A front-face 'SNi synthase' engineered from a retaining 'double-SN2' hydrolase.

SNi-like mechanisms, which involve front-face leaving group departure and nucleophile approach, have been observed experimentally and computationally in chemical and enzymatic substitution at α-glycosyl electrophiles. Since SNi-like, SN1 and SN2 substitution pathways can be energetically comparable, engineered switching could be feasible. Here, engineering of Sulfolobus solfataricus ß-glycosidase, which originally catalyzed double SN2 substitution, changed its mode to SNi-like. Destruction of the first SN2 nucleophile through E387Y mutation created a ß-stereoselective catalyst for glycoside synthesis from activated substrates, despite lacking a nucleophile. The pH profile, kinetic and mutational analyses, mechanism-based inactivators, X-ray structure and subsequent metadynamics simulations together suggest recruitment of substrates by π-sugar interaction and reveal a quantum mechanics-molecular mechanics (QM/MM) free-energy landscape for the substitution reaction that is similar to those of natural, SNi-like glycosyltransferases. This observation of a front-face mechanism in a ß-glycosyltransfer enzyme highlights that SNi-like pathways may be engineered in catalysts with suitable environments and suggests that 'ß-SNi' mechanisms may be feasible for natural glycosyltransfer enzymes.

STRU-cloning: a fast, inexpensive and efficient cloning procedure applicable to both small scale and structural genomics size cloning.

We have developed a Single-Tube Restriction-based Ultrafiltration (STRU) cloning procedure that updates traditional ligation-dependent cloning to challenge the newer, faster and more efficient ligation-free techniques and could make it the method of choice. STRU-cloning employs centrifugal filter units with membrane of suitable cut off to remove small unwanted DNA fragments created during restriction of plasmids or PCR products. Heat inactivation, of restriction enzymes, followed by DNA ligation is then performed on the filtrate. By removing the agarose gel electrophoresis DNA purification step from the traditional protocol, which is time consuming and is known to be the cause of ligation problems, STRU-cloning becomes fast, very efficient, inexpensive and offers the highest degree of cloning flexibility by using restriction sites and can be performed in a single tube. This novel agarose gel-free cloning procedure provides benefits for both small and large scale cloning projects. Unlike traditional cloning it can be easily implemented as a fully automated process at very low costs.

Inhibition of MptpB phosphatase from Mycobacterium tuberculosis impairs mycobacterial survival in macrophages.

OBJECTIVES: The secreted Mycobacterium tuberculosis protein tyrosine phosphatase (MptpB) is a virulence factor for M. tuberculosis and contributes to its survival within host macrophages. The aim of this study was to identify potent selective inhibitors of MptpB and to determine the efficacy of these compounds in mycobacterium-infected macrophages. METHODS: The inhibitory effect of a small library of compounds on MptpB was first examined in vitro. The efficacy of these compounds was further examined in mycobacterium-infected macrophages. RESULTS: We have identified a new family of double-site isoxazole-based compounds that are potent selective inhibitors of MptpB. Importantly, the inhibitors substantially reduce mycobacterial survival in infected macrophages. In contrast with current anti-tubercular drugs, these MptpB inhibitors do not have bactericidal action but rather, severely impair mycobacterial growth within macrophages. Docking analysis suggests a double-site binding mechanism of inhibition with the isoxazole head in the active site and a salicylate group in a secondary binding pocket that is a unique structural feature of MptpB. CONCLUSIONS: These results provide the first evidence that inhibition of phosphatases can be exploited against mycobacterial infections. The cell activity of the inhibitors together with the lack of MptpB human orthologues suggests a strong potential for these compounds to be developed as drug candidates against tuberculosis and promises a new therapeutic strategy to tackle clearance and reduce the persistence of M. tuberculosis infection.

MptpB, a virulence factor from Mycobacterium tuberculosis, exhibits triple-specificity phosphatase activity.

