Wipa Suginta

Crystal structures of Vibrio harveyi chitinase A complexed with chitooligosaccharides: implications for the catalytic mechanism

This research describes four X-ray structures of Vibrio harveyi chitinase A and its catalytically inactive mutant (E315M) in the presence and absence of substrates. The overall structure of chitinase A is that of a typical family-18 glycosyl hydrolase comprising three distinct domains: (i) the amino-terminal chitin-binding domain; (ii) the main catalytic (alpha/beta)(8) TIM-barrel domain; and (iii) the small (alpha+beta) insertion domain. The catalytic cleft of chitinase A has a long, deep groove, which contains six chitooligosaccharide ring-binding subsites (-4)(-3)(-2)(-1)(+1)(+2). The binding cleft of the ligand-free E315M is partially blocked by the C-terminal (His)(6)-tag. Structures of E315M-chitooligosaccharide complexes display a linear conformation of pentaNAG, but a bent conformation of hexaNAG. Analysis of the final 2F(o)-F(c) omit map of E315M-NAG6 reveals the existence of the linear conformation of the hexaNAG at a lower occupancy with respect to the bent conformation. These crystallographic data provide evidence that the interacting sugars undergo conformational changes prior to hydrolysis by the wild-type enzyme.

Enzymatic properties of wild-type and active site mutants of chitinase A from Vibrio carchariae, as revealed by HPLC-MS

The enzymatic properties of chitinase A from Vibrio carchariae have been studied in detail by using combined HPLC and electrospray MS. This approach allowed the separation of α and β anomers and the simultaneous monitoring of chitooligosaccharide products down to picomole levels. Chitinase A primarily generated β‐anomeric products, indicating that it catalyzed hydrolysis through a retaining mechanism. The enzyme exhibited endo characteristics, requiring a minimum of two glycosidic bonds for hydrolysis. The kinetics of hydrolysis revealed that chitinase A had greater affinity towards higher Mr chitooligomers, in the order of (GlcNAc)6 > (GlcNAc)4 > (GlcNAc)3, and showed no activity towards (GlcNAc)2 and pNP‐GlcNAc. This suggested that the binding site of chitinase A was probably composed of an array of six binding subsites. Point mutations were introduced into two active site residues – Glu315 and Asp392 – by site‐directed mutagenesis. The D392N mutant retained significant chitinase activity in the gel activity assay and showed ≈ 20% residual activity towards chitooligosaccharides and colloidal chitin in HPLC‐MS measurements. The complete loss of substrate utilization with the E315M and E315Q mutants suggested that Glu315 is an essential residue in enzyme catalysis. The recombinant wild‐type enzyme acted on chitooligosaccharides, releasing higher quantities of small oligomers, while the D392N mutant favored the formation of transient intermediates. Under standard hydrolytic conditions, all chitinases also exhibited transglycosylation activity towards chitooligosaccharides and pNP‐glycosides, yielding picomole quantities of synthesized chitooligomers. The D392N mutant displayed strikingly greater efficiency in oligosaccharide synthesis than the wild‐type enzyme.

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

BackgroundSince chitin is a highly abundant natural biopolymer, many attempts have been made to convert this insoluble polysaccharide into commercially valuable products using chitinases and β-N-acetylglucosaminidases (GlcNAcases). We have previously reported the structure and function of chitinase A from Vibrio harveyi 650. This study t reports the identification of two GlcNAcases from the same organism and their detailed functional characterization.ResultsThe genes encoding two new members of family-20 GlcNAcases were isolated from the genome of V. harveyi 650, cloned and expressed at a high level in E. coli. Vh Nag1 has a molecular mass of 89 kDa and an optimum pH of 7.5, whereas Vh 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, Vh Nag2 being ten-fold more active than Vh Nag1. Product analysis by TLC and quantitative HPLC suggested that Vh 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 Vh Nag2 contains four GlcNAc binding subsites, designated (-1),(+1),(+2), and (+3).ConclusionsTwo 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.

