Epoxide hydrolase-catalyzed resolution of ethyl 3-phenylglycidate using whole cells of Pseudomonas sp.

A Pseudomonas sp. was isolated with enantioselective epoxide hydrolase activity to ethyl 3-phenylglycidate. Cells grown on sucrose and suspended in 10% (v/v) dimethyl formamide as co-solvent produced (2R,3S) ethyl 3-phenylglycidate with 95% ee and 26% yield in 12 h from 0.2% (w/v) of the racemate.     

Cloning and functional expression of alkaline alpha-galactosidase from melon fruit: similarity to plant SIP proteins uncovers a novel family of plant glycosyl hydrolases

Raffinose and stachyose are ubiquitous galactosyl-sucrose oligosaccharides in the plant kingdom which play major roles, second only to sucrose, in photoassimilate translocation and seed carbohydrate storage. These sugars are initially metabolised by alpha-galactosidases (alpha-gal). We report the cloning and functional expression of the first genes, CmAGA1 and CmAGA2, encoding for plant alpha-gals with alkaline pH optima from melon fruit (Cucumis melo L.), a raffinose and stachyose translocating species. The alkaline alpha-gal genes show very high sequence homology with a family of undefined 'seed imbibition proteins' (SIPs) which are present in a wide range of plant families. In order to confirm the function of SIP proteins, a representative SIP gene, from tomato, was expressed and shown to have alkaline alpha-gal activity. Phylogenetic analysis based on amino acid sequences shows that the family of alkaline alpha-gals shares little homology with the known prokaryotic and eukaryotic alpha-gals of glycosyl hydrolase families 27 and 36, with the exception of two cross-family conserved sequences containing aspartates which probably function in the catalytic step. This previously uncharacterised, plant-specific alpha-gal family of glycosyl hydrolases, with optimal activity at neutral-alkaline pH likely functions in key processes of galactosyl-oligosaccharide metabolism, such as during seed germination and translocation of RFO photosynthate.     

Acid hydrolase activity of cultured bovine oligodendrocytes

We measured the activity of several acid hydrolases of cultured oligodendrocytes prepared from adult bovine brain white matter to clarify the biochemical basis of bovine oligodendrocytes in vitro. Lysosomal enzyme activities were assayed by using 4-methylumbelliferyl glycosides as substrates. Lysosomal enzyme activities became higher at 8–11 days in vitro (DIV) than 4 DIV. The enrichment in acid hydrolase specific activities in oligodendrocytes may be associated with lysosomal origin of myelin-like membranes.     

The regulatory complex of Drosophila melanogaster 26S proteasomes. Subunit composition and localization of a deubiquitylating enzyme

Drosophila melanogaster embryos are a source for homogeneous and stable 26S proteasomes suitable for structural studies. For biochemical characterization, purified 26S proteasomes were resolved by two-dimensional (2D) gel electrophoresis and subunits composing the regulatory complex (RC) were identified by amino acid sequencing and immunoblotting, before corresponding cDNAs were sequenced. 17 subunits from Drosophila RCs were found to have homologues in the yeast and human RCs. An additional subunit, p37A, not yet described in RCs of other organisms, is a member of the ubiquitin COOH-terminal hydrolase family (UCH). Analysis of EM images of 26S proteasomes-UCH-inhibitor complexes allowed for the first time to localize one of the RC's specific functions, deubiquitylating activity.The masses of 26S proteasomes with either one or two attached RCs were determined by scanning transmission EM (STEM), yielding a mass of 894 kD for a single RC. This value is in good agreement with the summed masses of the 18 identified RC subunits (932 kD), indicating that the number of subunits is complete.     

