ABSTRACT
The opportunistic pathogen Clostridium perfringens possesses the ability to colonize the protective mucin layer in the gastrointestinal tract. To assist this, the C. perfringens genome contains a battery of genes encoding glycoside hydrolases (GHs) that are likely active on mucin glycans, including four genes encoding family 84 GHs: CpGH84A (NagH), CpGH84B (NagI), CpGH84C (NagJ) and CpGH84D (NagK). To probe the potential advantage gained by the expansion of GH84 enzymes in C. perfringens, we undertook the structural and functional characterization of the CpGH84 catalytic modules. Here, we show that these four CpGH84 catalytic modules act as ß-N-acetyl-D-glucosaminidases able to hydrolyze N- and O-glycan motifs. CpGH84A and CpGH84D displayed a substrate specificity restricted to terminal ß-1,2- and ß-1,6-linked N-acetyl-D-glucosamine (GlcNAc). CpGH84B and CpGH84C appear more promiscuous with activity on terminal ß-1,2-, ß-1,3- and ß-1,6-linked GlcNAc; both possess some activity toward ß-1,4-linked GlcNAc, but this is dependent upon which monosaccharide it is linked to. Furthermore, all the CpGH84s have different optimum pHs ranging from 5.2 to 7.0. Consistent with their ß-N-acetyl-D-glucosaminidase activities, the structures of the four catalytic modules revealed similar folds with a catalytic site including a conserved -1 subsite that binds GlcNAc. However, nonconserved residues in the vicinity of the +1 subsite suggest different accommodation of the sugar preceding the terminal GlcNAc, resulting in subtly different substrate specificities. This structure-function comparison of the four GH84 catalytic modules from C. perfringens reveals their different biochemical properties, which may relate to how they are deployed in the bacterium's niche in the host.
Subject(s)
Clostridium perfringens/enzymology , Glycoside Hydrolases/chemistry , Glycoside Hydrolases/metabolism , Biocatalysis , Crystallography, X-Ray , Glycoside Hydrolases/genetics , Humans , Models, Molecular , Protein ConformationABSTRACT
Family 81 glycoside hydrolases (GHs), which are known to cleave ß-1,3-glucans, are found in archaea, bacteria, eukaryotes, and viruses. Here we examine the structural and functional features of the GH81 catalytic module, BhGH81, from the Bacillus halodurans protein BH0236 to probe the molecular basis of ß-1,3-glucan recognition and cleavage. BhGH81 displayed activity on laminarin, curdlan, and pachyman, but not scleroglucan; the enzyme also cleaved ß-1,3-glucooligosaccharides as small as ß-1,3-glucotriose. The crystal structures of BhGH81 in complex with various ß-1,3-glucooligosaccharides revealed distorted sugars in the -1 catalytic subsite and an arrangement consistent with an inverting catalytic mechanism having a proposed conformational itinerary of 2S0 â 2,5B â 5S1. Notably, the architecture of the catalytic site, location of an adjacent ancillary ß-1,3-glucan binding site, and the surface properties of the enzyme indicate the likely ability to recognize the double and/or triple-helical quaternary structures adopted by ß-1,3-glucans.
Subject(s)
Bacillus/enzymology , Glycoside Hydrolases/chemistry , Glycoside Hydrolases/metabolism , beta-Glucans/metabolism , Bacillus/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Catalytic Domain , Crystallography, X-Ray , Models, Molecular , Multigene Family , Protein Conformation , Substrate SpecificityABSTRACT
BH0236 from Bacillus halodurans is a multimodular ß-1,3-glucanase comprising an N-terminal family 81 glycoside hydrolase catalytic module, an internal family 6 carbohydrate-binding module (CBM) that binds the nonreducing end of ß-1,3-glucan chains, and an uncharacterized C-terminal module classified into CBM family 56. Here, we determined that this latter CBM, BhCBM56, bound the soluble ß-1,3-glucan laminarin with a dissociation constant (Kd ) of â¼26 µm and displayed higher affinity for insoluble ß-1,3-glucans with Kd values of â¼2-10 µm but lacked affinity for ß-1,3-glucooligosaccharides. The X-ray crystal structure of BhCBM56 and NMR-derived chemical shift mapping of the binding site revealed a ß-sandwich fold, with the face of one ß-sheet possessing the ß-1,3-glucan-binding surface. On the basis of the functional and structural properties of BhCBM56, we propose that it binds a quaternary polysaccharide structure, most likely the triple helix adopted by polymerized ß-1,3-glucans. Consistent with the BhCBM56 and BhCBM6/56 binding profiles, deletion of the CBM56 from BH0236 decreased activity of the enzyme on the insoluble ß-1,3-glucan curdlan but not on soluble laminarin; additional deletion of the CBM6 also did not affect laminarin degradation but further decreased curdlan hydrolysis. The pseudo-atomic solution structure of BH0236 determined by small-angle X-ray scattering revealed structural insights into the nature of avid binding by the BhCBM6/56 pair and how the orientation of the active site in the catalytic module factors into recognition and degradation of ß-1,3-glucans. Our findings reinforce the notion that catalytic modules and their cognate CBMs have complementary specificities, including targeting of polysaccharide quaternary structure.