Chitoporin from Vibrio harveyi: a channel with exceptional sugar specificity

Background: Vibrio harveyi chitoporin (VhChiP) was recently identified as a pore-forming channel responsible for chitooligosaccharide uptake through the outer membrane of V. harveyi. Results: Kinetic analysis revealed that VhChiP was several orders of magnitude more active than other known sugar-specific porins. Conclusion: VhChiP is a channel with exceptional sugar specificity. Significance: The high activity of VhChiP reflects an evolutionary adaptation required for V. harveyi to thrive under extreme aquatic conditions. Chitoporin (VhChiP) is a sugar-specific channel responsible for the transport of chitooligosaccharides through the outer membrane of the marine bacterium Vibrio harveyi. Single channel reconstitution into black lipid membrane allowed single chitosugar binding events in the channel to be resolved. VhChiP has an exceptionally high substrate affinity, with a binding constant of K = 5.0 × 106 m−1 for its best substrate (chitohexaose). The on-rates of chitosugars depend on applied voltages, as well as the side of the sugar addition, clearly indicating the inherent asymmetry of the VhChiP lumen. The binding affinity of VhChiP for chitohexaose is 1–5 orders of magnitude larger than that of other known sugar-specific porins for their preferred substrates. Thus, VhChiP is the most potent sugar-specific channel reported to date, with its high efficiency presumably reflecting the need for the bacterium to take up chitin-containing nutrients promptly under turbulent aquatic conditions to exploit them efficiently as its sole source of energy.

Expression and refolding of Omp38 from Burkholderia pseudomallei and Burkholderia thailandensis, and its function as a diffusion porin

In the present paper, we describe cloning and expression of two outer membrane proteins, BpsOmp38 (from Burkholderia pseudomallei) and BthOmp38 (from Burkholderia thailandensis) lacking signal peptide sequences, using the pET23d(+) expression vector and Escherichia coli host strain Origami(DE3). The 38 kDa proteins, expressed as insoluble inclusion bodies, were purified, solubilized in 8 M urea, and then subjected to refolding experiments. As seen on SDS/PAGE, the 38 kDa band completely migrated to approximately 110 kDa when the purified monomeric proteins were refolded in a buffer system containing 10% (w/v) Zwittergent 3-14, together with a subsequent heating to 95 degrees C for 5 min. CD spectroscopy revealed that the 110 kDa proteins contained a predominant beta-sheet structure, which corresponded completely to the structure of the Omp38 proteins isolated from B. pseudomallei and B. thailandensis. Immunoblot analysis using anti-BpsOmp38 polyclonal antibodies and peptide mass analysis by MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS confirmed that the expressed proteins were BpsOmp38 and BthOmp38. The anti-BpsOmp38 antibodies considerably exhibited the inhibitory effects on the permeation of small sugars through the Omp38-reconstituted liposomes. A linear relation between relative permeability rates and M(r) of neutral sugars and charged antibiotics suggested strongly that the in vitro re-assembled Omp38 functioned fully as a diffusion porin.

Molecular Uptake of Chitooligosaccharides through Chitoporin from the Marine Bacterium Vibrio harveyi

Background Chitin is the most abundant biopolymer in marine ecosystems. However, there is no accumulation of chitin in the ocean-floor sediments, since marine bacteria Vibrios are mainly responsible for a rapid turnover of chitin biomaterials. The catabolic pathway of chitin by Vibrios is a multi-step process that involves chitin attachment and degradation, followed by chitooligosaccharide uptake across the bacterial membranes, and catabolism of the transport products to fructose-6-phosphate, acetate and NH3. Principal Findings This study reports the isolation of the gene corresponding to an outer membrane chitoporin from the genome of Vibrio harveyi. This porin, expressed in E. coli, (so called VhChiP) was found to be a SDS-resistant, heat-sensitive trimer. Immunoblotting using anti-ChiP polyclonal antibody confirmed the expression of the recombinant ChiP, as well as endogenous expression of the native protein in the V. harveyi cells. The specific function of VhChiP was investigated using planar lipid membrane reconstitution technique. VhChiP nicely inserted into artificial membranes and formed stable, trimeric channels with average single conductance of 1.8±0.13 nS. Single channel recordings at microsecond-time resolution resolved translocation of chitooligosaccharides, with the greatest rate being observed for chitohexaose. Liposome swelling assays showed no permeation of other oligosaccharides, including maltose, sucrose, maltopentaose, maltohexaose and raffinose, indicating that VhChiP is a highly-specific channel for chitooligosaccharides. Conclusion/Significance We provide the first evidence that chitoporin from V. harveyi is a chitooligosaccharide specific channel. The results obtained from this study help to establish the fundamental role of VhChiP in the chitin catabolic cascade as the molecular gateway that Vibrios employ for chitooligosaccharide uptake for energy production.

