A front-face 'Si synthase' engineered from a retaining 'double-S2' hydrolase.

Si-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 Si-like, S1 and S2 substitution pathways can be energetically comparable, engineered switching could be feasible. Here, engineering of Sulfolobus solfataricus β-glycosidase, which originally catalyzed double S2 substitution, changed its mode to Si-like.

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.

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.

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).

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.

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.

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).

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.

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.

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.

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.