Structural insights into the substrate-binding mechanism for a novel chitosanase

Abstract

Chitosanase is able to specifically cleave β-1,4-glycosidic bond linkages in chitosan to produce a chito-oligomer product, which has found a variety of applications in many areas, including functional food and cancer therapy. Although several structures for chitosanase have been determined, the substrate-binding mechanism for this enzyme has not been fully elucidated because of the lack of a high-resolution structure of the chitosanase-substrate complex. In the present study we show the crystal structure of a novel chitosanase OU01 from Microbacterium sp. in complex with its substrate hexa-glucosamine (GlcN)6, which belongs to the GH46 (glycoside hydrolyase 46) family in the Carbohydrate Active Enzymes database (http://www.cazy.org/). This structure allows precise determination of the substrate-binding mechanism for the first time. The chitosanase-(GlcN)6 complex structure demonstrates that, from the -2 to +1 position of the (GlcN)6 substrate, the pyranose rings form extensive interactions with the chitosanase-binding cleft. Several residues (Ser27, Tyr37, Arg45, Thr58, Asp60, His203 and Asp235) in the binding cleft are found to form important interactions required to bind the substrate. Site-directed mutagenesis of these residues showed that mutations of Y37F and H203A abolish catalytic activity. In contrast, the mutations T58A and D235A only lead to a moderate loss of catalytic activity, whereas the S27A mutation retains ~80% of the enzymatic activity. In combination with previous mutagenesis studies, these results suggest that the -2, -1 and +1 subsites play a dominant role in substrate binding and catalysis. DSF (differential scanning fluorimetry) assays confirmed that these mutations had no significant effect on protein stability. Taken together, we present the first mechanistic interpretation for the substrate (GlcN)6 binding to chitosanase, which is critical for the design of novel chitosanase used for biomass conversion.

Characterization of a New Chitosanase from a Marine Bacillus sp. and the Anti-Oxidant Activity of Its Hydrolysate

“…N106 [GenBank: AAA63405], formed a bigger cluster, whereas the families GH75 and GH80 members formed other two clusters. As shown by multiple sequence alignment (Figure 2), the conserved region labeled by triangles in CsnQ was related to the substrate binding and catalytic activities of the family GH46 [20][21][22][23][24]. These results indicated that CsnQ was a new member of the family GH46.…”

Chitosanases from Family 46 of Glycoside Hydrolases: From Proteins to Phenotypes

“…This is due to a relatively large proportion of glutamate and aspartate among substrate binding residues ( Table 1 B), most of which interact with the amino groups of chitosan substrate. This abundance of acidic residues in the substrate binding cleft is thought to be responsible for the high (even if not absolute) specificity of GH46 enzymes as chitosanases and their poor recognition of chitinous, highly N -acetylated substrates [ 32 , 33 ].…”

“…(referred as Pseudomonas sp. LL2 in Protein Data Bank) [ 33 ]. Chitosanases from Streptomyces coelicolor A3(2) (named ScCsn46A or CsnA or SCO0677) [ 34 , 35 ], Pseudomonas sp.…”

Crystal structure of a family 80 chitosanase from Mitsuaria chitosanitabida

“…GH‐I clan enzymes share an invariant core comprising the surrounding region of one β‐sheet consisting of β1, β2 and β3 in the upper domain, and the two helices (α1 and α8) that form the base of subsites in the lower domain. In the OU01 enzyme, Ser27 (corresponding to Gly43 in McChoA), Tyr37 (Tyr55), Arg45 (Arg70), Thr58 (Gly83), Asp60 (Asp84), His203 (no equivalent in McChoA) and Asp235 (no equivalent in McChoA) form the −2, −1 and +1 subsites that are essential for substrate binding and catalysis . Superposition of the structures indicated that Arg70 and Asp84 of McChoA are located in the −2 subsite (Fig.…”

“…However, the Cys297Gly mutant did exhibit slight but significant catalytic activity, and the possibility that other disulphides may form cannot be excluded at this stage . Although the −3 subsite is not considered to be essential for chitosan hydrolysis in GH46 , the role of the C‐terminal residue in GH80 should be investigated by mutagenesis and enzymatic assays.…”