Jan Stout

Development and application of a screening assay for glycoside phosphorylases

Glycoside phosphorylases (GPs) are interesting enzymes for the glycosylation of chemical molecules. They require only a glycosyl phosphate as sugar donor and an acceptor molecule with a free hydroxyl group. Their narrow substrate specificity, however, limits the application of GPs for general glycoside synthesis. Although an enzymes substrate specificity can be altered and broadened by protein engineering and directed evolution, this requires a suitable screening assay. Such a screening assay has not yet been described for GPs. Here we report a screening procedure for GPs based on the measurement of released inorganic phosphate in the direction of glycoside synthesis. It appeared necessary to inhibit endogenous phosphatase activity in crude Escherichia coli cell extracts with molybdate, and inorganic phosphate was measured with a modified phosphomolybdate method. The screening system is general and can be used to screen GP enzyme libraries for novel donor and acceptor specificities. It was successfully applied to screen a residue E649 saturation mutagenesis library of Cellulomonas uda cellobiose phosphorylase (CP) for novel acceptor specificity. An E649C enzyme variant was found with novel acceptor specificity toward alkyl beta-glucosides and phenyl beta-glucoside. This is the first report of a CP enzyme variant with modified acceptor specificity.

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 β‐glucosides. Triple mutants containing donor (T508I/N667A) as well as acceptor mutations (E649C or E649G) also display a broader acceptor specificity than any of the parent enzymes. Moreover, further broadening of the acceptor specificity has been achieved by site‐saturation mutagenesis of residues near the active site entrance. The best enzyme variant contains the additional N156D and N163D mutations and is active towards various alkyl β‐glucosides, methyl α‐glucoside and cellobiose. In comparison with the wild‐type C. uda CP enzyme, which cannot accept anomerically substituted glucosides at all, the obtained increase in substrate specificity is significant. The described CP enzyme variants should be useful for the synthesis of cellobiosides and other glycosides with prebiotic and pharmaceutical properties. Biotechnol. Bioeng. 2010;107: 413–420.

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. In at least one case an intriguing space-group transition from a primitive monoclinic to a primitive orthorhombic lattice was observed during data collection. The structure of CPCuda was determined by maximum-likelihood molecular replacement, thus establishing a starting point for an investigation of the structural and mechanistic determinants of disaccharide phosphorylase activity.

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. Our structures reveal how a canonical bacterial GshA module that catalyzes the condensation of L-glutamate and L-cysteine to γ-glutamylcysteine is linked to a novel ATP-grasp-like module responsible for the ensuing formation of glutathione from γ-glutamylcysteine and glycine. Notably, we identify an unprecedented subdomain in the ATP-grasp module of GshF at the interface of the GshF dimer, which is poised to mediate intersubunit communication and allosteric regulation of enzymatic activity. Comparison of the two GshF structures and mapping of structure-function relationships reveal that the bifunctional GshF structural platform operates as a dynamic dimeric assembly.

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. Here, we report the crystal structures of a stand‐alone SoxY protein of C. limicola f. thiosulfatophilum, solved at 2.15 Å and 2.40 Å resolution using X‐ray diffraction data collected at 100 K and room temperature, respectively. The structure reveals a monomeric Ig‐like protein, with an N‐terminal α‐helix, that oligomerizes into a tetramer via conserved contact regions between the monomers. The tetramer can be described as a dimer of dimers that exhibits one large hydrophobic contact region in each dimer and two small hydrophilic interface patches in the tetramer. At the tetramer interface patch, two conserved redox‐active C‐terminal cysteines form an intersubunit disulfide bridge. Intriguingly, SoxY exhibits a dimer/tetramer equilibrium that is dependent on the redox state of the cysteines and on the type of sulfur substrate component bound to them. Taken together, the dimer/tetramer equilibrium, the specific interactions between the subunits in the tetramer, and the significant conservation level of the interfaces strongly indicate that these SoxY oligomers are biologically relevant.

