Inhibition of Mycobacterium tuberculosis InhA by 3-nitropropanoic acid.

3-Nitropropanoic acid (3NP), a bioactive fungal natural product, was previously demonstrated to inhibit growth of Mycobacterium tuberculosis. Here we demonstrate that 3NP inhibits the 2-trans-enoyl-acyl carrier protein reductase (InhA) from Mycobacterium tuberculosis with an IC50 value of 71 µM, and present the crystal structure of the ternary InhA-NAD+ -3NP complex. The complex contains the InhA substrate-binding loop in an ordered, open conformation with Tyr158, a catalytically important residue whose orientation defines different InhA substrate/inhibitor complex conformations, in the "out" position. 3NP occupies a hydrophobic binding site adjacent to the NAD+ cofactor and close to that utilized by the diphenyl ether triclosan, but binds predominantly via electrostatic and water-mediated hydrogen-bonding interactions with the protein backbone and NAD+ cofactor. The identified mode of 3NP binding provides opportunities to improve inhibitory activity toward InhA.

Enzymatic electrochemical biosensor for glyphosate detection based on acid phosphatase inhibition.

A novel enzymatic electrochemical biosensor was fabricated for the indirect detection of glyphosate-based acid phosphatase inhibition. The biosensor was constructed on a screen-printed carbon electrode modified with silver nanoparticles, decorated with electrochemically reduced graphene oxide, and chemically immobilized with acid phosphatase via glutaraldehyde cross-linking. We measured the oxidation current by chronoamperometry. The current arose from the enzymatic reaction of acid phosphatase and the enzyme-substrate disodium phenyl phosphate. The biosensing response is a decrease in signal resulting from inhibition of acid phosphatase in the presence of glyphosate inhibitor. The inhibition of acid phosphatase by glyphosate was investigated as a reversible competitive-type reaction based on the Lineweaver-Burk equation. Computational docking confirmed that glyphosate was the inhibitor bound in the substrate-binding pocket of acid phosphatase and that it was able to inhibit the enzyme efficiently. Additionally, the established method was applied to the selective analysis of glyphosate in actual samples with satisfactory results following a standard method.

Discovery of New and Potent InhA Inhibitors as Antituberculosis Agents: Structure-Based Virtual Screening Validated by Biological Assays and X-ray Crystallography.

The enoyl-acyl carrier protein reductase InhA of Mycobacterium tuberculosis is an attractive, validated target for antituberculosis drug development. Moreover, direct inhibitors of InhA remain effective against InhA variants with mutations associated with isoniazid resistance, offering the potential for activity against MDR isolates. Here, structure-based virtual screening supported by biological assays was applied to identify novel InhA inhibitors as potential antituberculosis agents. High-speed Glide SP docking was initially performed against two conformations of InhA differing in the orientation of the active site Tyr158. The resulting hits were filtered for drug-likeness based on Lipinski's rule and avoidance of PAINS-like properties and finally subjected to Glide XP docking to improve accuracy. Sixteen compounds were identified and selected for in vitro biological assays, of which two (compounds 1 and 7) showed MIC of 12.5 and 25 µg/mL against M. tuberculosis H37Rv, respectively. Inhibition assays against purified recombinant InhA determined IC50 values for these compounds of 0.38 and 0.22 µM, respectively. A crystal structure of the most potent compound, compound 7, bound to InhA revealed the inhibitor to occupy a hydrophobic pocket implicated in binding the aliphatic portions of InhA substrates but distant from the NADH cofactor, i.e., in a site distinct from those occupied by the great majority of known InhA inhibitors. This compound provides an attractive starting template for ligand optimization aimed at discovery of new and effective compounds against M. tuberculosis that act by targeting InhA.

Characterization of the binding of a glycosylated serine protease from Euphorbia cf. lactea latex to human fibrinogen.

