Understanding How Diverse β-Mannanases Recognize Heterogeneous Substrates

Abstract

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.

A Novel Glycoside Hydrolase Family 113 Endo-β-1,4-Mannanase from Alicyclobacillus sp. Strain A4 and Insight into the Substrate Recognition and Catalytic Mechanism of This Family

“…Another finding is that Man113A also had greater capacity to degrade glucomannan (konjac flour, 370.4 U/mg) than galactomannan (locust bean gum, 298.8 U/mg). This property is similar to that of BaMan5A from Bacillus agaradhaerens, with a higher k cat /K m value on konjac flour (2.6 ϫ 10 4 /min/M) than on locust bean gum (1.1 ϫ 10 4 /min/M) (22). The polar residue Asn127 of BaMan5A at the ϩ2 subsite may contact the 2-OH group of the substrate in either an axial (as in mannose) or an equatorial (as in glucose) configuration.…”

Tracing Determinants of Dual Substrate Specificity in Glycoside Hydrolase Family 5

“…Mannan and glucan sugars differ in the configuration of the hydroxyl group at the C2 sugar, with mannan units having an axial configuration and glucan units having an equatorial configuration (supplemental Fig. S2) (13). With mannan present in the active site, Asn-20 forms two hydrogen bonds with the axial OH-C2 group at Ϫ2 subsite (Fig.…”

The Biochemistry and Structural Biology of Plant Cell Wall Deconstruction

“…Recognition of Man and Glc at subsites distal to 21 is highly variable, although some general trends are emerging that point to a divergence in specificity between GH5 and GH26 mannanases. GH5 mannanases are able to accommodate Glc at the 22 and +1 subsites (Tailford et al, 2009) and are thus able to hydrolyze mannosidic linkages flanked by Man or Glc. Indeed, one of these enzymes, BaMan5A, does not recognize O2 as a specificity determinant at any subsite distal to 21.…”

“…Indeed, one of these enzymes, BaMan5A, does not recognize O2 as a specificity determinant at any subsite distal to 21. Thus, while BaMan5A hydrolyzes only mannosidic bonds, the topographical features of the substrate-binding cleft of this enzyme are optimized to utilize glucomannan as its preferred substrate (Tailford et al, 2009). The relaxed specificity for Glc or Man, apart from the critical 21 subsite, is a feature shared with the other GH5 mannanases, where structural information is available.…”