Novel approach for structural identification of protein family: glyoxalase I

Glyoxalase is one of two enzymes of the glyoxalase detoxification system against methylglyoxal and other aldehydes, the metabolites derived from glycolysis. The glyoxalase system is found almost in all living organisms: bacteria, protozoa, plants, and animals, including humans, and is related to the class of ‘life essential proteins’. The enzyme belongs to the expanded Glyoxalase/Bleomycin resistance protein/Dioxygenase superfamily. At present the GenBank contains about 700 of amino acid sequences of this enzyme type, and the Protein Data Bank includes dozens of spatial structures. We have offered a novel approach for structural identification of glyoxalase I protein family, which is based on the selecting of basic representative proteins with known structures. On this basis, six new subfamilies of these enzymes have been derived. Most populated subfamilies A1 and A2 were based on representative human Homo sapiens and bacterial Escherichia coli enzymes. We have found that the principle feature, which defines the subfamilies’ structural differences, is conditioned by arrangement of N- and C-domains inside the protein monomer. Finely, we have deduced the structural classification for the glyoxalase I and assigned about 460 protein sequences distributed among six new subfamilies. Structural similarities and specific differences of all the subfamilies have been presented. This approach can be used for structural identification of thousands of the so-called hypothetical proteins with the known PDB structures allowing to identify many of already existing atomic coordinate entrees.     

A reinvestigation of inhibitors of glyoxalase I

It has been reported earlier that nucleotides, nucleosides and a series of structurally related compounds as well as compounds based on transition state analogy inhibit yeast glyoxalase I. In our study on the metabolic regulation of glyoxalase I, we have found that nucleotides such as ATP, GTP and different classes of other reagents based on transition state analogy (D-isoascorbate, dihydroxyfumaric acid, rhodizonic acid) do not inhibit yeast or goat liver glyoxalase I. The reported inhibition of glyoxalase I by these compounds has been found to be due to the interference of these compounds with the absorbancy at 240 nm of S-D-lactoylglutathione formed by the glyoxalase I reaction. Glyoxalase I from goat liver has been found to be strongly and competitively inhibited by lactaldehyde. But, lactaldehyde has very little inhibitory effect on yeast glyoxalase I. Lactaldehyde is formed from methylglyoxal, the substrate for glyoxalase I by the enzyme methylglyoxal reductase. D-Lactaldehyde inhibits the liver enzyme more strongly than L-lactaldehyde.     

Human red cell glyoxalase I polymorphism

Human erythrocyte glyoxalase I has been subjected to starch gel electrophoresis, and its isoenzymatic forms have been visualized by a new positive staining procedure. The enzyme exhibits polymorphism and holds promise as a useful new genetic marker.     

Oltipraz protects the passive smoke induced changes in renal glyoxalase system of rats

The present study was performed to investigate the effect of oltipraz on passive smoke-induced alteration in renal glyoxalase system of rats. Adult Sprague-Dawley rats were exposed daily to passive cigarette smoke in a whole-body exposure chamber 6 h per day for 2, 4 and 12 weeks. The animals being sacrificed after 2 and 12 weeks were maintained on control diet, powdered 4% Teklad rat chow (Harlan Teklad, Madison, WI, USA). The 4 weeks group was divided into three subgroups, one receiving control diet, other two receiving control diet supplemented with two doses of oltipraz (either 167 or 500 ppm), starting 1 week prior to initiation of smoke exposure until the end of the experiment. The activity of glyoxalase I was higher in animals exposed for 4 and 12 weeks of passive smoke than those exposed for 2 weeks. There was no significant difference between 4 and 12 weeks. Glyoxalase II activity was lower in animals exposed to passive smoke for 4 weeks than those exposed for 2 weeks. However, the activity approached the basal level after 12 weeks of exposure. Furthermore, oltipraz treatment maintained the activity of both glyoxalase closer to the basal levels.     

