Understanding How Diverse β-Mannanases Recognize Heterogeneous Substrates

摘要

The mechanism by which polysaccharide-hydrolyzing enzymes manifest specificity towardnheterogeneous substrates, in which the sequence of sugars is variable, is unclear. An excellent example ofnsuch heterogeneity is provided by the plant structural polysaccharide glucomannan, which comprises anbackbone of β-1,4-linked glucose and mannose units. β-Mannanases, located in glycoside hydrolase (GH)nfamilies 5 and 26, hydrolyze glucomannan by cleaving the glycosidic bond of mannosides at the -1 subsite.nThe mechanism by which these enzymes select for glucose or mannose at distal subsites, which is critical tondefining their substrate specificity on heterogeneous polymers, is currently unclear. Here we report thenbiochemical properties and crystal structures of both a GH5 mannanase and a GH26 mannanase and describenthe contributions to substrate specificity in these enzymes. The GH5 enzyme, BaMan5A, derived fromnBacillus agaradhaerens, can accommodate glucose or mannose at both its-2 and+1subsites, while the GH26nBacillus subtilis mannanase, BsMan26A, displays tight specificity for mannose at its negative binding sites.nThe crystal structure of BaMan5A reveals that a polar residue at the -2 subsite can make productive contactnwith the substrate 2-OH group in either its axial (as in mannose) or its equatorial (as in glucose) configuration,nwhile other distal subsites do not exploit the 2-OH group as a specificity determinant. Thus, BaMan5A is ablento hydrolyze glucomannan in which the sequence of glucose and mannose is highly variable. The crystalnstructure of BsMan26A in light of previous studies on the Cellvibrio japonicus GH26 mannanases CjMan26Anand CjMan26C reveals that the tighter mannose recognition at the -2 subsite is mediated by polarninteractions with the axial 2-OH group of a 4C1 ground state mannoside. Mutagenesis studies showed thatnvariants of CjMan26A, from which these polar residues had been removed, do not distinguish between Mannand Glc at the -2 subsite, while one of these residues, Arg 361, confers the elevated activity displayed by thenenzyme against mannooligosaccharides. The biological rationale for the variable recognition of Man- andnGlc-configured sugars by β-mannanases is discussed.