Preliminary X-ray crystallographic analysis of 2-methylcitrate synthase from Salmonella typhimurium

Analysis of the genomic sequences of Escherichia coli and Salmonella typhimurium has revealed the presence of several homologues of the well studied citrate synthase (CS). One of these homologues has been shown to code for 2-methylcitrate synthase (2-MCS) activity. 2-MCS catalyzes one of the steps in the 2-methylcitric acid cycle found in these organisms for the degradation of propionate to pyruvate and succinate. In the present work, the gene coding for 2-MCS from S. typhimurium (StPrpC) was cloned in pRSET-C vector and overexpressed in E. coli. The protein was purified to homogeneity using Ni-NTA affinity chromatography. The purified protein was crystallized using the microbatch-under-oil method. The StPrpC crystals diffracted X-rays to 2.4 A resolution and belonged to the triclinic space group P1, with unit-cell parameters a = 92.068, b = 118.159, c = 120.659 A, alpha = 60.84, beta = 67.77, gamma = 81.92 degrees. Computation of rotation functions using the X-ray diffraction data shows that the protein is likely to be a decamer of identical subunits, unlike CSs, which are dimers or hexamers.

Metabolic enzymes as potential drug targets in Plasmodium falciparum.

Plasmodium falciparum causes the most severe form of malaria that is fatal in many cases. Emergence of drug resistant strains of P. falciparum requires that new drug targets be-identified. This review considers in detail enzymes of the glycolytic pathway, purine salvage pathway, pyrimidine biosynthesis and proteases involved in catabolism of haemoglobin. Structural features of P. falciparum triosephosphate isomerase which could be exploited for parasite specific drug development have been highlighted. Utility of P. falciparum hypoxanthine-guanine-phosphoribosyltransferase, adenylosuccinate synthase, dihydroorotate dehydrogenase, thymidylate synthase-dihydrofolate reductase, cysteine and aspartic proteases have been elaborated in detail. The review also briefly touches upon other potential targets in P. falciparum

Ultra high throughput sequencing excludes MDH1 as candidate gene for RP28-linked retinitis pigmentosa

Purpose: Mutations in IDH3B, an enzyme participating in the Krebs cycle, have recently been found to cause autosomal recessive retinitis pigmentosa (arRP). The MDH1 gene maps within the RP28 arRP linkage interval and encodes cytoplasmic malate dehydrogenase, an enzyme functionally related to IDH3B. As a proof of concept for candidate gene screening to be routinely performed by ultra high throughput sequencing (UHTs), we analyzed MDH1 in a patient from each of the two families described so far to show linkage between arRP and RP28. Methods: With genomic long-range PCR, we amplified all introns and exons of the MDH1 gene (23.4 kb). PCR products were then sequenced by short-read UHTs with no further processing. Computer-based mapping of the reads and mutation detection were performed by three independent software packages. Results: Despite the intrinsic complexity of human genome sequences, reads were easily mapped and analyzed, and all algorithms used provided the same results. The two patients were homozygous for all DNA variants identified in the region, which confirms previous linkage and homozygosity mapping results, but had different haplotypes, indicating genetic or allelic heterogeneity. None of the DNA changes detected could be associated with the disease.

Studies on iron metabolism in Neurospora crassa

Neurospora crassa Em 5297a secretes an ironbinding compound (X) when grown under conditions of iron deficiency. Decreasing the concentration of iron in the medium results in an increase of X and a corresponding fall in catalase activity. Under iron-deficient conditions the production of X precedes the fall in catalase activity. The iron complex of the iron-binding compound (XFe) can act as a good iron source to the organism to maintain normal growth and catalase activity, even though the iron is held very firmly in the chemical sense. While ferrichrome is as potent as XFe, as an iron source to N. crassa, ferrichrome A and ferric acethydroxamate are only partially beneficial. XFe, when provided as the sole iron source, also influences nonheme iron enzyme activities like succinic dehydrogenase and aconitase. XFe is permeable to N. crassa mycelia and is incorporated at a much faster rate compared with that from a simple chelate such as ferric citrate.

