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 enzyme's 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.

Oligosaccharide binding in family 8 glycosidases: crystal structures of active-site mutants of the beta-1,4-xylanase pXyl from Pseudoaltermonas haloplanktis TAH3a in complex with substrate and product.

The structures of inactive mutants D144A and E78Q of the glycoside hydrolase family 8 (GH-8) endo-beta-1,4-d-xylanase (pXyl) from the Antarctic bacterium Pseudoalteromonas haloplanktis TAH3a in complex with its substrate xylopentaose (at 1.95 A resolution) and product xylotriose (at 1.9 A resolution) have been determined by X-ray crystallography. A detailed comparative analysis of these with the apo-enzyme and with other GH-8 structures indicates an induced fit mechanism upon ligand binding whereby a number of conformational changes and, in particular, a repositioning of the proton donor into a more catalytically competent position occurs. This has also allowed for the description of protein-ligand interactions in this enzyme and for the demarcation of subsites -3 to +3. An in-depth analysis of each of these subsites gives an insight into the structure-function relationship of this enzyme and the basis of xylose/glucose discrimination in family 8 glycoside hydrolases. Furthermore, the structure of the -1/+1 subsite spanning complex reveals that the substrate is distorted from its ground state conformation. Indeed, structural analysis and in silico docking studies indicate that substrate hydrolysis in GH-8 members is preceded by a conformational change, away from the substrate ground-state chair conformation, to a pretransition state local minimum (2)S(O) conformation.

Study of the active site residues of a glycoside hydrolase family 8 xylanase.

Site-directed mutagenesis and a comparative characterisation of the kinetic parameters, pH dependency of activity and thermal stability of mutant and wild-type enzymes have been used in association with crystallographic analysis to delineate the functions of several active site residues in a novel glycoside hydrolase family 8 xylanase. Each of the residues investigated plays an essential role in this enzyme: E78 as the general acid, D281 as the general base and in orientating the nucleophilic water molecule, Y203 in maintaining the position of the nucleophilic water molecule and in structural integrity and D144 in sugar ring distortion and transition state stabilization. Interestingly, although crystal structure analyses and the pH-activity profiles clearly identify the functions of E78 and D281, substitution of these residues with their amide derivatives results in only a 250-fold and 700-fold reduction in their apparent k(cat) values, respectively. This, in addition to the observation that the proposed general base is not conserved in all glycoside hydrolase family 8 enzymes, indicates that the mechanistic architecture in this family of inverting enzymes is more complex than is conventionally believed and points to a diversity in the identity of the mechanistically important residues as well as in the arrangement of the intricate microenvironment of the active site among members of this family.

Glutathione reductase of the malarial parasite Plasmodium falciparum: crystal structure and inhibitor development.

The malarial parasite Plasmodium falciparum is known to be sensitive to oxidative stress, and thus the antioxidant enzyme glutathione reductase (GR; NADPH+GSSG+H(+) <==> NADP(+)+2 GSH) has become an attractive drug target for antimalarial drug development. Here, we report the 2.6A resolution crystal structure of P.falciparum GR. The homodimeric flavoenzyme is compared to the related human GR with focus on structural aspects relevant for drug design. The most pronounced differences between the two enzymes concern the shape and electrostatics of a large (450A(3)) cavity at the dimer interface. This cavity binds numerous non-competitive inhibitors and is a target for selective drug design. A 34-residue insertion specific for the GRs of malarial parasites shows no density, implying that it is disordered. The precise location of this insertion along the sequence allows us to explain the deleterious effects of a mutant in this region and suggests new functional studies. To complement the structural comparisons, we report the relative susceptibility of human and plasmodial GRs to a series of tricyclic inhibitors as well as to peptides designed to interfere with protein folding and dimerization. Enzyme-kinetic studies on GRs from chloroquine-resistant and chloroquine-sensitive parasite strains were performed and indicate that the structure reported here represents GR of P.falciparum strains in general and thus is a highly relevant target for drug development.

Flt3 ligand structure and unexpected commonalities of helical bundles and cystine knots.

Human Flt3 ligand (Flt3L) stimulates early hematopoiesis by activating a type III tyrosine kinase receptor on primitive bone marrow stem cells. The crystal structure of soluble Flt3L reveals that it is a homodimer of two short chain alpha-helical bundles. Comparisons of structure-function relationships of Flt3L with the homologous hematopoietic cytokines macrophage colony stimulating factor (MCSF) and stem cell factor (SCF) suggest that they have a common receptor binding mode that is distinct from the paradigm derived from the complex of growth hormone with its receptor. Furthermore, we identify recognition features common to all helical and cystine-knot protein ligands that activate type III tyrosine kinase receptors, and the closely related type V tyrosine kinase receptors.

Crystal structure of the hexameric traffic ATPase of the Helicobacter pylori type IV secretion system.

The type IV secretion system of Helicobacter pylori consists of 10--15 proteins responsible for transport of the transforming protein CagA into target epithelial cells. Secretion of CagA crucially depends on the hexameric ATPase, HP0525, a member of the VirB11-PulE family. We present the crystal structure of a binary complex of HP0525 bound to ADP. Each monomer consists of two domains formed by the N- and C-terminal halves of the sequence. ADP is bound at the interface between the two domains. In the hexamer, the N- and C-terminal domains form two rings, which together form a chamber open on one side and closed on the other. A model is proposed in which HP0525 functions as an inner membrane pore, the closure and opening of which is regulated by ATP binding and ADP release.

