Fragment Screening Hit Draws Attention to a Novel Transient Pocket Adjacent to the Recognition Site of the tRNA-Modifying Enzyme TGT.

Fragment-based lead discovery was applied to tRNA-guanine transglycosylase, an enzyme modifying post-transcriptionally tRNAs in Shigella, the causative agent of shigellosis. TGT inhibition prevents translation of Shigella's virulence factor VirF, hence reducing pathogenicity. One discovered fragment opens a transient subpocket in the preQ1-recognition site by pushing back an aspartate residue. This step is associated with reorganization of further amino acids structurally transforming a loop adjacent to the recognition site by duplicating the volume of the preQ1-recognition pocket. We synthesized 6-carboxamido-, 6-hydrazido-, and 4-guanidino-benzimidazoles to target the opened pocket, including a dihydro-imidazoquinazoline with a propyn-1-yl exit vector pointing into the transient pocket and displacing a conserved water network. MD simulations and hydration-site analysis suggest water displacement to contribute favorably to ligand binding. A cysteine residue, exclusively present in bacterial TGTs, serves as gatekeeper of the transient subpocket. It becomes accessible upon pocket opening for selective covalent attachment of electrophilic ligands in eubacterial TGTs.

Arabidopsis thaliana RGXT1 and RGXT2 encode Golgi-localized (1,3)-alpha-D-xylosyltransferases involved in the synthesis of pectic rhamnogalacturonan-II.

Two homologous plant-specific Arabidopsis thaliana genes, RGXT1 and RGXT2, belong to a new family of glycosyltransferases (CAZy GT-family-77) and encode cell wall (1,3)-alpha-d-xylosyltransferases. The deduced amino acid sequences contain single transmembrane domains near the N terminus, indicative of a type II membrane protein structure. Soluble secreted forms of the corresponding proteins expressed in insect cells showed xylosyltransferase activity, transferring d-xylose from UDP-alpha-d-xylose to l-fucose. The disaccharide product was hydrolyzed by alpha-xylosidase, whereas no reaction was catalyzed by beta-xylosidase. Furthermore, the regio- and stereochemistry of the methyl xylosyl-fucoside was determined by nuclear magnetic resonance to be an alpha-(1,3) linkage, demonstrating the isolated glycosyltransferases to be (1,3)-alpha-d-xylosyltransferases. This particular linkage is only known in rhamnogalacturonan-II, a complex polysaccharide essential to vascular plants, and is conserved across higher plant families. Rhamnogalacturonan-II isolated from both RGXT1 and RGXT2 T-DNA insertional mutants functioned as specific acceptor molecules in the xylosyltransferase assay. Expression of RGXT1- and RGXT2-enhanced green fluorescent protein constructs in Arabidopsis revealed that both fusion proteins were targeted to a Brefeldin A-sensitive compartment and also colocalized with the Golgi marker dye BODIPY TR ceramide, consistent with targeting to the Golgi apparatus. Taken together, these results suggest that RGXT1 and RGXT2 encode Golgi-localized (1,3)-alpha-d-xylosyltransferases involved in the biosynthesis of pectic rhamnogalacturonan-II.

Differential localization of alternatively spliced hypoxanthine-xanthine-guanine phosphoribosyltransferase isoforms in Toxoplasma gondii.

A unique feature of the Toxoplasma gondii purine salvage pathway is the expression of two isoforms of the hypoxanthine-xanthine-guanine phosophoribosyltransferase (HXGPRT) of the parasite encoded by a single genetic locus. These isoforms differ in the presence or absence of a 49-amino acid insertion (which is specified by a single differentially spliced exon) but exhibit similar substrate specificity, kinetic characteristics, and temporal expression patterns. To examine possible functional differences between the two HXGPRT isoforms, fluorescent protein fusions were expressed in parasites lacking the endogenous hxgprt gene. Immunoblot analysis of fractionated cell extracts and fluorescence microscopy indicated that HXGPRT-I (which lacks the 49-amino acid insertion) is found in the cytosol, whereas HXGPRT-II (which contains the insertion) localizes to the inner membrane complex (IMC) of the parasite. Simultaneous expression of both isoforms resulted in the formation of hetero-oligomers, which distributed between the cytosol and IMC. Chimeric constructs expressing N-terminal peptides from either isoform I (11 amino acids) or isoform II (60 amino acids) fused to a chloramphenicol acetyl transferase (CAT) reporter demonstrated that the N-terminal domain of isoform II is both necessary and sufficient for membrane association. Metabolic labeling experiments with transgenic parasites showed that isoform II or an isoform II-CAT fusion protein (but not isoform I or isoform I-CAT) incorporate [(3)H]palmitate. Mutation of three adjacent cysteine residues within the isoform II-targeting domain to serines blocked both palmitate incorporation and IMC attachment without affecting enzyme activity, demonstrating that acylation of N-terminal isoform II cysteine residues is responsible for the association of HXGPRT-II with the IMC.

Analysis of the DXD motifs in human xylosyltransferase I required for enzyme activity.