Erythrocyte glyoxalase I polymorphism in the owl monkey,Aotus

Three erythrocyte glyoxalase I phenotypes were observed in a sample of 235 karyotypically defined New World owl monkeys, Aotus. The selective distribution of glyoxalase I allele (GLO¹, GLO²) is related to the karyotype of each animal. Owl monkeys with a karyotype VI had an equal distribution of GLO¹ and GLO² genes in the population. Aotus with karyotype II, III, IV, or V had, exclusively, the GLO² allele (expressed as the fast electrophoretic phenotype), in contrast with monkeys with karyotype I or VII, which had only the GLO¹ allele (expressed as the slow electrophoretic phenotype).     

Structural investigation into the inhibitory mechanisms of indomethacin and its analogues towards human glyoxalase I

In the present work, a combined study of kinetic analysis, molecular docking, and molecular dynamics simulations on indomethacin and its analogues is performed to better understand their inhibitory mechanisms towards human glyoxalase I (GLOI). A remarkable correlation (R² = 0.974) was observed for six inhibitors including indomethacin between their experimental inhibitory affinities and predicted binding free energy parameter (ΔG_bind,pred). This suggests that ΔG_bind,pred of a GLOI/inhibitor complex can be efficiently used to interpolate the experimental inhibitory affinity of a ligand of similar nature in the GLOI enzyme system. Energetic analyses revealed that electrostatic contribution plays an important role in their inhibitory mechanisms, which reflects the significant contribution of the coordination bond between zinc and ligands. The present work highlights that indomethacin is a promising lead as GLOI inhibitors for further development since it may bind all subsites in the active site pocket of GLOI and stabilize the flexible loop (152-159).     

Radiomodification of glyoxalase I in the liver and spleen of mice: Adaptive response and split-dose effect

Glyoxalase system, particularly glyoxalase I (Gly I) plays an important role in regulation of cell division and is considered to be a metabolic indicator of cell proliferation. The glyoxalase system is likely to have a close link with cellular radiosensitivity. Therefore, we have examined the effect of adaptive and split-dose of -rays on the activity of Gly I in the liver and spleen of mice. For the adaptive response studies, mice pre-treated with a conditioning dose of 0.5 Gy were given a challenging dose of 4 Gy at varying time intervals. In the split-dose studies, a dose of 4 Gy was delivered into two equal fractions and spaced at different time intervals. The results show that pre-exposure to a conditioning dose or the fractionation of total dose decreased the specific activity of Gly I in the liver and spleen of mice. The decreased activity of Gly I was suggestive of protective action induced by the conditioning dose and fractionation of dose. The similar pattern of radiation response of Gly I probably supported the possibility of involvement of a common pathway in the radiation-induced adaptive and split-dose effect. From these observations a close link between the Gly I and the adaptive-response as well as the split-dose effect is speculated. Since, the glyoxalase system is vital for a variety of biological functions including cell division and repair, the present findings may have relevance in understanding the dose-fractionation as well as the biological defence induced by low doses of radiations.     

Crystal structure of type II peptide deformylase from Staphylococcus aureus

The first crystal structure of Class II peptide deformylase has been determined. The enzyme from Staphylococcus aureus has been overexpressed and purified in Escherichia coli and the structure determined by x-ray crystallography to 1.9 A resolution. The purified iron-enriched form of S. aureus peptide deformylase enzyme retained high activity over many months. In contrast, the iron-enriched form of the E. coli enzyme is very labile. Comparison of the two structures details many differences; however, there is no structural explanation for the dramatic activity differences we observed. The protein structure of the S. aureus enzyme reveals a fold similar, but not identical to, the well characterized E. coli enzyme. The most striking deviation of the S. aureus from the E. coli structure is the unique conformation of the C-terminal amino acids. The distinctive C-terminal helix of the latter is replaced by a strand in S. aureus which wraps around the enzyme, terminating near the active site. Although there are no differences at the amino acid level near the active site metal ion, significant changes are noted in the peptide binding cleft which may play a role in the design of general peptide deformylase inhibitors.     

