, Sweden.
Anna M
AM
Larsson
Lars
L
Anderson
Bingze
B
Xu
Inés G
IG
Muñoz
Isabel
I
Usón
Jan-Christer
JC
Janson
Henrik
H
Stålbrand
Jerry
J
Ståhlberg
D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006868:Hydrolysis; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008361:Mannosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D049880:Mytilus edulis / Q000201:enzymology*; D009844:Oligosaccharides / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
D007527:Isoenzymes; D009844:Oligosaccharides; D008361:Mannosidases; C041867:endo-1,4-beta-D-mannanase