Proposed amino acid sequence and the 1.63 A X-ray crystal structure of a plant cysteine protease,ervatamin B: some insights into the structural basis of its stability and substrate specificity

The crystal structure of a cysteine protease ervatamin B, isolated from the medicinal plant Ervatamia coronaria, has been determined at 1.63 A. The unknown primary structure of the enzyme could also be traced from the high-quality electron density map. The final refined model, consisting of 215 amino acid residues, 208 water molecules, and a thiosulfate ligand molecule, has a crystallographic R-factor of 15.9% and a free R-factor of 18.2% for F > 2sigma(F). The protein belongs to the papain superfamily of cysteine proteases and has some unique properties compared to other members of the family. Though the overall fold of the structure, comprising two domains, is similar to the others, a few natural substitutions of conserved amino acid residues at the interdomain cleft of ervatamin B are expected to increase the stability of the protein. The substitution of a lysine residue by an arginine (residue 177) in this region of the protein may be important, because Lys --> Arg substitution is reported to increase the stability of proteins. Another substitution in this cleft region that helps to hold the domains together through hydrogen bonds is Ser36, replacing a conserved glycine residue in the others. There are also some substitutions in and around the active site cleft. Residues Tyr67, Pro68, Val157, and Ser205 in papain are replaced by Trp67, Met68, Gln156, and Leu208, respectively, in ervatamin B, which reduces the volume of the S2 subsite to almost one-fourth that of papain, and this in turn alters the substrate specificity of the enzyme.     

Attenuating functions of the C terminus of lambda integrase

The tyrosine family site-specific recombinases, in contrast to the related type I topoisomerases, which act as monomers on a single DNA molecule, rely on multi-protein complexes to synapse partner DNAs and coordinate two sequential strand exchanges involving four nicking-closing reactions. Here, we analyze three mutants of the catalytic domain of lambda integrase (Int), A241V, I353M and W350ter that are defective for normal recombination, but possess increased topoisomerase activity. The mutant enzymes can carry out individual DNA strand exchanges using truncated substrates or Holliday junctions, and they show more DNA-cleavage activity than wild-type Int on isolated att sites. Structural modeling predicts that the substituted residues may destabilize interactions between the C-terminal beta-strand (beta7) of Int and the core of the protein. The cleavage-competent state of Int requires the repositioning of the nucleophile (Y342) located on beta6 and the catalyst K235 located on the flexible beta2-beta3 loop, relative to their positions in a crystal structure of the inactive conformation. We propose that the anchoring of beta7 against the protein core restrains the movement of Tyr342 and/or Lys235, causing an attenuation of cleavage activity in most contexts. Within a bona fide recombination complex, the release of strand beta7 would allow Tyr342 and Lys235 to assume catalytically active conformations in coordination with other Int protomers in the complex. The loss of beta7 packing by misalignment or truncation in the mutant proteins described here causes a loss of regulated activity, thereby favoring DNA cleavage activity in monomeric complexes and forfeiting the coordination of strand-exchange necessary for efficient recombination.     

Binding of specific DNA base-pair mismatches by N-methylpurine-DNA glycosylase and its implication in initial damage recognition

Most DNA glycosylases including N-methylpurine-DNA glycosylase (MPG), which initiate DNA base excision repair, have a wide substrate range of damaged or altered bases in duplex DNA. In contrast, uracil-DNA glycosylase (UDG) is specific for uracil and excises it from both single-stranded and duplex DNAs. Here we show by DNA footprinting analysis that MPG, but not UDG, bound to base-pair mismatches especially to less stable pyrimidine-pyrimidine pairs, without catalyzing detectable base cleavage. Thermal denaturation studies of these normal and damaged (e.g. 1,N(6)-ethenoadenine, varepsilonA) base mispairs indicate that duplex instability rather than exact fit of the flipped out damaged base in the catalytic pocket is a major determinant in the initial recognition of damage by MPG. Finally, based on our determination of binding affinity and catalytic efficiency we conclude that the initial recognition of substrate base lesions by MPG is dependent on the ease of flipping of the base from unstable pairs to a flexible catalytic pocket.     

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.     

Passive cigarette smoke and renal glyoxalase system

Cigarette smoking is associated with a number of fatal diseases, including cancer of different organs. A number of oxoaldehydes are found in cigarette smoke, among which methylglyoxal (MG) is known to cause toxicity to cells upon accumulation. In biological systems, MG is converted to s-d-lactoylglutathione by glyoxalase I with reduced glutathine (GSH) as a cofactor, and s-d-lactoylglutathione is converted to D-lactic acid with simultaneous regeneration of GSH, by glyoxalase II. In the present study, we have investigated the status of the glyoxalase enzymes in kidney tissues from rats exposed to passive cigarette smoke. No significant change has been noted in glyoxalase I activity. Glyoxalase II was decreased during 1 and 2 weeks of exposure, and after that the activity was increased. The initial decrease in the activity of gly II may be due to the excess amount of methylglyoxal generated due to smoke exposure or the adduct formed by MG and GSH which known to inhibit gly II activity. Both enzymes help in the detoxification of cigarette smoke induced chemicals and biochemicals.     