Molecular docking analysis of Enterotoxin I from Staphylococcus aureus with Nafcillin analogues

Staphylococcal enterotoxins (SEs) are liked with food poisoning and other related infections. Nafcillin is an antibiotic used to treat S. aureus. Therefore, it is of interest to study the molecular interactions of 25 nafcillin analogues with enterotoxin I using molecular docking analysis. The analysis shows optimal interaction features of Nafcillin analogues with Enterotoxin I from Staphylococcus aureus for further consideration.     

Crystal structure of the capsular polysaccharide synthesizing protein CapE of Staphylococcus aureus

Enzymes synthesizing the bacterial CP (capsular polysaccharide) are attractive antimicrobial targets. However, we lack critical information about the structure and mechanism of many of them. In an effort to reduce that gap, we have determined three different crystal structures of the enzyme CapE of the human pathogen Staphylococcus aureus. The structure reveals that CapE is a member of the SDR (short-chain dehydrogenase/reductase) super-family of proteins. CapE assembles in a hexameric complex stabilized by three major contact surfaces between protein subunits. Turnover of substrate and/or coenzyme induces major conformational changes at the contact interface between protein subunits, and a displacement of the substrate-binding domain with respect to the Rossmann domain. A novel dynamic element that we called the latch is essential for remodelling of the protein–protein interface. Structural and primary sequence alignment identifies a group of SDR proteins involved in polysaccharide synthesis that share the two salient features of CapE: the mobile loop (latch) and a distinctive catalytic site (MxxxK). The relevance of these structural elements was evaluated by site-directed mutagenesis.     

Crystal structure of interleukin-19 defines a new subfamily of helical cytokines

Interleukin-19 (IL-19) is a novel cytokine that was initially identified during a sequence data base search aimed at finding potential IL-10 homologs. IL-19 shares a receptor complex with IL-20, indicating that the biological activities of these two cytokines overlap and that both may play an important role in regulating development and proper functioning of the skin. We determined the crystal structure of human recombinant IL-19 and refined it at 1.95-A resolution to an R-factor of 0.157. Unlike IL-10, which forms an intercalated dimer, the molecule of IL-19 is a monomer made of seven amphipathic helices, A-G, creating a unique helical bundle. On the basis of the observed structure, we propose that IL-19, IL-20, and other putative members of the proposed IL-10 family together form a distinct subfamily of helical cytokines.     

Crystal structure of Staphylococcus aureus tRNA adenosine deaminase TadA in complex with RNA

Bacterial tRNA adenosine deaminases (TadAs) catalyze the hydrolytic deamination of adenosine to inosine at the wobble position of tRNA(Arg2), a process that enables this single tRNA to recognize three different arginine codons in mRNA. In addition, inosine is also introduced at the wobble position of multiple eukaryotic tRNAs. The genes encoding these deaminases are essential in bacteria and yeast, demonstrating the importance of their biological activity. Here we report the crystallization and structure determination to 2.0 A of Staphylococcus aureus TadA bound to the anticodon stem-loop of tRNA(Arg2) bearing nebularine, a non-hydrolyzable adenosine analog, at the wobble position. The cocrystal structure reveals the basis for both sequence and structure specificity in the interactions of TadA with RNA, and it additionally provides insight into the active site architecture that promotes efficient hydrolytic deamination.     

Discovery of a new type inhibitor of human glyoxalase I by myricetin-based 4-point pharmacophore

The human glyoxalase I (hGLO I), which is a rate-limiting enzyme in the pathway for detoxification of apoptosis-inducible methylglyoxal (MG), has been expected as an attractive target for the development of new anti-cancer drugs. We have previously identified a natural compound myricetin as a substrate transition-state (Zn²⁺-bound MG-glutathione (GSH) hemithioacetal) mimetic inhibitor of hGLO I. Here, we constructed a hGLO I/inhibitor 4-point pharmacophore based on the binding mode of myricetin to hGLO I. Using this pharmacophore, in silico screening of chemical library was performed by docking study. Consequently, a new type of compound, which has a unique benzothiazole ring with a carboxyl group, named TLSC702, was found to inhibit hGLO I more effectively than S-p-bromobenzylglutathione (BBG), a well-known GSH analog inhibitor. The computational simulation of the binding mode indicates the contribution of Zn²⁺-chelating carboxyl group of TLSC702 to the hGLO I inhibitory activity. This implies an important scaffold-hopping of myricetin to TLSC702. Thus, TLSC702 may be a valuable seed compound for the generation of a new lead of anti-cancer pharmaceuticals targeting hGLO I.     