In this study, the binding of a glycosylated serine protease (EuP-82) with human fibrinogen was investigated by isothermal titration calorimetry (ITC). ITC analysis indicated that the binding of EuP-82 to fibrinogen in the conditions with or without the activator (Ca2+ ) was an exothermic reaction (dominant negative enthalpy), which tended to be driven by hydrogen bonding and van der Waals interactions. In contrast, the binding of fibrinogen-EuP-82 in the condition with the inhibitor (Zn2+ ) was an unfavorable endothermic reaction. EuP-82 could not inhibit the platelet activity in citrated whole blood via the ADP-receptor pathways (mainly, P2Y1 and P2Y12), but it could enhance the platelet aggregation. The ITC together with whole blood platelet aggregation suggested that EuP-82 provided multiple fibrinogen-binding sites that were not related to the arginine-glycine-aspartate (RGD) and the dodecapeptide sequences of fibrinogen. In addition, EuP-82 had neither thrombin-like activity nor anticoagulant activity. The SR-FTIR spectra revealed that EuP-82 was a glycoprotein. Deglycosylation of EuP-82 did not affect its proteolytic activity. Moreover, EuP-82 did not exhibit any toxicity to the living cells (NIH-3T3). This study supports that EuP-82 may be useful for wound-healing material through stabilizing the clot via the platelet induction for the first process.

Overproduction of the N-terminal anticodon-binding domain of the non-discriminating aspartyl-tRNA synthetase from Helicobacter pylori for crystallization and NMR measurements.

Aminoacyl-tRNA synthetases (aaRSs) covalently attach an amino acid to its cognate tRNA isoacceptors through an ester bond. The standard set of 20 amino acids implies 20 aaRSs for each pair of amino acid/tRNA isoacceptors. However, the genomes of all archaea and some bacteria do not encode for a complete set of 20 aaRSs. For the human pathogenic bacterium Helicobacter pylori, a gene encoding asparaginyl-tRNA synthetase (AsnRS) is absent whilst an aspartyl-tRNA synthetase (AspRS) aminoacylates both tRNA(Asp) and tRNA(Asn) with aspartate. The structural and functional basis for this non-discriminatory behavior is not well understood. Here we report the over-production of the N-terminal anticodon-binding domain of H. pylori ND-AspRS using Escherichia coli BL21(DE3) host cells. Prolonged expression of this protein resulted in a toxic phenotype, limiting the expression period to just 30min. Purified protein was monomeric in solution by gel filtration chromatography and stable up to 42°C as observed in temperature-dependent dynamic light scattering measurements. Circular dichroism indicated a mixture of α-helix and ß-sheet secondary structure at 20°C and predominantly ß-sheet at 70°C. Optimized crystallization conditions at pH 5.6 with PEG 4000 as a co-precipitant produced well-formed crystals and (1)H NMR spectrum showed a well dispersed chemical shift envelope characteristic of a folded protein.

A novel immunosensor based on cobalt oxide nanocomposite modified single walled carbon nanohorns for the selective detection of aflatoxin B1.

We developed a novel, sensitive, and selective platform for the specific determination of aflatoxin B1 (AFB1). Single-walled carbon nanohorns decorated by a cobalt oxide composite and gold nanoparticles were created to provide facile electron transfer and improve the sensor's sensitivity. In addition, we attributed the selectivity of the proposed sensor to the specific binding property of the anti-aflatoxin B1 antibody. We clarified the specific interaction of the proposed immunosensor to AFB1 using homology modeling combined with molecular docking. In the presence of AFB1, the current signal of the modified electrode reduced; this involved specific antibody-antigen binding, including hydrophobic hydrogen bonding and pi-pi stack interactions. The new AFB1 sensor platform showed two linearity ranges of 0.01-1 ng mL-1 and 1-100 ng mL-1, with the limit of detection at 0.0019 ng mL-1. We investigated the proposed immunosensor in real samples, including peanuts, certified reference material of a peanut sample (labeled 206 µg kg-1 AFB1), corn, and chicken feed. The sensor's accuracy was 86.1-104.4% recovery, which agrees with the reference HPLC technique using paired t-test analysis. The present work shows excellent performance for AFB1 detection and could be applied for food quality control or modified to detect other mycotoxins.

Crystallization and preliminary crystallographic analysis of ß-mannanase from Bacillus licheniformis.