Bacterial pathogens have developed sophisticated mechanisms of evading the immune system to survive in infected host cells. Central to the pathogenesis of Mycobacterium tuberculosis is the arrest of phagosome maturation, partly through interference with PtdIns signalling. The protein phosphatase MptpB is an essential secreted virulence factor in M. tuberculosis. A combination of bioinformatics analysis, enzyme kinetics and substrate-specificity characterization revealed that MptpB exhibits both dual-specificity protein phosphatase activity and, importantly, phosphoinositide phosphatase activity. Mutagenesis of conserved residues in the active site signature indicates a cysteine-based mechanism of dephosphorylation and identifies two new catalytic residues, Asp165, essential in catalysis, and Lys164, apparently involved in substrate specificity. Sequence similarities with mammalian lipid phosphatases and a preference for phosphoinositide substrates suggests a potential novel role of MptpB in PtdIns metabolism in the host and reveals new perspectives for the role of this phosphatase in mycobacteria pathogenicity.

Small-angle X-ray scattering reveals the solution structure of a bacteriophytochrome in the catalytically active Pr state.

Phytochromes are light-sensing macromolecules that are part of a two component phosphorelay system controlling gene expression. Photoconversion between the Pr and Pfr forms facilitates autophosphorylation of a histidine in the dimerization domain (DHp). We report the low-resolution structure of a bacteriophytochrome (Bph) in the catalytic (CA) Pr form in solution determined by small-angle X-ray scattering (SAXS). Ab initio modeling reveals, for the first time, the domain organization in a typical bacteriophytochrome, comprising an chromophore binding and phytochrome (PHY) N terminal domain followed by a C terminal histidine kinase domain. Homologous high-resolution structures of the light-sensing chromophore binding domain (CBD) and the cytoplasmic part of a histidine kinase sensor allows us to model 75% of the structure with the remainder comprising the phytochrome domain which has no 3D representative in the structural database. The SAXS data reveal a dimeric Y shaped macromolecule and the relative positions of the chromophores (biliverdin), autophosphorylating histidine residues and the ATP molecules in the kinase domain. SAXS data were collected from a sample in the autophosphorylating Pr form and reveal alternate conformational states for the kinase domain that can be modeled in an open (no-catalytic) and closed (catalytic) state. This model suggests how light-induced signal transduction can stimulate autophosphorylation followed by phosphotransfer to a response regulator (RR) in the two-component system.

Unique regulation of the active site of the serine esterase S-formylglutathione hydrolase.

S-Formylglutathione hydrolases (SFGHs) are highly conserved thioesterases present in prokaryotes and eukaryotes, and form part of the formaldehyde detoxification pathway, as well as functioning as xenobiotic-hydrolysing carboxyesterases. As defined by their sensitivity to covalent modification, SFGHs behave as cysteine hydrolases, being inactivated by thiol alkylating agents, while being insensitive to inhibition by organophosphates such as paraoxon. As such, the enzyme has been classified as an esterase D in animals, plants and microbes. While SFGHs do contain a conserved cysteine residue that has been implicated in catalysis, sequence analysis also reveals the classic catalytic triad of a serine hydrolase. Using a combination of selective protein modification and X-ray crystallography, AtSFGH from Arabidopsis thaliana has been shown to be a serine hydrolase rather than a cysteine hydrolase. Uniquely, the conserved reactive cysteine (Cys59) previously implicated in catalysis lies in close proximity to the serine hydrolase triad, serving a gate-keeping function in comprehensively regulating access to the active site. Thus, any covalent modification of Cys59 inhibited all hydrolase activities of the enzyme. When isolated from Escherichia coli, a major proportion of recombinant AtSFGH was recovered with the Cys59 forming a mixed disulfide with glutathione. Reversible disulfide formation with glutathione could be demonstrated to regulate hydrolase activity in vitro. The importance of Cys59 in regulating AtSFGH in planta was demonstrated in transient expression assays in Arabidopsis protoplasts. As determined by fluorescence microscopy, the Cys59Ser mutant enzyme was shown to rapidly hydrolyse 4-methylumbelliferyl acetate in paraoxon-treated cells, while the native enzyme was found to be inactive. Our results clarify the classification of AtSFGHs as hydrolases and suggest that the regulatory and conserved cysteine provides an unusual redox-sensitive regulation to an enzyme functioning in both primary and xenobiotic metabolism in prokaryotes and eukaryotes.

