Chitinase B of "Microbulbifer degradans" 2-40 contains two catalytic domains with different chitinolytic activities

Abstract

Chitinase B of "Microbulbifer degradans" 2-40 is a modular protein that is predicted to contain two glycoside hydrolase family 18 (GH18) catalytic domains, two polyserine domains, and an acidic repeat domain. Each of the GH18 domains was shown to be catalytically active against chitin. Activity assays reveal that the amino-terminal catalytic domain (GH18(N)) releases methylumbelliferone from 4'-methylumbelliferyl-N,N'-diacetylchitobiose 13.6-fold faster than the carboxy-terminal catalytic domain (GH18(C)) and releases chitobiose from the nonreducing end of chitooligosaccharides, therefore functioning as an exochitinase. GH18(C) releases methylumbelliferone from 4'-methylumbelliferyl-N,N',N"-triacetylchitotriose 2.7-fold faster than GH18(N) and cleaves chitooligosaccharides at multiple bonds, consistent with endochitinolytic activity. Each domain was maximally active from 30 to 37 degrees C and from pH 7.2 to 8.0 and was not affected by Mg(2+), Mn(2+), Ca(2+), K(+), EDTA, EGTA, or 1.0 M NaCl. The activity of each domain was moderately inhibited by Ni(2+), Sr(2+), and Cu(2+), while Hg(2+) completely abolished activity. When the specific activities of various recombinant portions of ChiB were calculated by using native chitin as a substrate, the polypeptide containing the endo-acting domain was twofold more active on native chitin than the other containing the exo-acting domain. The presence of both domains in a single reaction increased the amount of reducing sugars released from native chitin to 140% above the theoretical combined rate, indicating that the domains function cooperatively to degrade chitin. These data demonstrate that the GH18 domains of ChiB have different activities on the same substrate and function cooperatively to enhance chitin depolymerization.

FIG. 1.

Comparison of the domain architectures of T. kodakaraensis KOD1 ChiA and “M. degradans” strain 2-40 ChiB. Gray boxes, type II secretion signal; black boxes, predicted lipobox; SSS, polyserine domains; hatched box, acidic repeat domain; crosshatched boxes, chitin binding domains. Black bars indicate the truncated portions of ChiB created for this work. GH18_N is located between amino acids 221 and 605 of ChiB. GH18_C is located between amino acids 860 and 1254.

FIG. 2.

The predicted GH18 domains of ChiB are catalytically active against chitin. The nucleotide sequence corresponding to each predicted catalytic domain and some flanking sequence (GH18_N, codons 156 to 641; GH18_C, codons 837 to 1266) was amplified by PCR, ligated into the pETBlue2 expression vector, induced by IPTG (isopropyl-β-d-thiogalactopyranoside), and purified from cell lysates on an Ni-NTA agarose column. Equal amounts of each recombinant protein (20 μg) were fractionated by electrophoresis in a glycol chitin zymogram and refolded as described in Materials and Methods. After staining with Calcofluor, zones of activity appear as dark bands against a bright background. The sizes of the active bands are in good agreement with the predicted masses of the recombinant proteins.

FIG. 3.

GH18_N and GH18_C have similar pH and temperature optima. To determine the pH optimum (top panel) and temperature optimum (bottom panel) for each catalytic domain, GH18_N (triangles) and GH18_C (squares) were purified as described in Materials and Methods and incubated with MUF-diNAG or MUF-triNAG, respectively, as described in footnote a of Table 1. The activity under reaction conditions that permitted maximum activity was assigned a value of 100%. The data are the means from three replicates, and each experiment was repeated three times with similar results. Standard errors are indicated by error bars.

FIG. 4.

GH18_N and GH18_C exhibit exo- and endochitinase activities, respectively. GH18_N and GH18_C were purified as described in Materials and Methods and incubated with GlcNAc₄ (A), GlcNAc₅ (B), and GlcNAc₆ (C). Standards and reaction products were labeled with 2-aminobenzoic acid and fractionated by electrophoresis in a 15% polyacrylamide gel (8). Labeled chitooligosaccharides were visualized by using a UV transilluminator. Standards, from bottom, are chitobiose, chitotriose, chitotetrose, and chitopentose.

FIG. 5.

Model of chitin depolymerization by ChiB. ChiB is likely to attach to the surface of the cell via a lipoprotein anchor (black box). Activity of the endochitinolytic GH18_C releases chitooligosaccharides from polymeric chitin (stippled box). Free chitooligosaccharides (circles) are then acted upon by the exochitinolytic GH18_N that processively releases chitobiose from the nonreducing end. Free chitobiose would then be taken up by the cell and metabolized. The polyserine linkers (SSS) may provide flexibility to the enzyme and optimize interaction with substrates.