A bacterial glycosidase enables mannose-6-phosphate modification and improved cellular uptake of yeast-produced recombinant human lysosomal enzymes.

Lysosomal storage diseases are treated with human lysosomal enzymes produced in mammalian cells. Such enzyme therapeutics contain relatively low levels of mannose-6-phosphate, which is required to target them to the lysosomes of patient cells. Here we describe a method for increasing mannose-6-phosphate modification of lysosomal enzymes produced in yeast.

Glutathione biosynthesis in bacteria by bifunctional GshF is driven by a modular structure featuring a novel hybrid ATP-grasp fold.

Glutathione is an intracellular redox-active tripeptide thiol with a central role in cellular physiology across all kingdoms of life. Glutathione biosynthesis has been traditionally viewed as a conserved process relying on the sequential activity of two separate ligases, but recently, an enzyme (GshF) that unifies both necessary reactions in one platform has been identified and characterized in a number of pathogenic and free-living bacteria. Here, we report crystal structures of two prototypic GshF enzymes from Streptococcus agalactiae and Pasteurella multocida in an effort to shed light onto the structural determinants underlying their bifunctionality and to provide a structural framework for the plethora of biochemical and mutagenesis studies available for these enzymes.

Construction of cellobiose phosphorylase variants with broadened acceptor specificity towards anomerically substituted glucosides.

The general application of glycoside phosphorylases such as cellobiose phosphorylase (CP) for glycoside synthesis is hindered by their relatively narrow substrate specificity. We have previously reported on the creation of Cellulomonas uda CP enzyme variants with either modified donor or acceptor specificity. Remarkably, in this study it was found that the donor mutant also displays broadened acceptor specificity towards several beta-glucosides.

Crystallization and X-ray diffraction studies of inverting trehalose phosphorylase from Thermoanaerobacter sp.

Disaccharide phosphorylases are attractive enzymatic platforms for tailor-made sugar synthesis owing to their ability to catalyze both the synthesis and the breakdown of disaccharides. Trehalose phosphorylase from Thermoanaerobacter sp. (TP) is a glycoside hydrolase family 65 enzyme which catalyzes the reversible breakdown of trehalose [D-glucopyranosyl-alpha(1,1)alpha-D-glucopyranose] to beta-D-glucose 1-phosphate and D-glucose.

Crystallization and X-ray diffraction studies of cellobiose phosphorylase from Cellulomonas uda.

Disaccharide phosphorylases are able to catalyze both the synthesis and the breakdown of disaccharides and have thus emerged as attractive platforms for tailor-made sugar synthesis. Cellobiose phosphorylase from Cellulomonas uda (CPCuda) is an enzyme that belongs to glycoside hydrolase family 94 and catalyzes the reversible breakdown of cellobiose [beta-D-glucopyranosyl-(1,4)-D-glucopyranose] to alpha-D-glucose-1-phosphate and D-glucose. Crystals of ligand-free recombinant CPCuda and of its complexes with substrates and reaction products yielded complete X-ray diffraction data sets to high resolution using synchrotron radiation but suffered from significant variability in diffraction quality.

Towards structural studies of the old yellow enzyme homologue SYE4 from Shewanella oneidensis and its complexes at atomic resolution.

Shewanella oneidensis is an environmentally versatile Gram-negative gamma-proteobacterium that is endowed with an unusually large proteome of redox proteins. Of the four old yellow enzyme (OYE) homologues found in S. oneidensis, SYE4 is the homologue most implicated in resistance to oxidative stress.

Expression, purification, crystallization and structure determination of two glutathione S-transferase-like proteins from Shewanella oneidensis.

Genome analysis of Shewanella oneidensis, a Gram-negative bacterium with an unusual repertoire of respiratory and redox capabilities, revealed the presence of six glutathione S-transferase-like genes (sogst1-sogst6). Glutathione S-transferases (GSTs; EC 2.5.

X-ray crystallographic analysis of the sulfur carrier protein SoxY from Chlorobium limicola f. thiosulfatophilum reveals a tetrameric structure.

Dissimilatory oxidation of thiosulfate in the green sulfur bacterium Chlorobium limicola f. thiosulfatophilum is carried out by the ubiquitous sulfur-oxidizing (Sox) multi-enzyme system. In this system, SoxY plays a key role, functioning as the sulfur substrate-binding protein that offers its sulfur substrate, which is covalently bound to a conserved C-terminal cysteine, to another oxidizing Sox enzyme.

Crystallization, preliminary crystallographic analysis and phasing of the thiosulfate-binding protein SoxY from Chlorobium limicola f. thiosulfatophilum.

The 22 kDa SoxYZ protein complex from the green sulfur bacterium Chlorobium limicola f. thiosulfatophilum is a central player in the sulfur-oxidizing (Sox) enzyme system of the organism by activating thiosulfate for oxidation by SoxXA and SoxB. It has been proposed that SoxYZ exists as a heterodimer or heterotetramer, but the properties and role of the individual components of the complex thus far remain unknown.

Psychological characteristics and factors related to willingness to donate again among anonymous oocyte donors.

Objective: To assess post-donation psychological status of a large sample of professionally recruited, paid anonymous donors and to determine whether there were any differences between those who were willing to donate again and those who were not.

Design: Cross-sectional survey.

Setting: Healthy donors who were recruited by two private groups.