The effects of the surface-exposed residues on the binding and hydrolytic activities of Vibrio carchariae chitinase A

BackgroundVibrio carchariae chitinase A (EC3.2.1.14) is a family-18 glycosyl hydrolase and comprises three distinct structural domains: i) the amino terminal chitin binding domain (ChBD); ii) the (α/β)8 TIM barrel catalytic domain (CatD); and iii) the α + β insertion domain. The predicted tertiary structure of V. carchariae chitinase A has located the residues Ser33 & Trp70 at the end of ChBD and Trp231 & Tyr245 at the exterior of the catalytic cleft. These residues are surface-exposed and presumably play an important role in chitin hydrolysis.ResultsPoint mutations of the target residues of V. carchariae chitinase A were generated by site-directed mutagenesis. With respect to their binding activity towards crystalline α-chitin and colloidal chitin, chitin binding assays demonstrated a considerable decrease for mutants W70A and Y245W, and a notable increase for S33W and W231A. When the specific hydrolyzing activity was determined, mutant W231A displayed reduced hydrolytic activity, whilst Y245W showed enhanced activity. This suggested that an alteration in the hydrolytic activity was not correlated with a change in the ability of the enzyme to bind to chitin polymer. A mutation of Trp70 to Ala caused the most severe loss in both the binding and hydrolytic activities, which suggested that it is essential for crystalline chitin binding and hydrolysis. Mutations varied neither the specific hydrolyzing activity against p NP-[GlcNAc]2, nor the catalytic efficiency against chitohexaose, implying that the mutated residues are not important in oligosaccharide hydrolysis.ConclusionOur data provide direct evidence that the binding as well as hydrolytic activities of V. carchariae chitinase A to insoluble chitin are greatly influenced by Trp70 and less influenced by Ser33. Though Trp231 and Tyr245 are involved in chitin hydrolysis, they do not play a major role in the binding process of crystalline chitin and the guidance of the chitin chain into the substrate binding cleft of the enzyme.

Substrate binding modes and anomer selectivity of chitinase A from Vibrio harveyi

High-performance liquid chromatography mass spectrometry (HPLC MS) was employed to assess the binding behaviors of various substrates to Vibrio harveyi chitinase A. Quantitative analysis revealed that hexaNAG preferred subsites −2 to +2 over subsites −3 to +2 and pentaNAG only required subsites −2 to +2, while subsites −4 to +2 were not used at all by both substrates. The results suggested that binding of the chitooligosaccharides to the enzyme essentially occurred in compulsory fashion. The symmetrical binding mode (−2 to +2) was favored presumably to allow the natural form of sugars to be utilized effectively. Crystalline α chitin was initially hydrolyzed into a diverse ensemble of chitin oligomers, providing a clear sign of random attacks that took place within chitin chains. However, the progressive degradation was shown to occur in greater extent at later time to complete hydrolysis. The effect of the reducing-end residues were also investigated by means of HPLC MS. Substitutions of Trp275 to Gly and Trp397 to Phe significantly shifted the anomer selectivity of the enzyme toward β substrates. The Trp275 mutation modulated the kinetic property of the enzyme by decreasing the catalytic constant (kcat) and the substrate specificity (kcat/Km) toward all substrates by five- to tenfold. In contrast, the Trp397 mutation weakened the binding strength at subsite (+2), thereby speeding up the rate of the enzymatic cleavage toward soluble substrates but slowing down the rate of the progressive degradation toward insoluble chitin.