The mannan endo-1,4-ß-mannosidase (ManB) from Bacillus licheniformis strain DSM13 was overexpressed in Escherichia coli. Purification of the thermostable and alkali-stable recombinant mannanase yielded approximately 50 mg enzyme per litre of culture. Crystals were grown by hanging-drop vapour diffusion using a precipitant solution consisting of 12%(w/v) PEG 8000, 0.2 M magnesium acetate tetrahydrate and 0.1 M MES pH 6.5. The protein crystallized in the monoclinic space group P2(1), with two molecules per asymmetric unit and unit-cell parameters a = 48.58, b = 91.75, c = 89.55 Å, ß = 98.29°, and showed diffraction to 2.3 Šresolution.

Efficient recombinant expression and secretion of a thermostable GH26 mannan endo-1,4-beta-mannosidase from Bacillus licheniformis in Escherichia coli.

BACKGROUND: Mannans are one of the key polymers in hemicellulose, a major component of lignocellulose. The Mannan endo-1,4-beta-mannosidase or 1,4-beta-D-mannanase (EC 3.2.1.78), commonly named beta-mannanase, is an enzyme that can catalyze random hydrolysis of beta-1,4-mannosidic linkages in the main chain of mannans, glucomannans and galactomannans. The enzyme has found a number of applications in different industries, including food, feed, pharmaceutical, pulp/paper industries, as well as gas well stimulation and pretreatment of lignocellulosic biomass for the production of second generation biofuel. Bacillus licheniformis is a Gram-positive endospore-forming microorganism that is generally non-pathogenic and has been used extensively for large-scale industrial production of various enzymes; however, there has been no previous report on the cloning and expression of mannan endo-1,4-beta-mannosidase gene (manB) from B. licheniformis. RESULTS: The mannan endo-1,4-beta-mannosidase gene (manB), commonly known as beta-mannanase, from Bacillus licheniformis strain DSM13 was cloned and overexpressed in Escherichia coli. The enzyme can be harvested from the cell lysate, periplasmic extract, or culture supernatant when using the pFLAG expression system. A total activity of approximately 50,000 units could be obtained from 1-l shake flask cultures. The recombinant enzyme was 6 x His-tagged at its C-terminus, and could be purified by one-step immobilized metal affinity chromatography (IMAC) to apparent homogeneity. The specific activity of the purified enzyme when using locust bean gum as substrate was 1672 +/- 96 units/mg. The optimal pH of the enzyme was between pH 6.0 - 7.0; whereas the optimal temperature was at 50 - 60 degrees C. The recombinant beta-mannanase was stable within pH 5 - 12 after incubation for 30 min at 50 degrees C, and within pH 6 - 9 after incubation at 50 degrees C for 24 h. The enzyme was stable at temperatures up to 50 degrees C with a half-life time of activity (tau1/2) of approximately 80 h at 50 degrees C and pH 6.0. Analysis of hydrolytic products by thin layer chromatography revealed that the main products from the bioconversion of locus bean gum and mannan were various manno-oligosaccharide products (M2 - M6) and mannose. CONCLUSION: Our study demonstrates an efficient expression and secretion system for the production of a relatively thermo- and alkali-stable recombinant beta-mannanase from B. licheniformis strain DSM13, suitable for various biotechnological applications.

The crystal structure of benzophenone synthase from Garcinia mangostana L. pericarps reveals the basis for substrate specificity and catalysis.

Benzophenone synthase (BPS) catalyzes the production of 2,4,6-trihydroxybenzophenone via the condensation of benzoyl-CoA and three units of malonyl-CoA. The biosynthetic pathway proceeds with the formation of the prenylated xanthone α-mangostin from 2,4,6-trihydroxybenzophenone. Structural elucidation was performed to gain a better understanding of the structural basis of the function of Garcinia mangostana L. (mangosteen) BPS (GmBPS). The structure reveals the common core consisting of a five-layer αßαßα fold as found in other type III polyketide synthase enzymes. The three residues Met264, Tyr266 and Gly339 are proposed to have a significant impact on the substrate-binding specificity of the active site. Crystallographic and docking studies indicate why benzoyl-CoA is preferred over 4-coumaroyl-CoA as the substrate for GmBPS. Met264 and Tyr266 in GmBPS are properly oriented for accommodation of the 2,4,6-trihydroxybenzophenone product but not of naringenin. Gly339 offers a minimal steric hindrance to accommodate the extended substrate. Moreover, the structural arrangement of Thr133 provides the elongation activity and consequently facilitates extension of the polyketide chain. In addition to its impact on the substrate selectivity, Ala257 expands the horizontal cavity and might serve to facilitate the initiation/cyclization reaction. The detailed structure of GmBPS explains its catalytic function, facilitating further structure-based engineering to alter its substrate specificity and obtain the desired products.