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. Recombinant purified protein was produced in Escherichia coli and crystallized in space group P2(1)2(1)2(1). Crystals of recombinant TP were obtained in their native form and were soaked with glucose, with n-octyl-beta-D-glucoside and with trehalose. The crystals presented a number of challenges including an unusually large unit cell, with a c axis measuring 420 A, and variable diffraction quality. Crystal-dehydration protocols led to improvements in diffraction quality that were often dramatic, typically from 7-8 to 3-4 A resolution. The structure of recombinant TP was determined by molecular replacement to 2.8 A resolution, thus establishing a starting point for investigating the structural and mechanistic determinants of the disaccharide phosphorylase activity. To the best of our knowledge, this is the first crystal structure determination of an inverting trehalose phosphorylase.

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. SYE4 was recombinantly expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1) and were moderately pseudo-merohedrally twinned, emulating a P422 metric symmetry. The native crystals of SYE4 were of exceptional diffraction quality and provided complete data to 1.10 A resolution using synchrotron radiation, while crystals of the reduced enzyme and of the enzyme in complex with a wide range of ligands typically led to high-quality complete data sets to 1.30-1.60 A resolution, thus providing a rare opportunity to dissect the structure-function relationships of a good-sized enzyme (40 kDa) at true atomic resolution. Here, the attainment of a number of experimental milestones in the crystallographic studies of SYE4 and its complexes are reported, including isolation of the elusive hydride-Meisenheimer complex.

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 2.5.1.18) are found in all kingdoms of life and are involved in phase II detoxification processes by catalyzing the nucleophilic attack of reduced glutathione on diverse electrophilic substrates, thereby decreasing their reactivity. Structure-function studies of prokaryotic GST-like proteins are surprisingly underrepresented in the scientific literature when compared with eukaryotic GSTs. Here, the production and purification of recombinant SoGST3 (SO_1576) and SoGST6 (SO_4697), two of the six GST-like proteins in S. oneidensis, are reported and preliminary crystallographic studies of crystals of the recombinant enzymes are presented. SoGST3 was crystallized in two different crystal forms in the presence of GSH and DTT that diffracted to high resolution: a primitive trigonal form in space group P3(1) that exhibited merohedral twinning with a high twin fraction and a primitive monoclinic form in space group P2(1). SoGST6 yielded primitive orthorhombic crystals in space group P2(1)2(1)2(1) from which diffraction data could be collected to medium resolution after application of cryo-annealing protocols. Crystal structures of both SoGST3 and SoGST6 have been determined based on marginal search models by maximum-likelihood molecular replacement as implemented in the program Phaser.

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. Here, the heterologous expression, purification, and the crystallization of stable tetrameric SoxY are reported. Crystals of SoxY diffract to 2.15 A resolution and belong to space group C222(1), with unit-cell parameters a = 41.22, b = 120.11, c = 95.30 A. MIRAS data from Pt(2+)- and Hg(2+)-derivatized SoxY crystals resulted in an interpretable electron-density map at 3 A resolution after density modification.

Structural insights into component SoxY of the thiosulfate-oxidizing multienzyme system of Chlorobaculum thiosulfatiphilum

We discuss the crystal structure of component SoxY of the SoxYZ complex that is known to play a key role in the sulfur-oxidizing multienzyme system of the green sulfur bacterium Chlorobaculum thiosulfatiphilum. The protein appears to be structurally similar to a monomeric immunoglobulin-like protein that oligomerizes into a tetramer via conserved contact regions between the monomers. The tetramer is a dimer of dimers and exhibits one large hydrophobic contact region in each dimer, and two small hydrophilic interface patches between the dimers. At the tetramer interface patch, two conserved redox-active C-terminal cysteines form an intersubunit disulfide bridge. Depending on the redox state of the cysteines, the tetramer is in equilibrium with the dimers, each one of which is a candidate to covalently bind a thiosulfate molecule by means of a thiol-disulfide exchange reaction with the interprotein disulfide bonds. The significant conservation level of the interfaces, the specific interactions between the subunits in the tetramer, and the dimer-tetramer equilibrium suggest that these SoxY oligomers are biologically relevant. A possible role for these protomers in the mechanism of the Sox-system is proposed.