Detection and recovery of sublethally-injured enterotoxigenic Staphylococcus aureus

AIMS: To determine whether sublethally-injured (acid- or heat-shocked) Staphylococcus aureus cells are recoverable using selective agar overlays. METHODS AND RESULTS: Brain Heart Infusion (BHI) Agar overlaid with either Baird-Parker Agar (BPA) or Gram-Positive Agar (GPA) was compared in the ability to resuscitate heat- and acid-shocked enterotoxigenic Staph. aureus. BHI/BPA overlays allowed for greater recovery of both heat- and acid-shocked cells than BHI/GPA, although the former was not selective and allowed growth of bacteria other than Staph. aureus. No significant difference existed in percent recovery of heat- and acid-shocked cells between the two overlay approaches. Significant differences were noted in counts on BHI/GPA plates and straight selective GPA/GPA plates, however. Viability of heat- and acid-shocked Staph. aureus was also examined using fluorescence microscopy, the relative counts of which correlated well to the calculated percent recovery on selective agar overlays. CONCLUSIONS: This work has shown that an improved agar overlay technique increases the sensitivity of the standard plate count while enumerating sublethally-injured enterotoxigenic Staph. aureus compared with direct plating onto selective media. SIGNIFICANCE AND IMPACT OF THE STUDY: These data emphasize the need to develop practical and cost-effective methods that reliably detect and enumerate sublethally-injured pathogens such as Staph. aureus.     

Crystal structure of Sa240: a ribose pyranase homolog with partial active site from Staphylococcus aureus

Ribose is transported into cells in its pyranose form and must be rearranged to its furanose form for further utilization. Ribose pyranase RbsD catalyzes the conversion of ribose from the pyranose to furanose form. This is the key step for substrate supply to ribokinase RbsK, which converts ribose to ribose-5-phosphate for further metabolism. Sequence analysis indicated Sa240 from Staphylococcus aureus was a ribose pyranase homolog. Here we showed that Sa240 formed dimeric structure both in solution and in crystal. S240-ribose complex structure showed a ribose binding site formed by an incomplete active site compared with RbsD. Because the catalytic activity of ribose pyranase depends on its oligomeric state, we propose Sa240 is catalytically inactive in its dimeric structure.     

Crystal structure of a novel prolidase from Deinococcus radiodurans identifies new subfamily of bacterial prolidases

Xaa‐Pro peptidases (XPP) are dinuclear peptidases of MEROPS M24B family that hydrolyze Xaa‐Pro iminopeptide bond with a trans‐proline at the second position of the peptide substrate. XPPs specific towards dipeptides are called prolidases while those that prefer longer oligopeptides are called aminopeptidases P. Though XPPs are strictly conserved in bacterial and archaeal species, the structural and sequence features that distinguish between prolidases and aminopeptidases P are not always clear. Here, we report 1.4 Å resolution crystal structure of a novel XPP from Deinococcus radiodurans (XPPdr). XPPdr forms a novel dimeric structure via unique dimer stabilization loops of N‐terminal domains such that their C‐terminal domains are placed far apart from each other. This novel dimerization is also the consequence of a different orientation of N‐terminal domain in XPPdr monomer than those in other known prolidases. The enzymatic assays show that it is a prolidase with broad substrate specificity. Our structural, mutational, and molecular dynamics simulation analyses show that the conserved Arg46 of N‐terminal domain is important for the dipeptide selectivity. Our BLAST search found XPPdr orthologs with conserved sequence motifs which correspond to unique structural features of XPPdr, thus identify a new subfamily of bacterial prolidases.