Enzyme inactivation through sulfhydryl oxidation by physiologic NO-carriers.

Nitric oxide (NO) is a pluripotent regulatory molecule, yet the molecular mechanisms by which it exerts its effects are largely unknown. Few physiologic target molecules of NO have been identified, and even for these, the modifications caused by NO remain uncharacterized. Human glutathione reductase (hGR), a central enzyme of cellular antioxidant defense, is inhibited by S-nitrosoglutathione (GSNO) and by diglutathionyl-dinitroso-iron (DNIC-[GSH]2), two in vivo transport forms of NO. Here, crystal structures of hGR inactivated by GSNO and DNIC-[GSH]2 at 1.7 A resolution provide the first picture of enzyme inactivation by NO-carriers: in GSNO-modified hGR, the active site residue Cys 63 is oxidized to an unusually stable cysteine sulfenic acid (R-SOH), whereas modification with DNIC-[GSH]2 oxidizes Cys 63 to a cysteine sulfinic acid (R-SO2H). Our results illustrate that various forms of NO can mediate distinct chemistry, and that sulfhydryl oxidation must be considered as a major mechanism of NO action.

Charge is the major discriminating factor for glutathione reductase versus trypanothione reductase inhibitors.

Benson et al. (Biochem. J. 1992, 286, 9) reported three novel competitive inhibitors of trypanothione reductase (TR), which were selected to complement a hydrophobic region identified on the TR structure which was not present on human glutathione reductase (hGR). Benson et al. also noted that chlorpromazine, a tricyclic antidepressant known to have trypanocidal activity, was an inhibitor of TR. Here we show that chlorpromazine is a competitive inhibitor of TRs from Crithidia fasciculata (Ki = 14 microM) and Trypanosoma cruzi (Ki = 10 microM), but the drug binds > 50-fold more weakly (Ki = 762 microM) to hGR. Analogues of chlorpromazine differing in the length of the side chain carrying the positively charged R-group are also selective TR inhibitors whereas, a tricyclic structure carrying a negatively charged side chain is a competitive inhibitor with selectivity for hGR (K(hGR)i = 165 microM vs. K(TR)i = 1400 microM). This finding suggests that simple charge characteristics, rather than differences in hydrophobicity, may account for a significant portion of the selectivity of this series of inhibitors for these two enzymes. Electrostatic analysis of the structures of TR and hGR thus provides a rationale for these results, and offers a new principle for inhibitor design. The principle gains further support from the observation that all known tricyclic competitive inhibitors of TR are positively charged. In order to investigate the in vivo relevance of our findings we have examined the effect of chlorpromazine and its negatively charged analogue on the growth of C. fasciculata parasites. Consistent with our kinetics, chlorpromazine (50 microM) inhibited the growth of parasites by 50%, while no measurable decrease in parasite growth rate was noted in the presence of the negatively charged inhibitor (400 microM). Furthermore, the highly similar inhibitory profiles of C. fasciculata TR and T. cruzi TR suggest that drug-design studies using the structurally better-studied C. fasciculata TR are also relevant to the human pathogen T. cruzi.

Kinetics and crystallographic analysis of human glutathione reductase in complex with a xanthene inhibitor.

We have determined the crystal structure of a complex between the noncompetitive inhibitor (Kis = 27 microM, Kii = 48 microM with respect to oxidized glutathione (GSSG) and Kis = 144 microM, Kii = 176 microM with respect to NADPH) 6-hydroxy-3-oxo-3H-xanthene-9-propionic acid (XAN) and human glutathione reductase (hGR). The structure, refined to an R-factor of 0.158 at 2.0 A resolution, reveals XAN bound in the large cavity present at the hGR dimer interface where it does not overlap the glutathione binding site. The inhibitor binding causes extensive local structural changes that primarily involve amino acid residues from a 30-residue alpha-helix that lines the cavity and contributes to the active site of hGR. Despite the lack of physical overlap of XAN with the GSSG binding site, no GSSG binding is seen in soaks carried out with high XAN and GSSG concentrations, suggesting that some subtle interaction between the sites exists. An earlier crystallographic analysis on the complex between hGR and 3,7-diamino-2,8-dimethyl-5-phenyl-phenazinium chloride (safranin) showed that safranin bound at this same site. We have found that safranin also inhibits hGR in a noncompetitive fashion, but it binds about 16 times less tightly (Kis = 453 microM, Kii = 586 microM with respect to GSSG) than XAN and does not preclude the binding of GSSG in the crystal. Although in structure-based drug design competitive inhibitors are usually targetted, XAN's binding to a well defined site that is unique to glutathione reductase suggests that noncompetitive inhibitors could also serve as lead compounds for structure-based drug design, in particular as components of chimeric inhibitors.

Overexpression of Crithidia fasciculata trypanothione reductase and crystallization using a novel geometry.

TR1, a previously cloned gene for Crithidia fasciculata trypanothione reductase (TR), has been overexpressed in Escherichia coli strain SG5 to produce about 20 mg enzyme 1(-l) of culture. Since natural C. fasciculata TR is heterogeneous, this expression system provides an important source of homogeneous C. fasciculata TR for use in structural studies and drug design. Steady-state kinetic constants of the purified recombinant enzyme are K(m) = 56 micro M and k(cat) = 10 500 min(-1). Four crystal forms of TR1 were grown using this preparation. Synchrotron radiation was crucial to discover the high level of order present in crystal form IV, which diffracts to about 1.4 A resolution. To optimize growth and handling of form IV crystals, a novel crystallization setup called the 'plug drop' was developed.