Human xylosyltransferase I (XT-I) is the initial enzyme involved in the biosynthesis of the glycosaminoglycan linker region in proteoglycans. Here, we tested the importance of the DXD motifs at positions 314-316 and 745-747 for enzyme activity and the nucleotide binding capacity of human XT-I. Mutations of the 314DED316 motif did not have any effect on enzyme activity, whereas alterations of the 745DWD747 motif resulted in reduced XT-I activity. Loss of function was observed after exchange of the highly conserved aspartic acid at position 745 with glycine. However, mutation of Asp745 to glutamic acid retained full enzyme activity, indicating the importance of an acidic amino acid at this position. Reduced substrate affinity was observed for mutants D747G (Km=6.9 microm) and D747E (Km=4.4 microm) in comparison with the wild-type enzyme (Km=0.9 microm). Changing the central tryptophan to a neutral, basic, or acidic amino acid resulted in a 6-fold lower Vmax, with Km values comparable with those of the wild-type enzyme. Despite the major effect of the DWD motif on XT-I activity, nucleotide binding was not abolished in the D745G and D747G mutants, as revealed by UDP-bead binding assays. Ki values for inhibition by UDP were determined to be 1.9-24.6 microm for the XT-I mutants. The properties of binding of XT-I to heparin-beads, the Ki constants for noncompetitive inhibition by heparin, and the activation by protamine were not altered by the generated mutations.

A gene encoding the cytokinin enzyme zeatin O-xylosyltransferase of Phaseolus vulgaris.

Zeatin is the most active and ubiquitous form of the naturally occurring cytokinins. Glycosyl conjugates of zeatin are found in many plant tissues and are considered important for storage and protection against degradative enzymes. Two enzymes catalyzing the formation of O-glycosyl derivatives of zeatin have been characterized, O-glucosyltransferase and O-xylosyltransferase, occurring in seeds of lima bean (Phaseolus lunatus) and bean (Phaseolus vulgaris), respectively. Recently, the ZOG1 gene (zeatin O-glucosyltansferase) was isolated from P. lunatis (). Based on the ZOG1 sequence, the ZOX1 gene (zeatin O-xylosyltransferase) was cloned from P. vulgaris. ZOX1 contains an open reading frame of 1362 bp that codes for a 454-amino acid peptide of 51 kD. The recombinant protein has properties identical to the native enzyme: it catalyzes O-xylosylzeatin formation with UDP-Xyl as a glycosyl donor but does not recognize UDP-Glucose as a substrate. The ZOX1 and ZOG1 genes exhibit 93% identity at the nucleotide level and 90% similarity at the amino acid level. Neither gene contains introns. These zeatin-specific genes and their promoters will be useful for studies of the regulation of active versus storage forms of cytokinins. Comparison of sequences encoding similar enzymes with distinct substrate specificity may lead to identification of epitopes specific to cytokinin and glycosyl donor molecules.

Characterization of five human cDNAs with homology to the yeast SIR2 gene: Sir2-like proteins (sirtuins) metabolize NAD and may have protein ADP-ribosyltransferase activity.

The yeast Sir2 protein regulates epigenetic gene silencing and as a possible antiaging effect it suppresses recombination of rDNA. Studies involving cobB, a bacterial SIR2-like gene, have suggested it could encode a pyridine nucleotide transferase. Here five human sirtuin cDNAs are characterized. The SIRT1 sequence has the closest homology to the S. cerevisiae Sir2p. The SIRT4 and SIRT5 sirtuins more closely resemble prokaryotic sirtuin sequences. The five human sirtuins are widely expressed in fetal and adult tissues. Recombinant E. coli cobT and cobB proteins each showed a weak NAD-dependent mono-ADP-ribosyltransferase activity using 5, 6-dimethylbenzimidazole as a substrate. Recombinant E. coli cobB and human SIRT2 sirtuin proteins were able to cause radioactivity to be transferred from [32P]NAD to bovine serum albumin (BSA). When a conserved histidine within the human SIRT2 sirtuin was converted to a tyrosine, the mutant recombinant protein was unable to transfer radioactivity from [32P]NAD to BSA. These results suggest that the sirtuins may function via mono-ADP-ribosylation of proteins.

Protein processing and auxin response in transgenic tobacco harboring a putative cDNA of zeatin O-xylosyltransferase from Phaseolus vulgaris.

Zeatin is rapidly metabolized to O-xylosylzeatin in Phaseolus vulgaris seeds. The zeatin O-xylosyltransferase mediating this conversion, a 50 kDa protein, occurs mainly in the endosperm, both in the cytoplasm and the nuclei. A monoclonal antibody specific to the enzyme was used to isolate cDNAs from an expression library derived from P. vulgaris seeds. Two highly homologous, full-length cDNAs were isolated. The ORFs encode proteins of 69 and 67 kDa, respectively, with 90% homology at the amino acid level. cDNA-encoded protein obtained from in vitro transcription/translation was processed to protein of 50 kDa by bean endosperm extract. Transgenic tobacco plants harboring the larger ORF under the control of the CaMV35S promoter were more sensitive to the auxin NAA than control plants. The symptoms included leaf chlorosis, restriction of root elongation, and eventual cessation of growth. The antigenic preprotein was processed, and labeled zeatin was converted to O-xylosylzeatin in transgenic plants grown on NAA-containing medium. Analyses of independently transformed families indicated that the presence of the transgene coincided with the increased auxin sensitivity and protein processing correlated with the manifestation of auxin-induced damage. These results suggest that posttranslational processing regulates enzyme activity, and offer the possibility that cytokinin-auxin balance may be affected by stimulation of cytokinin metabolic enzyme activity by auxin.