Structure-function study of AKR4C14, an aldo-keto reductase from Thai jasmine rice (Oryza sativa L. ssp. indica cv. KDML105).

Aldo-keto reductases (AKRs) are NADPH/NADP+-dependent oxidoreductase enzymes that metabolize an aldehyde/ketone to the corresponding alcohol. AKR4C14 from rice exhibits a much higher efficiency in metabolizing malondialdehyde (MDA) than do the Arabidopsis enzymes AKR4C8 and AKR4C9, despite sharing greater than 60% amino-acid sequence identity. This study confirms the role of rice AKR4C14 in the detoxification of methylglyoxal and MDA, and demonstrates that the endogenous contents of both aldehydes in transgenic Arabidopsis ectopically expressing AKR4C14 are significantly lower than their levels in the wild type. The apo structure of indica rice AKR4C14 was also determined in the absence of the cofactor, revealing the stabilized open conformation. This is the first crystal structure in AKR subfamily 4C from rice to be observed in the apo form (without bound NADP+). The refined AKR4C14 structure reveals a stabilized open conformation of loop B, suggesting the initial phase prior to cofactor binding. Based on the X-ray crystal structure, the substrate- and cofactor-binding pockets of AKR4C14 are formed by loops A, B, C and ß1α1. Moreover, the residues Ser211 and Asn220 on loop B are proposed as the hinge residues that are responsible for conformational alteration while the cofactor binds. The open conformation of loop B is proposed to involve Phe216 pointing out from the cofactor-binding site and the opening of the safety belt. Structural comparison with other AKRs in subfamily 4C emphasizes the role of the substrate-channel wall, consisting of Trp24, Trp115, Tyr206, Phe216, Leu291 and Phe295, in substrate discrimination. In particular, Leu291 could contribute greatly to substrate selectivity, explaining the preference of AKR4C14 for its straight-chain aldehyde substrate.

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.

Mutations of Trp275 and Trp397 altered the binding selectivity of Vibrio carchariae chitinase A.

Point mutations of the active-site residues Trp168, Tyr171, Trp275, Trp397, Trp570 and Asp392 were introduced to Vibrio carchariae chitinase A. The modeled 3D structure of the enzyme illustrated that these residues fully occupied the substrate binding cleft and it was found that their mutation greatly reduced the hydrolyzing activity against pNP-[GlcNAc](2) and colloidal chitin. Mutant W397F was the only exception, as it instead enhanced the hydrolysis of the pNP substrate to 142% and gave no activity loss towards colloidal chitin. The kinetic study with the pNP substrate demonstrated that the mutations caused impaired K(m) and k(cat) values of the enzyme. A chitin binding assay showed that mutations of the aromatic residues did not change the binding equilibrium. Product analysis by thin layer chromatography showed higher efficiency of W275G and W397F in G4-G6 hydrolysis over the wild type enzyme. Though the time course of colloidal chitin hydrolysis displayed no difference in the cleavage behavior of the chitinase variants, the time course of G6 hydrolysis exhibited distinct hydrolytic patterns between wild-type and mutants W275G and W397F. Wild type initially hydrolyzed G6 to G4 and G2, and finally G2 was formed as the major end product. W275G primarily created G2-G5 intermediates, and later G2 and G3 were formed as stable products. In contrast, W397F initially produced G1-G5, and then the high-M(r) intermediates (G3-G5) were broken down to G1 and G2 end products. This modification of the cleavage patterns of chitooligomers suggested that residues Trp275 and Trp397 are involved in defining the binding selectivity of the enzyme to soluble substrates.