Effect of casein diet on gonadotropin releasing hormone antagonist induced changes in adrenal gonadal functions in male rats

Adult male rats received daily injections (sc) of gonadotropin releasing hormone antagonist (0.2 mg/kg(-1) x day(-1)) for 21 days when they were sacrificed on day 22, adrenal weight, adrenal A5-3beta (delta 5-3beta) hydroxysteroid dehydrogenase (Delta5-3beta-HSD) activity and serum level of corticosterone were increased significantly while testicular 17beta (17beta) hydroxysteroid dehydrogenase (17beta-HSD) activity and serum level of testosterone and spermatogenesis were decreased in the rats fed on 5% casein diet. GnRH antagonist treated rats fed on 20% casein diet, resulted significant decrease in adrenal weight, serum corticosterone and adrenal A5-3beta-HSD activity while testicular 17beta-HSD activity serum testosterone levels and the weights of sex organs were increased with respect to anti GnRH treated rats fed on 5% casein diet. But the GnRH antagonist treated rats fed on 20% casein diet showed decreased spermatogenesis quantitatively and sperm count appeared similar to anti GnRH treated rats fed on 5% casein diet. These results indicate that high casein diet protects adrenocortical activity and stimulates testosterone synthesis without effecting spermatogenic arrest in GnRH antagonist treated rats. It may be concluded that GnRH antagonist in presence of high milk protein diet may be considered to be a suitable antihormone in the development of an ideal male contraceptive.     

High affinity association of myo-inositol trisphosphates with phytase and its effect upon the catalytic potential of the enzyme

A neutral phytase from germinating mung bean (Vigna radiata) seeds dephosphorylates myo-inositol hexakisphosphate sequentially to myo-inositol. The enzyme also binds with higher affinity to myo-inositol trisphosphates (1,4,5), (2,4,5), and (1,3,4) isomers without catalysis. The high affinity complex elicits Ca(2+) mobilization in vitro from microsomes/vacuoles via the formation of a ternary complex with the receptor for Ins(1,4,5)P(3). As a sequel to our previous report, we have carried out a detailed characterization of the two sites and examined the mutual interactions between them. Presaturation of the high affinity site leads to an increase in the affinity of the enzyme for phytic acid and its rate of dephosphorylation as well. From the products of limited tryptic cleavage of phytase, two peptides, each with one activity, have been isolated. The larger peptide ( approximately 66 kDa) contains the catalytic site, and the smaller peptide ( approximately 5 kDa) has the high affinity myo-inositol trisphosphate-binding site. The interaction between the dual activities of phytase has been observed also at the level of the two peptides. A sequence homology search using N-terminal 12 amino acid residues of the 5-kDa fragment has revealed significant homology with the Homer class of proteins implicated in signaling pathways involving metabotropic glutamate receptor and myo-inositol 1,4,5-trisphosphate receptor. These results indicate a second role of phytase in Ca(2+) mobilization during germination of mung been seed via a salvage pathway that involves allosteric activation by myo-inositol trisphosphate.     

Cell volume changes affect gluconeogenesis in the perfused liver of the catfish<Emphasis Type="Italic">Clarias batrachus</Emphasis>

In addition to lactate and pyruvate, some amino acids were found to serve as potential gluconeogenic substrates in the perfused liver ofClarias batrachus. Glutamate was found to be the most effective substrate, followed by lactate, pyruvate, serine, ornithine, proline, glutamine, glycine, and aspartate. Four gluconeogenic enzymes, namely phosphoenolpyruvate carboxykinase (PEPCK), pyruvate carboxylase (PC), fructose 1,6-bisphosphatase (FBPase) and glucose 6-phosphatase (G6Pase) could be detected mainly in liver and kidney, suggesting that the latter are the two major organs responsible for gluconeogenic activity in this fish. Hypo-osmotically induced cell swelling caused a significant decrease of gluconeogenic efflux accompanied with significant decrease of activities of PEPCK, FBPase and G6Pase enzymes in the perfused liver. Opposing effects were seen in response to hyperosmotically induced cell shrinkage. These changes were partly blocked in the presence of cycloheximide, suggesting that the aniso-osmotic regulations of gluconeogenesis possibly occurs through an inverse regulation of enzyme proteins and/or a regulatory protein synthesis in this catfish. In conclusion, gluconeogenesis appears to play a vital role inC. batrachus in maintaining glucose homeostasis, which is influenced by cell volume changes possibly for proper energy supply under osmotic stress.