Oxidation of vitamin A1 and vitamin A2 aldehydes to the corresponding acids by enzymes from pig and rat livers

VITAMIN A is stored in rat liver largely as its ester with small amounts of the alcohol, but is transported in the normal circulating blood in the latter form1. Although it was generally believed that the alcohol form is the more physiological state of the vitamin, since the work of Dowling and Wald2, it is being recognized that vitamin A acid and not the alcohol may be nearer to the 'active vitamin A'. If this were to be so, it would be important to demonstrate that a mechanism exists in the rat for the production of vitamin A acid from vitamin A alcohol through the intermediate, the aldehyde. Regarding the formation of the aldehyde, it has been well established that the alcohol dehydrogenase can bring about the conversion of vitamin A alcohol to retinene3. The presence of an enzyme in rat and pig liver catalysing the oxidation of retinene1 and retinene2 to the corresponding acids has been demonstrated in the present work and the partially purified enzyme preparation shown to be completely devoid of alcohol dehydrogenase activity.

Synthesis and identification of a new class of (S)-2,6-diamino-4,5,6,7-tetrahydrobenzo[d]thiazole derivatives as potent antileukemic agents

Benzothiazoles are multitarget agents with broad spectrum of biological activity. Among the antitumor agents discovered in recent years, the identification of various 2-(4-aminophenyl) benzothiazoles as potent and selective antitumor drugs against different cancer cell lines has stimulated remarkable interest. Some of the benzothiazoles are known to induce cell cycle arrest, activation of caspases and interaction with DNA molecule. Based on these interesting properties of benzothiazoles and to obtain new biologically active agents, a series of novel 4,5,6,7-tetrahydrobenzo[d]thiazole derivatives 5(a-i) were synthesized and evaluated for their efficacy as antileukemic agents in human leukemia cells (K562 and Reh). The chemical structures of the synthesized compounds were confirmed by H-1 NMR, LCMS and IR analysis. The cytotoxicity of these compounds were determined using trypan blue exclusion, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Results showed that, these compounds mediate a significant cytotoxic response to cancer cell lines tested. We found that the compounds having electron withdrawing groups at different positions of the phenyl ring of the thiourea moiety displayed significant cytotoxic effect with IC50 value less than 60 mu M. To rationalize the role of electron withdrawing group in the induction of cytotoxicity, we have chosen molecule 5g (IC50 similar to 15 mu M) which is having chloro substitution at ortho and para positions. Flow cytometric analysis of annexin V-FITC/ propidium iodide (PI) double staining and DNA fragmentation suggest that 5g can induce apoptosis.

A binderless, bulk modified, renewable surface amperometric sensor for NADH and ethanol

Graphite particles are exfoliated and subsequently functionalized with toluidine blue. The resulting covalently modified graphite particles are restacked without any binder to form a surface-renewable, bulk-modified electrode. Electrocatalytic oxidation of NADH and its application in the amperometric biosensing of ethanol using alcohol dehydrogenase enzyme have been demonstrated with this material.

Synthesis and Antileukemic Activity of 1-((S)-2-Amino-4,5,6,7-tetrahydrobenzo[d]thiazol-6-yl)-3-(substituted phenyl)urea Derivatives

Heterocyclic urea derivatives play an important role as anticancer agents because of their good inhibitory activity against receptor tyrosine kinases (RTKs), raf kinases, protein tyrosine kinases (PTKs), and NADH oxidase, which play critical roles in many aspects of tumorigenesis. Benzothiazole moiety constitutes an important scaffold of drugs, possessing several pharmacological functions, mainly the anticancer activity. Based on these interesting properties of benzothiazoles and urea moiety to obtain new biologically active agents, we synthesized a series of novel 1-((S)-2-amino-4,5,6.7-tetrahydrobenzo[d]thiazol-6-yl)-3-(substituted phenyl)urea derivatives and evaluated for their efficacy as antileukemic agents against two human leukemic cell lines (K562 and Reh). These compounds showed good and moderate cytotoxic effect to cancer cell lines tested. Compounds with electron-withdrawing chloro and fluoro substituents on phenyl ring showed good activity and compounds with electron-donating methoxy group showed moderate activity. Compound with electron-withdrawing dichloro substitution on phenyl ring of aryl urea showed good activity. Further, lactate dehydrogenase (LDH) assay, flow cytometric analysis of annexin V-FITC/propidium iodide (PI) double staining and DNA fragmentation studies showed that compound with dichloro substitution on phenyl ring of aryl urea can induce apoptosis.

