Substrate binding to the inactive mutants of Streptomyces sp. N174 chitosanase: indirect evaluation from the thermal unfolding experiments

AbstractOligosaccharide binding to chitosanase from Streptomyces sp. N174 was indirectly evaluated from thermal unfolding experiments of the protein. Thermal unfolding curves were obtained by fluorescence spectroscopy in the presence of d-glucosamine oligosaccharides ((GlcN)n, n=3,4,5, and 6) using the inactive mutant chitosanase in which the catalytic residue, Glu22, is mutated to glutamine (E22Q), aspartic acid (E22D), or alanine (E22A). The midpoint temperature of the unfolding transition (Tm) of E22Q was found to be 44.4°C at pH 7.0. However, the Tm increased upon the addition of (GlcN)n by 1.3°C (n=3), 2.5°C (n=4), 5.2°C (n=5), or 7.6°C (n=6). No appreciable change in Tm was observed when (GlcNAc)6 was added to E22Q. The effect of (GlcN)n on the thermal stability was examined using the other protein, RNaseT1, but the oligosaccharide did not affect Tm of the protein. Thus, we concluded that the stabilization effect of (GlcN)n on the chitosanase results from specific binding of the oligosaccharides to the substrate binding cleft. When E22D or E22A was used instead of E22Q, the increases in Tm induced by (GlcN)6 binding were 2.7°C for E22D and 4.2°C for E22A. In E22D or E22A, interaction with (GlcN)6 seems to be partly disrupted by a conformational distortion in the catalytic cleft

Novel β-N-acetylglucosaminidases from Vibrio harveyi 650: Cloning, expression, enzymatic properties, and subsite identification

<p>Abstract</p> <p>Background</p> <p>Since chitin is a highly abundant natural biopolymer, many attempts have been made to convert this insoluble polysaccharide into commercially valuable products using chitinases and <it>β</it>-<it>N</it>-acetylglucosaminidases (GlcNAcases). We have previously reported the structure and function of chitinase A from <it>Vibrio harveyi </it>650. This study t reports the identification of two GlcNAcases from the same organism and their detailed functional characterization.</p> <p>Results</p> <p>The genes encoding two new members of family-20 GlcNAcases were isolated from the genome of <it>V. harveyi </it>650, cloned and expressed at a high level in <it>E. coli</it>. <it>Vh</it>Nag1 has a molecular mass of 89 kDa and an optimum pH of 7.5, whereas <it>Vh</it>Nag2 has a molecular mass of 73 kDa and an optimum pH of 7.0. The recombinant GlcNAcases were found to hydrolyze all the natural substrates, <it>Vh</it>Nag2 being ten-fold more active than <it>Vh</it>Nag1. Product analysis by TLC and quantitative HPLC suggested that <it>Vh</it>Nag2 degraded chitooligosaccharides in a sequential manner, its highest activity being with chitotetraose. Kinetic modeling of the enzymic reaction revealed that binding at subsites (-2) and (+4) had unfavorable (positive) binding free energy changes and that the binding pocket of <it>Vh</it>Nag2 contains four GlcNAc binding subsites, designated (-1),(+1),(+2), and (+3).</p> <p>Conclusions</p> <p>Two novel GlcNAcases were identified as exolytic enzymes that degraded chitin oligosaccharides, releasing GlcNAc as the end product. In living cells, these intracellular enzymes may work after endolytic chitinases to complete chitin degradation. The availability of the two GlcNAcases, together with the previously-reported chitinase A from the same organism, suggests that a systematic development of the chitin-degrading enzymes may provide a valuable tool in commercial chitin bioconversion.</p

Complete subsite mapping of a “loopful” GH19 chitinase from rye seeds based on its crystal structure

AbstractCrystallographic analysis of a mutated form of “loopful” GH19 chitinase from rye seeds a double mutant RSC-c, in which Glu67 and Trp72 are mutated to glutamine and alanine, respectively, (RSC-c-E67Q/W72A) in complex with chitin tetrasaccharide (GlcNAc)4 revealed that the entire substrate-binding cleft was completely occupied with the sugar residues of two (GlcNAc)4 molecules. One (GlcNAc)4 molecule bound to subsites −4 to −1, while the other bound to subsites +1 to +4. Comparisons of the main chain conformation between liganded RSC-c-E67Q/W72A and unliganded wild type RSC-c suggested domain motion essential for catalysis. This is the first report on the complete subsite mapping of GH19 chitinase

NMR Line Shape Analysis of a Multi-state Ligand Binding Mechanism in Chitosanase

Chitosan interaction with chitosanase was examined through analysis of spectral line shapes in the NMR HSQC titration experiments. We established that the substrate, chitosan hexamer, binds to the enzyme through the three-state induced-fit mechanism with fast formation of the encounter complex followed by slow isomerization of the bound-state into the final conformation. Mapping of the chemical shift perturbations in two sequential steps of the mechanism highlighted involvement of the substrate-binding subsites and the hinge region in the binding reaction. Equilibrium parameters of the three-state model agreed with the overall thermodynamic dissociation constant determined by ITC. This study presented the first kinetic evidence of the induced-fit mechanism in the glycoside hydrolases

Thermodynamic Analysis for Binding of 4-O-β-tri-N-acetylchitotriosyl Moranoline, a Transition State Analogue Inhibitor for Hen Egg White Lysozyme