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

BACKGROUND: Vibrio 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 (alpha/beta)8 TIM barrel catalytic domain (CatD); and iii) the alpha + beta 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. RESULTS: Point mutations of the target residues of V. carchariae chitinase A were generated by site-directed mutagenesis. With respect to their binding activity towards crystalline alpha-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 pNP-[GlcNAc]2, nor the catalytic efficiency against chitohexaose, implying that the mutated residues are not important in oligosaccharide hydrolysis. CONCLUSION: Our 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.

Crystal structure of the N-terminal anticodon-binding domain of the nondiscriminating aspartyl-tRNA synthetase from Helicobacter pylori.

The N-terminal anticodon-binding domain of the nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) plays a crucial role in the recognition of both tRNAAsp and tRNAAsn. Here, the first X-ray crystal structure of the N-terminal domain of this enzyme (ND-AspRS1-104) from the human-pathogenic bacterium Helicobacter pylori is reported at 2.0 Šresolution. The apo form of H. pylori ND-AspRS1-104 shares high structural similarity with the N-terminal anticodon-binding domains of the discriminating aspartyl-tRNA synthetase (D-AspRS) from Escherichia coli and ND-AspRS from Pseudomonas aeruginosa, allowing recognition elements to be proposed for tRNAAsp and tRNAAsn. It is proposed that a long loop (Arg77-Lys90) in this H. pylori domain influences its relaxed tRNA specificity, such that it is classified as nondiscriminating. A structural comparison between D-AspRS from E. coli and ND-AspRS from P. aeruginosa suggests that turns E and F (78GAGL81 and 83NPKL86) in H. pylori ND-AspRS play a crucial role in anticodon recognition. Accordingly, the conserved Pro84 in turn F facilitates the recognition of the anticodons of tRNAAsp (34GUC36) and tRNAAsn (34GUU36). The absence of the amide H atom allows both C and U bases to be accommodated in the tRNA-recognition site.

Purification, characterization, cloning and structural analysis of Crocodylus siamensis ovotransferrin for insight into functions of iron binding and autocleavage.

Ovotransferrin (OTf), the major protein constituent of egg white, is of great interest due to its pivotal role in biological iron transport and storage processes and its spontaneous autocleavage into peptidic fragments with alternative biological properties, such as antibacterial and antioxidant activities. However, despite being well-investigated in avian, a detailed elucidation of the structure-function relationship of ovotransferrins in the closely related order of Crocodilia has not been reported to date. In this study, electron paramagnetic resonance (EPR) confirmed the presence of two spectroscopically distinct ferric iron binding sites in Crocodylus siamensis OTf (cOTf), but implied a five-fold lower quantity of bound iron than in hen OTf (hOTf). In addition, quantitative estimation of free sulfhydryl groups revealed slight differences to hOTf. To gain a better structural understanding of cOTf, we found a cOTf gene consisting of an open reading frame of 2040bp and encoding a protein of 679 amino acids. In silico prediction of the three-dimensional structure of cOTf and comparison with hOTf revealed four evolutionarily conserved iron-binding sites in both N- and C-lobes, as well as the presence of only 13 of the 15 disulfide bonds in hOTf. This evolutionary loss of disulfide linkages in conjunction with the lack of hydrogen bonding from a dilysine trigger in the C-lobe are presumed to affect the iron binding and autocleavage character of cOTf. As a result, cOTf may be capable of exerting a more diverse array of functions compared to its avian counterparts; for instance, ion buffering, antioxidant and antimicrobial activities.

OmpA signal peptide leads to heterogenous secretion of B. subtilis chitosanase enzyme from E. coli expression system.

