The modular architecture of Cellvibrio japonicus mannanases in glycoside hydrolase families 5 and 26 points to differences in their role in mannan degradation

Abstract

beta-1,4-Mannanases (mannanases), which hydrolyse mannans and glucomannans, are located in glycoside hydrolase families (GHs) 5 and 26. To investigate whether there are fundamental differences in the molecular architecture and biochemical properties of GH5 and GH26 mannanases, four genes encoding these enzymes were isolated from Cellvibrio japonicus and the encoded glycoside hydrolases were characterized. The four genes, man5A, man5B, man5C and man26B, encode the mannanases Man5A, Man5B, Man5C and Man26B, respectively. Man26B consists of an N-terminal signal peptide linked via an extended serine-rich region to a GH26 catalytic domain. Man5A, Man5B and Man5C contain GH5 catalytic domains and non-catalytic carbohydrate-binding modules (CBMs) belonging to families 2a, 5 and 10; Man5C in addition contains a module defined as X4 of unknown function. The family 10 and 2a CBMs bound to crystalline cellulose and ivory nut crystalline mannan, displaying very similar properties to the corresponding family 10 and 2a CBMs from Cellvibrio cellulases and xylanases. CBM5 bound weakly to these crystalline polysaccharides. The catalytic domains of Man5A, Man5B and Man26B hydrolysed galactomannan and glucomannan, but displayed no activity against crystalline mannan or cellulosic substrates. Although Man5C was less active against glucomannan and galactomannan than the other mannanases, it did attack crystalline ivory nut mannan. All the enzymes exhibited classic endo-activity producing a mixture of oligosaccharides during the initial phase of the reaction, although their mode of action against manno-oligosaccharides and glucomannan indicated differences in the topology of the respective substrate-binding sites. This report points to a different role for GH5 and GH26 mannanases from C. japonicus. We propose that as the GH5 enzymes contain CBMs that bind crystalline polysaccharides, these enzymes are likely to target mannans that are integral to the plant cell wall, while GH26 mannanases, which lack CBMs and rapidly release mannose from polysaccharides and oligosaccharides, target the storage polysaccharide galactomannan and manno-oligosaccharides.

X4 Modules Represent a New Family of Carbohydrate-binding Modules That Display Novel Properties

“…The plasmid pGP1, encoding Man5C-CBM35, was generated by amplifying nucleotides 589 -981 of man5C using the primers 5Ј-GCGCATATGAT-GGCAGTACCGGAAGGC-3Јand 5Ј-GCGCTCGAGGGCTGGCGAGCG-GATGGTC-3Ј, which contain NdeI and XhoI restriction sites, respectively, and the resultant DNA was cloned into similarly restricted and pET22b. Construction of pDH25 encoding the glycoside hydrolase family 5 (GH5) catalytic module of Man5C was described previously (21). The recombinant plasmid pDH26, which encodes Man5C-CBM35-GH5, was generated by amplifying the region of man5C comprising nucleotides 589 -2495 using the primers 5Ј-GCGCATATGATGGCAGTACCG-GAAGGC-3Ј and 5Ј-GCGGGATCCTTATTGCATCAGCGACCGG-3Ј, which contain NdeI and BamHI restriction sites, respectively.…”

“…The Man5C derivatives (50 nM) were incubated with 5 mg/ml ivory nut mannan or insoluble DGM in 50 mM sodium phosphate/12 mM citrate (PC) buffer, pH 6.5 at 37°C for up to 5 h in a total volume of 0.5 ml. At regular time intervals, a 40-l aliquot was removed, the enzyme was inactivated by boiling for 10 min, and mannotetraose, the primary reaction product, was quantified by HPLC following the method of Hogg et al (21). To determine the rate of mannohexaose hydrolysis, the hexasaccharide (0.6 mM) was incubated with 40 nM enzyme in PC buffer, pH 6.5 at 37°C for up to 30 min in a total volume of 0.4 ml, and the release of mannotetraose was quantified as described above.…”

“…This is exemplified by three plant cell wall-degrading enzymes from Cellvibrio japonicus, Xyn10B (xylanase), Abf62A (arabinofuranosidase), and Est1A (acetyl xylan esterase) that contain an identical ϳ150-amino acid module, termed X4, whose role in enzyme function is unclear (19,20). This X4 module is also present in a mannanase (Man5C) from the same organism (21). To understand the mechanism by which glycoside hydrolases attack the complex composite structure that comprises the * The costs of publication of this article were defrayed in part by the payment of page charges.…”

The Biochemistry and Structural Biology of Plant Cell Wall Deconstruction

“…In general, the ligand specificity of CBMs reflects the substrate cleaved by the cognate enzyme (discussed further below). Many of these enzymes, however, also contain a CBM that binds to crystalline cellulose (Kellett et al, 1990;McKie et al, 2001;Hogg et al, 2003). It has been suggested that once bound, these type A modules are able to slide across the surface of cellulose (Jervis et al, 1997), enabling the substrate-specific type B and type C CBMs to lock onto its ligand and thus direct the enzyme to its target glycosidic bonds (Kellett et al, 1990).…”

“…Thus, CBMs acting in trans have only a minor influence on the access problem, although this might reflect the dissociation of the targeting and (possible) disrupting function of these modules. Of potential significance is the location of crystalline cellulose-specific CBMs in many enzymes that display no cellulase activity (Kellett et al, 1990;McKie et al, 2001;Hogg et al, 2003;Vincent et al, 2010), which argues against these modules having a specialized function in cellulose degradation. A more likely explanation for the capacity of CBMs to increase the activity of glycoside hydrolases against insoluble substrates is that they reduce the "accessibility problem" by bringing the appended catalytic modules into intimate and prolonged association with their target substrate, thereby enhancing catalytic efficiency.…”