4-O-β-tri-N-acetylchitotriosyl moranoline (GN3M) is a transition-state analogue for hen egg white lysozyme (HEWL) and identified as the most potent inhibitor till date. Isothermal titration calorimetry experiments provided the thermodynamic parameters for binding of GN3M to HEWL and revealed that the binding is driven by a favorable enthalpy change (ΔH° = −11.0 kcal/mol) with an entropic penalty (−TΔS° = 2.6 kcal/mol), resulting in a free energy change (ΔG°) of −8.4 kcal/mol [Ogata et al. (2013) 288, 6,072–6,082]. Dissection of the entropic term showed that a favorable solvation entropy change (−TΔSsolv° = −9.2 kcal/mol) is its sole contributor. The change in heat capacity (ΔCp°) for the binding of GN3M was determined to be −120.2 cal/K·mol. These results indicate that the bound water molecules play a crucial role in the tight interaction between GN3M and HEWL

Site-directed Mutagenesis of Evolutionary Conserved Carboxylic Amino Acids in the Chitosanase from Streptomyces sp. N174 Reveals Two Residues Essential for Catalysis

The comparison of four sequences of prokaryotic chitosanases, belonging to the family 46 of glycosyl hydrolases, revealed a conserved N-terminal module of 50 residues, including five invariant carboxylic residues. To verify if some of these residues are important for catalytic activity in the chitosanase from Streptomyces sp. N174, these 5 residues were replaced by site-directed mutagenesis. Substitutions of Glu-22 or Asp-40 with sterically conservative (E22Q, D40N) or functionally conservative (E22D, D40E) residues reduced drastically specific activity and kcat, while Km− was only slightly changed. The other residues examined, Asp-6, Glu-36, and Asp-37, retained significant activity after mutation. Circular dichroism studies of the mutant chitosanases confirmed that the observed effects are not due to changes in secondary structure. These results suggested that Glu-22 and Asp-40 are directly involved in the catalytic center of the chitosanase and the other residues are not essential for catalytic activity

A structural model for (GlcNAc)2 translocation via a periplasmic chitooligosaccharide-binding protein from marine Vibrio bacteria

VhCBP is a periplasmic chitooligosaccharide-binding protein mainly responsible for translocation of the chitooligosaccharide (GlcNAc)2 across the double membranes of marine bacteria. However, structural and thermodynamic understanding of the sugar-binding/-release processes of VhCBP is relatively less. VhCBP displayed the greatest affinity toward (GlcNAc)2, with lower affinity for longer-chain chitooligosaccharides [(GlcNAc)3–4]. (GlcNAc)4 partially occupied the closed sugar-binding groove, with two reducing-end GlcNAc units extending beyond the sugar-binding groove and barely characterized by weak electron density. Mutation of three conserved residues (Trp363, Asp365, and Trp513) to Ala resulted in drastic decreases in the binding affinity toward the preferred substrate (GlcNAc)2, indicating their significant contributions to sugar binding. The structure of the W513A–(GlcNAc)2 complex in a ‘half-open’ conformation unveiled the intermediary step of the (GlcNAc)2 translocation from the soluble CBP in the periplasm to the inner membrane–transporting components. Isothermal calorimetry data suggested that VhCBP adopts the high-affinity conformation to bind (GlcNAc)2, while its low-affinity conformation facilitated sugar release. Thus, chitooligosaccharide translocation, conferred by periplasmic VhCBP, is a crucial step in the chitin catabolic pathway, allowing Vibrio bacteria to thrive in oceans where chitin is their major source of nutrients

production of chitooligosaccharides from Rhizopus oligosporus NRRL2710 cells by chitosanase digestion

The intact cells of Rhizopus oligosporus NRRL2710, whose cell walls are abundant source of N-acetylglu- cosamine (GlcNAc) and glucosamine (GlcN), were digested with three chitinolytic enzymes, a GH-46 chitosanase from Streptomyces sp. N174 (CsnN174), a chitinase from Pyrococcus furiosus, and a chitinase from Trichoderma viride, respectively. Solubilization of the intact cells by CsnN174 was found to be the most efficient from solid state CP/MAS 13C NMR spectroscopy. Chitosanase products from Rhizopus cells were purified by cation exchange chromatography on CM-Sephadex C-25 and gel-filtration on Cellulofine Gcl-25 m. NMR and MALDI-TOF-MS analyses of the purified products revealed that GlcN–GlcNAc, (GlcN)2–GlcNAc, and (GlcN)2 were produced by the enzymatic digestion of the intact cells. The chitosan- ase digestion of Rhizopus cells was found to be an excellent system for the conversion of fungal biomass without any environmental impact

Kinetic analysis of the reaction catalyzed by chitinase A1 from Bacillus circulans WL-12 toward the novel substrates, partially N-deacetylated 4-methylumbelliferyl chitobiosides

AbstractThe kinetic behavior of chitinase A1 from Bacillus circulans WL-12 was investigated using the novel fluorogenic substrates, N-deacetylated 4-methylumbelliferyl chitobiosides [GlcN-GlcNAc-UMB (2), GlcNAc-GlcN-UMB (3), and (GlcN)2-UMB (4)], and the results were compared with those obtained using 4-methylumbelliferyl N,N′-diacetylchitobiose [(GlcNAc)2-UMB (1)] as the substrate. The chitinase did not release the UMB moiety from compound 4, but successfully released UMB from the other substrates. kcat/Km values determined from the releasing rate of the UMB moiety were: 145.3 for 1, 8.3 for 2, and 0.1 s−1 M−1 for 3. The lack of an N-acetyl group at subsite (−1) reduced the activity to a level 0.1% of that obtained with compound 1, while the absence of the N-acetyl group at subsite (−2) reduced the relative activity to 5.7%. These observations strongly support the theory that chitinase A1 catalysis occurs via a ‘substrate-assisted’ mechanism. Using these novel fluorogenic substrates, we were able to quantitatively evaluate the recognition specificity of subsite (−2) toward the N-acetyl group of the substrate sugar residue. The (−2) subsite of chitinase A1 was found to specifically recognize an N-acetylated sugar residue, but this specificity was not as strict as that found in subsite (−1)