The production of secreted recombinant proteins from E. coli is pivotal to the biotechnological industry because it reduces the cost of downstream processing. Proteins destined for secretion contain an N-terminal signal peptide that is cleaved by secretion machinery in the plasma membrane. The resulting protein is released in an active mature form. In this study, Bacillus subtilis chitosanase (Csn) was used as a model protein to compare the effect of two signal peptides on the secretion of heterologous recombinant protein. The results showed that the E. coli secretion machinery could recognize both native bacillus and E. coli signal peptides. However, only the native bacillus signal peptide could generate the same N-terminal sequence as in the wild type bacteria. When the recombinant Csn constructs contained the E. coli OmpA signal peptide, the secreted enzymes were heterogeneous, comprising a mixed population of secreted enzymes with different N-terminal sequences. Nevertheless, the E. coli OmpA signal peptide was found to be more efficient for high expression and secretion of bacillus Csn. These findings may be used to help engineer other recombinant proteins for secretory production in E. coli.

Bacterial ß-Glucosidase Reveals the Structural and Functional Basis of Genetic Defects in Human Glucocerebrosidase 2 (GBA2).

Human glucosylcerebrosidase 2 (GBA2) of the CAZy family GH116 is responsible for the breakdown of glycosphingolipids on the cytoplasmic face of the endoplasmic reticulum and Golgi apparatus. Genetic defects in GBA2 result in spastic paraplegia and cerebellar ataxia, while cross-talk between GBA2 and GBA1 glucosylceramidases may affect Gaucher disease. Here, we report the first three-dimensional structure for any GH116 enzyme, Thermoanaerobacterium xylanolyticum TxGH116 ß-glucosidase, alone and in complex with diverse ligands. These structures allow identification of the glucoside binding and active site residues, which are shown to be conserved with GBA2. Mutagenic analysis of TxGH116 and structural modeling of GBA2 provide a detailed structural and functional rationale for pathogenic missense mutations of GBA2.

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 alpha and beta anomers and the simultaneous monitoring of chitooligosaccharide products down to picomole levels. Chitinase A primarily generated beta-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 approximately 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.

Expression, purification, crystallization and preliminary crystallographic analysis of chitinase A from Vibrio carchariae.

Chitinase A of Vibrio carchariae was expressed in Escherichia coli M15 host cells as a 575-amino-acid fragment with full enzymatic activity using the pQE60 expression vector. The yield of the highly purified recombinant protein was approximately 70 mg per litre of bacterial culture. The molecular mass of the expressed protein was determined by HPLC/ESI-MS to be 63 770, including the hexahistidine tag. Crystals of recombinant chitinase A were grown to a suitable size for X-ray structure analysis in a precipitant containing 10%(v/v) PEG 400, 0.1 M sodium acetate pH 4.6 and 0.125 M CaCl2. The crystals belonged to the tetragonal space group P422, with two molecules per asymmetric unit and unit-cell parameters a = b = 127.64, c = 171.42 A. A complete diffraction data set was collected to 2.14 A resolution using a Rigaku/MSC R-AXIS IV++ detector system mounted on an RU-H3R rotating-anode X-ray generator.

Purification, characterization, and crystallization of Crocodylus siamensis hemoglobin.

Crocodylus siamensis hemoglobin was purified by a size exclusion chromatography, Sephacryl S-100 with buffer containing dithiothreitol. The purified Hb was dissociated to be two forms (α chain and ß chain) which observed by SDS-PAGE, indicated that the C. siamensis Hb was an unpolymerized form. The unpolymerized Hb (composed of two α chains and two ß chains) showed high oxygen affinity at 3.13 mmHg (P(50)) and 1.96 (n value), and a small Bohr effect (δH(+) = -0.29) at a pH of 6.9-8.4. Adenosine triphosphate did not affect the oxygenation properties, whereas bicarbonate ions strongly depressed oxygen affinity. Crude C. siamensis Hb solutions were showed high O(2) affinity at P(50) of 2.5 mmHg which may assure efficient utilization of the lung O(2) reserve during breath holding and diving. The purified Hbs were changed to cyanmethemoglobin forms prior crystallization. Rod- and plate-shaped crystals were obtained by the sitting-drop vapor-diffusion method at 5 °C using equal volumes of protein solution (37 mg/ml) and reservoir [10-13 % (w/v) PEG 4000, with 0.1 M Tris buffer in present of 0.2 M MgCl(2)·6H(2)O] solution at a pH of 7.0-8.5.