Purification and characterization of an N-acetyl-D-galactosamine-specific lectin from the edible mushroom Schizophyllum commune.

An N-acetyl-D-galactosamine (GalNAc)-specific lectin was purified from the edible mushroom, Schizophyllum commune, using affinity chromatography on a porcine stomach mucin (PSM)-Sepharose 4B column. Under reducing and non-reducing conditions, SDS-polyacrylamide gel electrophoresis gave a major band of 31.5 kDa. The Schizophyllum commune lectin (SCL) showed high affinity toward rat erythrocytes and the sugar inhibition assay exhibited its sugar specificity highly toward lactose and N-acetyl-D-galactosamine. It was stable at 55 degrees C for 30 min and at pH 3-10 for 18-h test. The lectin was shown to be a glycoprotein with cytotoxic activity against human epidermoid carcinoma cells. The N-terminus of SCL was blocked but amino acid sequences of internal tryptic peptides showed moderately sequence similarities with some other fungal and plant lectins. Crystals of SCL were obtained by the sitting drop vapour-diffusion method using polyethylene glycol 8000 as the precipitant, and gave an X-ray diffraction pattern to approximately 3.8 angstroms resolution.

A bacteriophytochrome regulates the synthesis of LH4 complexes in Rhodopseudomonas palustris.

The non-sulphur purple bacterium Rhodopseudomonas palustris contains five pucAB genes for peripheral light-harvesting complexes. Bacteria grown under high-light conditions absorb at 800 and 850 nm but in low-light the 850 nm peak is almost absent and LH2 complexes are replaced by LH4. The genome contains six bacteriophytochromes (Bph). Bphs sense light in the red/far-red through a reversible Pr to Pfr transformation that controls gene expression. Bph3 (RPA1537) controls the expression of a cluster of photosynthetic genes, however most of the peripheral light harvesting complex genes are outside of this region. The pucAB-d genes encode LH4 peptides and are near two Bphs (RPA3015, RPA3016). We have characterised three Bphs and show that Bph4 RPA3015 and Bph3 RPA1537 have different dark stable states. It is known that Bph3 is active in its red absorbing Pr form and suggests a working hypothesis that Bph4 is active in the Pfr state. We show that LH4 expression can be induced with red light at the Pr absorption maximum (708 nm) of Bph4. The property of light transmission of water maybe an important factor in understanding this adaptation. Bph4 can sense the reduction in light intensity indirectly through an increase in ratio of transmitted red/far-red light. The red right activated Bph4 regulates the synthesis of LH4 which concentrates bacteriochlorophyll a pigment absorption at 800 nm to exploit a recovery in water light transmission in this region.

Redox regulation of a soybean tyrosine-specific protein phosphatase.

Plant protein tyrosine phosphatases (PTPs) are important in regulating cellular responses to redox change through their reversible inactivation under oxidative conditions. Studies on the soybean (Glycine max) GmPTP have shown that, compared with its mammalian counterparts, the plant enzyme is relatively insensitive to inactivation by H2O2 but hypersensitive (k(inact) = 559 M(-1) s(-1)) to S-glutathionylation (thiolation) promoted by the presence of oxidized glutathione (GSSG). Through a combination of chemical and mutational modification studies, three of the seven cysteine residues of GmPTP have been identified by mass spectrometry as being able to inactivate the enzyme when thiolated by GSSG or alkylated with iodoacetamide. Conserved Cys 266 was shown to be essential for catalysis but surprisingly resistant to S-modification, whereas the regulatory Cys 78 and Cys 176 were readily thiolated and/or alkylated. Mutagenesis of these cysteines showed that all three residues were in proximity of each other, regulating each's reactivity to S-modifying agents. Through a combination of protein modification and kinetic experiments, we conclude that the inactivation of GmPTP by GSSG is regulated at two levels. Cys 176 appears to be required to promote the formation of the reduced form of Cys 266, which is otherwise unreactive. When thiolated, Cys 176 immediately inactivates the enzyme, and this is followed by the thiolation of Cys 78, which undergoes a slow disulfide exchange with Cys 266 giving rise to a Cys 78-Cys 266 disulfide. We speculate that this two-tiered protection is required for regulation of GmPTP under highly oxidizing conditions.