Structure and function of S-adenosylhomocysteine hydrolase

In mammals, S-adenosylhomocysteine hydrolase (AdoHcyase) is the only known enzyme to catalyze the breakdown of S-adenosylhomocysteine (AdoHcy) to homocysteine and adenosine. AdoHcy is the product of all adenosylmethionine (AdoMet)-dependent biological transmethylations. These reactions have a wide range of products, and are common in all facets of biometabolism. As a product inhibitor, elevated levels of AdoHcy suppress AdoMet-dependent transmethylations. Thus, AdoHcyase is a regulator of biological transmethylation in general. The three-dimensional structure of AdoHcyase complexed with reduced nicotinamide adenine dinucleotide phosphate (NADH) and the inhibitor (1′R, 2′S, 3′R)-9-(2′,3′-dihyroxycyclopenten-1-yl)adenine (DHCeA) was solved by a combination of the crystallographic direct methods program, SnB, to determine the selenium atom substructure and by treating the multiwavelength anomalous diffraction data as a special case of multiple isomorphous replacement. The enzyme architecture resembles that observed for NAD-dependent dehydrogenases, with the catalytic domain and the cofactor binding domain each containing a modified Rossmann fold. The two domains form a deep active site cleft containing the cofactor and bound inhibitor molecule. A comparison of the inhibitor complex of the human enzyme and the structure of the rat enzyme, solved without inhibitor, suggests that a 17° rigid body movement of the catalytic domain occurs upon inhibitor/substrate binding.     

Neurotoxic Organophosphate Degradation with Polyvinyl Alcohol Gel-Immobilized Microbial Cells

A genetically engineered strain of Escherichia coli that expresses organophosphorus hydrolase (OPH) was immobilized in a polyvinyl alcohol (PVA) cryogel to form a porous biocatalyst that successfully degrades organophosphorus (OP) neurotoxins. The impacts of both diffusion and reaction on biocatalyst efficiency were determined to enable prediction and optimization of the biocatalyst performance. The kinetic rate parameters and activation energies of pure OPH, free cell suspensions, and the immobilized cell biocatalyst were compared. Diffusion was a determining factor for paraoxon hydrolysis because of the very rapid OPH kinetics for its model substrate. Both the paraoxon diffusion through the PVA matrix and the diffusion associated with microbial transport of paraoxon were shown to impact the biocatalyst reaction. However, the enhancement in storage stability resulting from diffusional limitations provides an advantage to diffusion-limited operation. This research may serve as a guide to define the influence of diffusion in biological reaction systems. The broad substrate specificity and hydrolytic efficiency of OPH coupled with the ability to genetically engineer the enzyme for specific target OP neurotoxins enhance the suitability of OPH-based technologies for detoxification of these compounds. Cryoimmobilization provides a suitable vehicle as a cost-effective, efficient technology for bioremediation of environmental media contaminated with OP compounds.     

Enzymatic systems involved in decomposition reflects the ecology and taxonomy of saprotrophic fungi

One hundred and eleven strains of Basidiomycota, 39 strains of Ascomycota and 2 strains of Mucoromycotina belonging to wood decomposers that cause white-rot (WR) or brown-rot (BR), other wood associated saprotrophs (WA), litter decomposing cord-forming Basidiomycota (LDF), and saprotrophic microfungi (SA), were screened for the production of hydrolytic enzymes and laccase. The presence of enzyme-encoding genes was also analysed in the published genomes of saprotrophic fungi. Several genes, including those for acidic phosphatase, β-glucosidase and N-acetylglucosaminidase, were common in the genomes with enzyme activity widely displayed by fungi, while other enzymes, such as certain hemicellulases or laccase, were produced less frequently. Enzyme production by saprotrophic fungi was shaped by the combination of their ecophysiology and taxonomy. Basidiomycota exhibited higher activities of all enzymes, except alkaline phosphatase, α-glucosidase, N-acetylglucosaminidase, α-mannosidase and α-fucosidase, than Ascomycota. The SA and BR fungi showed distinct enzyme production patterns, while the enzyme production by WR, LDF and WA was similar. Differences among species were typically reflected in the level of enzyme activity rather than in the absence of enzymes. Enzyme screening results showed that in several cases, fungi exhibited enzyme activity without the presence of the corresponding gene and vice versa. This indicates that the use of genome-derived information for the prediction of potential enzyme production has substantial limitations and cannot replace functional screening of fungal cultures.