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PDBsum entry 2whl
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References listed in PDB file
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Key reference
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Title
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Understanding how diverse beta-Mannanases recognize heterogeneous substrates.
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Authors
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L.E.Tailford,
V.M.Ducros,
J.E.Flint,
S.M.Roberts,
C.Morland,
D.L.Zechel,
N.Smith,
M.E.Bjørnvad,
T.V.Borchert,
K.S.Wilson,
G.J.Davies,
H.J.Gilbert.
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Ref.
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Biochemistry, 2009,
48,
7009-7018.
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PubMed id
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Abstract
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The mechanism by which polysaccharide-hydrolyzing enzymes manifest specificity
toward heterogeneous substrates, in which the sequence of sugars is variable, is
unclear. An excellent example of such heterogeneity is provided by the plant
structural polysaccharide glucomannan, which comprises a backbone of
beta-1,4-linked glucose and mannose units. beta-Mannanases, located in glycoside
hydrolase (GH) families 5 and 26, hydrolyze glucomannan by cleaving the
glycosidic bond of mannosides at the -1 subsite. The mechanism by which these
enzymes select for glucose or mannose at distal subsites, which is critical to
defining their substrate specificity on heterogeneous polymers, is currently
unclear. Here we report the biochemical properties and crystal structures of
both a GH5 mannanase and a GH26 mannanase and describe the contributions to
substrate specificity in these enzymes. The GH5 enzyme, BaMan5A, derived from
Bacillus agaradhaerens, can accommodate glucose or mannose at both its -2 and +1
subsites, while the GH26 Bacillus subtilis mannanase, BsMan26A, displays tight
specificity for mannose at its negative binding sites. The crystal structure of
BaMan5A reveals that a polar residue at the -2 subsite can make productive
contact with the substrate 2-OH group in either its axial (as in mannose) or its
equatorial (as in glucose) configuration, while other distal subsites do not
exploit the 2-OH group as a specificity determinant. Thus, BaMan5A is able to
hydrolyze glucomannan in which the sequence of glucose and mannose is highly
variable. The crystal structure of BsMan26A in light of previous studies on the
Cellvibrio japonicus GH26 mannanases CjMan26A and CjMan26C reveals that the
tighter mannose recognition at the -2 subsite is mediated by polar interactions
with the axial 2-OH group of a (4)C(1) ground state mannoside. Mutagenesis
studies showed that variants of CjMan26A, from which these polar residues had
been removed, do not distinguish between Man and Glc at the -2 subsite, while
one of these residues, Arg 361, confers the elevated activity displayed by the
enzyme against mannooligosaccharides. The biological rationale for the variable
recognition of Man- and Glc-configured sugars by beta-mannanases is discussed.
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