Developing promiscuous glycosidases for glycoside synthesis: residues W433 and E432 in Sulfolobus solfataricus beta-glycosidase are important glucoside- and galactoside-specificity determinants.

Two residues that have been implicated in determining the substrate specificity of the thermophilic beta-glycosidase from the archaeon Sulfolobus solfataricus (SsbetaG), a member of the glycosyl hydrolase family 1, have been mutated by site-directed mutagenesis so as to create more versatile catalysts for carbohydrate chemistry. The wild-type and mutated sequences were expressed in E. coli with a His(7)-tag to allow one-step chromatographic purification. The E432C and W433C mutations removed key interactions with the OH-4 and OH-3 of the sugar substrates, thus reducing the discrimination of glucose, galactose and fucose with respect to other glycosides. This resulted in two glycosidases with greatly broadened substrate specificities. Observed changes include a 24-fold increase in Man:Gal activity and an 18-fold increase in GalA:Gal activity. This promiscuous substrate tolerance was further illustrated by the parallel synthesis of a beta-glycoside library of glucose, galactose, xylose and mannose in one pot at 50 degrees C, in organic solvent. The synthetic potential of the catalysts was further evaluated through alkyl glycoside transglycosylation yields, including the first examples of synthesis of beta-mannosides and beta-xylosides with SsbetaG.

Purification, crystallization and preliminary X-ray diffraction analysis of S-formylglutathione hydrolase from Arabidopsis thaliana: effects of pressure and selenomethionine substitution on space-group changes.

S-Formylglutathione hydrolase (SFGH) has activity toward several xenobiotic carboxyesters and catalyses the final step of formaldehyde detoxification: the hydrolysis of S-formylglutathione to formate and glutathione. The Arabidopsis thaliana enzyme (AtSFGH) was crystallized in space group C2, with unit-cell parameters a = 128.5, b = 81.1, c = 94.3 A, beta = 93.3 degrees and three molecules in the asymmetric unit. A second crystal form of AtSFGH could be obtained by pressurizing the monoclinic crystals at 2 MPa for 30 min. The resulting space group is either P3(1)21 or P3(2)21, with unit-cell parameters a = 75.1, c = 92.8 A and one molecule in the asymmetric unit. Crystallographic data have been collected for both crystal forms to resolutions of 1.7 A for the monoclinic crystal and 1.6 A for the trigonal crystal. The structure has been solved by MAD phasing using a three-wavelength data set collected from a monoclinic crystal of selenomethionine-labelled AtSFGH.

A novel higher plant protein tyrosine phosphatase interacts with SNF1-related protein kinases via a KIS (kinase interaction sequence) domain.

A novel protein phosphatase in Arabidopsis thaliana was identified by database searching. This protein, designated AtPTPKIS1, contains a protein tyrosine phosphatase (PTP) catalytic domain and a kinase interaction sequence (KIS) domain. It is predicted to interact with plant SNF1-related kinases (SnRKs), representing central regulators of metabolic and stress responses. AtPTPKIS1 has close homologues in other plant species, both dicots and monocots, but is not found in other kingdoms. The tomato homologue of AtPTPKIS1 was expressed as a recombinant protein and shown to hydrolyse a generic phosphatase substrate, and phosphotyrosine residues in synthetic peptides. The KIS domain of AtPTPKIS1 was shown to interact with the plant SnRK AKIN11 both in vivo in the yeast two-hybrid system, and in vitro in a GST-fusion 'pull down' assay. The genomes of Arabidopsis and other plants contain further predicted proteins related to AtPTPKIS1, which could also interact with SnRKs and act in novel regulatory and signalling pathways.