Structural and Thermodynamic Insights into Chitooligosaccharide Binding to Human Cartilage Chitinase 3-like Protein 2 (CHI3L2 or YKL-39).

Background: Human YKL-39 is currently recognized as a biomarker for osteoarthritis. Results: Crystal structures of YKL-39 reveal that chitooligosaccharide induces local conformational changes to stabilize sugar·protein complexes and that the protein contains five binding subsites for sugars. Conclusion: YKL-39 binds to chitooligosaccharide through enthalpic reactions. Significance: Our findings suggest how YKL-39 interacts with GlcNAc moieties of the natural ligands, which may possibly activate local tissue inflammation. Four crystal structures of human YKL-39 were solved in the absence and presence of chitooligosaccharides. The structure of YKL-39 comprises a major (β/α)8 triose-phosphate isomerase barrel domain and a small α + β insertion domain. Structural analysis demonstrates that YKL-39 interacts with chitooligosaccharides through hydrogen bonds and hydrophobic interactions. The binding of chitin fragments induces local conformational changes that facilitate tight binding. Compared with other GH-18 members, YKL-39 has the least extended chitin-binding cleft, containing five subsites for sugars, namely (−3)(−2)(−1)(+1)(+2), with Trp-360 playing a prominent role in the sugar-protein interactions at the center of the chitin-binding cleft. Evaluation of binding affinities obtained from isothermal titration calorimetry and intrinsic fluorescence spectroscopy suggests that YKL-39 binds to chitooligosaccharides with Kd values in the micromolar concentration range and that the binding energies increase with the chain length. There were no significant differences between the Kd values of chitopentaose and chitohexaose, supporting the structural evidence for the five binding subsite topology. Thermodynamic analysis indicates that binding of chitooligosaccharide to YKL-39 is mainly driven by enthalpy.

Porin involvement in cephalosporin and carbapenem resistance of Burkholderia pseudomallei.

Background Burkholderia pseudomallei (Bps) is a Gram-negative bacterium that causes frequently lethal melioidosis, with a particularly high prevalence in the north and northeast of Thailand. Bps is highly resistant to many antimicrobial agents and this resistance may result from the low drug permeability of outer membrane proteins, known as porins. Principal Findings Microbiological assays showed that the clinical Bps strain was resistant to most antimicrobial agents and sensitive only to ceftazidime and meropenem. An E. coli strain defective in most porins, but expressing BpsOmp38, exhibited considerably lower antimicrobial susceptibility than the control strain. In addition, mutation of Tyr119, the most prominent pore-lining residue in BpsOmp38, markedly altered membrane permeability, substitution with Ala (mutant BpsOmp38Y119A) enhanced uptake of the antimicrobial agents, while substitution with Phe (mutant BpsOmp38Y119F) inhibited uptake. Channel recordings of BpsOmp38 reconstituted in a planar black lipid membrane (BLM) suggested that the higher permeability of BpsOmp38Y119A was caused by widening of the pore interior through removal of the bulky side chain. In contrast, the lower permeability of BpsOmp38Y119F was caused by introduction of the hydrophobic side chain (Phe), increasing the ‘greasiness’ of the pore lumen. Significantly, liposome swelling assays showed no permeation through the BpsOmp38 channel by antimicrobial agents to which Bps is resistant (cefoxitin, cefepime, and doripenem). In contrast, high permeability to ceftazidime and meropenem was observed, these being agents to which Bps is sensitive. Conclusion/Significance Our results, from both in vivo and in vitro studies, demonstrate that membrane permeability associated with BpsOmp38 expression correlates well with the antimicrobial susceptibility of the virulent bacterium B. pseudomallei, especially to carbapenems and cephalosporins. In addition, substitution of the residue Tyr119 affects the permeability of the BpsOmp38 channel to neutral sugars and antimicrobial agents.