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Periplasmic solute-binding proteins in bacteria are involved in the active transport of nutrients into the cytoplasm. In marine bacteria of the genus Vibrio, a chitooligosaccharide-binding protein (CBP) is thought to be the major solute-binding protein controlling the rate of chitin uptake in these bacteria. However, the molecular mechanism of the CBP involvement in chitin metabolism has not been elucidated. Here, we report the structure and function of a recombinant chitooligosaccharide-binding protein from Vibrio harveyi, namely VhCBP, expressed in Escherichia coli. Isothermal titration calorimetry revealed that VhCBP strongly binds shorter chitooligosaccharides ((GlcNAc)(n), where n = 2, 3, and 4) with affinities that are considerably greater than those for glycoside hydrolase family 18 and 19 chitinases but does not bind longer ones, including insoluble chitin polysaccharides. We also found that VhCBP comprises two domains with flexible linkers and that the domain–domain interface forms the sugar-binding cleft, which is not long extended but forms a small cavity. (GlcNAc)(2) bound to this cavity, apparently triggering a closed conformation of VhCBP. Trp-363 and Trp-513, which stack against the two individual GlcNAc rings, likely make a major contribution to the high affinity of VhCBP for (GlcNAc)(2). The strong chitobiose binding, followed by the conformational change of VhCBP, may facilitate its interaction with an active-transport system in the inner membrane of Vibrio species.

Enhancing the transglycosylation (TG) activity of glycoside hydrolases does not always result in the production of oligosaccharides with longer chains, because the TG products are often decomposed into shorter oligosaccharides. Here, we investigated the mutation strategies for obtaining chitooligosaccharides with longer chains by means of TG reaction catalyzed by family GH18 chitinase A from Vibrio harveyi (VhChiA). HPLC analysis of the TG products from incubation of chitooligosaccharide substrates, GlcNAcn, with several mutant VhChiAs suggested that mutant W570G (mutation of Trp570 to Gly) and mutant D392N (mutation of Asp392 to Asn) significantly enhanced TG activity, but the TG products were immediately hydrolyzed into shorter GlcNAcn. On the other hand, the TG products obtained from mutants D313A and D313N (mutations of Asp313 to Ala and Asn, respectively) were not further hydrolyzed, leading to the accumulation of oligosaccharides with longer chains. The data obtained from the mutant VhChiAs suggested that mutations of Asp313, the middle aspartic acid residue of the DxDxE catalytic motif, to Ala and Asn are most effective for obtaining chitooligosaccharides with longer chains.

Vibrio harveyi chitinase A (VhChiA) is a GH-18 glycosyl hydrolase with a structure containing three distinct domains: i) the N-terminal chitin-binding domain; ii) the (α/β)8 TIM barrel catalytic domain; and iii) the α+β insertion domain. In this study, we cloned the gene fragment encoding the chitin-binding domain of VhChiA, termed ChBDVhChiA. The recombinant ChBDVhChiA was heterologously expressed in E. coli BL21 strain Tuner(DE3)pLacI host cells, and purified to homogeneity. CD measurements suggested that ChBDVhChiA contained β-sheets as major structural components and fluorescence spectroscopy showed that the protein domain was folded correctly, and suitable for functional characterization. Chitin binding assays showed that ChBDVhChiA bound to both α- and β-chitins, with the greatest affinity for β-colloidal chitin, but barely bound to polymeric chitosan. These results identified the tandem N-acetamido functionality on chitin chains as the specific sites of enzyme-substrate interactions. The binding affinity of the isolated domain was significantly lower than that of intact VhChiA, suggesting that the catalytic domain works synergistically with the chitin-binding domain to guide the polymeric substrate into the substrate binding cleft. These data confirm the physiological role of the chitin-binding domain of the marine bacterial GH-18 chitinase A in chitin-chitinase interactions.

Three acidic residues in the DXDXE sequence motif are suggested to play a concerted role in the catalysis of Vibrio harveyi ChiA. An increase in the optimum pH of 0.8 units in mutant D313A/N indicates that Asp313 influences the pKa of the ionizing groups around the cleavage site. D313A showed greatly reduced kcat/Km and increased KD, suggesting that Asp313 participates in catalysis and ligand binding. Investigation of the enzyme-substrate interactions of V. harveyi ChiA and Serratia marcescens ChiB revealed two conformations of Asp313 and (-1)GlcNAc. The first conformation, likely to be the initial conformation, showed that the β-COOH of Asp313 only interacted with the -C=O of the N-acetyl group in the distorted sugar. The second conformation, formed from the first by concerted bond rotations, demonstrated hydrogen bonds between the Asp313 side chain and the -NH of the N-acetyl group and the γ-COOH of Glu315. Here we propose a further refinement of the catalytic cycle of chitin hydrolysis by family-18 chitinases that involves four steps: Step 1: Pre-priming. An acidic pair is formed between Asp311 and Asp313. Step 2: Substrate binding. The Asp313 side chain detaches from Asp311 and rotates to form a H-bond with the C=O of the 2-acetamido group of -1GlcNAc. Step 3: Bond cleavage. The side chain of Asp313 and the 2-acetamido group simultaneously rotate, permitting Asp313 to interact with the side chain of Glu315 and facilitating bond cleavage. Step 4: Formation of reaction intermediate. The transient (-1) C1-GlcNAc cation readily reacts with the 2-acetamido group, forming an oxazolinium ion intermediate. Further attack by a neighboring water results in retention of β-configuration of the degradation products.

Vibrio harveyi chitinase A or VhChiA (EC.3.2.1.14) is a member of GH-18 chitinases that catalyzes chitin degradation from marine biomaterials. Our earlier structural data of VhChiA suggested that Tyr-435 marks the ending of subsite +2 and may influence binding of the interacting substrate at the aglycone binding sites. This study reports the effects of Tyr-435 using site-directed mutagenesis technique. Mutation of Tyr-435 to Ala (mutant Y435A) enhanced both binding and catalytic efficiency of VhChiA, whereas substitution of Tyr-435 to Trp (mutant Y435W) lessened the ability of the enzyme to bind and hydrolyze chitin substrates. The increased activity of Y435A can be explained by partial removal of a steric clash around subsite (+2), thereby allowing a chitin chain to move beyond or to access the enzyme's active site from the aglycone side more straightforwardly.