Simultaneous effect of lead and cadmium on granulosa cells: A cellular model for ovarian toxicity

Lead (Pb) and cadmium (Cd) are known reproductive toxicants, which accumulate in granulosa cells of the ovary. Female Charles foster rats were treated with sodium acetate (control), lead acetate and cadmium acetate either alone or in combination at a dose 0.05 mg/kg body weight intra-peritoneally for 15 days daily. Animals were killed at proestrous stage and granulosa cells were isolated from the ovaries. Binding of I-125-luteinizing hormone (I-125-LH), I-125-follicle stimulating hormone (I-125-FSH) and 17 beta-hydroxysteroid dehydrogenase activity were measured. As these receptors are localized on the surface of the cell membrane, we also estimated the membrane parameters of these cells. Our results demonstrated that both lead and cadmium caused a significant reduction in gonadotropin binding, which altered steroidogenic enzyme activity of granulosa cells. These changes exhibited a positive correlation with membrane changes of the granulosa cells.

Using Water as a Design Element in Crystal Engineering. Host-Guest Compounds of Hydrated 3,5-Dihydroxybenzoic Acid

Thirteen host guest compounds of 3,5-dihydroxybenzoic acid (DHBA) have been structurally characterized. Water molecules occupy the peripheries of a hexagonal void, created with DHBA molecules, and act as ``hooks'' to connect the guest molecules with the host-framework via hydrogen bonding. The ``water hook'' is an OH group acting as a donor. Consequently, the guest molecules were chosen so that they contain good hydrogen bond acceptor functionalities. A number of multicomponent hydrates were isolated with stoichiometries (DHBA)(x)(H2O). (guest),. Of these, compounds with the following as guests were obtained as crystals that were good enough for single crystal work: ethyl acetate (EtOAc), diethyl oxalate, dimethyl oxalate, di(n-propyl) oxalate, diethyl malonate, diethyl succinate, chloroacetonitrile, N,N-dimethyl formamide (DMF), acetone, dimethyl sulfoxide (DMSO), 1-propanol, and 2-butanol. From 2-butanol, a hemihydrate, (DHBA)(2)(H2O), was also obtained concomitantly. Further to guest stabilization, water acts as a good mediator of effective crystal packing and also determines the topology of the host framework. En the present series of compounds, the role of water is wide ranging, and it is not easy to classify it specifically as a host or as a guest.

Triacylglycerol synthesis in developing seeds of groundnut (Arachis hypogaea): Pathway and properties of enzymes of sn-Glycerol 3-phosphate formation

The enzymatic pathway for the synthesis of sn-glycerol 3-phosphate was investigated in developing groundnut seeds (Arachis hypogaea). Glycerol-3-phosphate dehydrogenase was not detected in this tissue but an active glycerokinase was demonstrated in the cytosolic fraction. It showed an optimum pH at 8.6 and positive cooperative interactions with both glycerol and ATP. Triosephosphate isomerase and glyceraldehyde-3-phosphate phosphatase were observed mainly in the cytosolic fraction while an active glyceraldehyde reductase was found mainly in the mitochondrial and microsomal fractions. The glyceraldehyde 3-phosphate phosphatase showed specificity and positive cooperativity with respect to glyceraldehyde 3-phosphate. The glyceraldehyde reductase was active toward glucose and fructose but not toward formaldehyde and showed absolute specificity toward NADPH. It is concluded that in the developing groundnut seed, sn-glycerol 3-phosphate is synthesized essentially by the pathway dihydroxyacetone phosphate ? glyceraldehyde 3-phosphate ?Pi glyceraldehyde ?NADPH glycerol ?ATP glycerol 3-phosphate. All the enyzmes of this pathway showed activity profiles commensurate with their participation in triacylglycerol synthesis which is maximal during the period 15�35 days after fertilization. Glycerokinase appears to be the rate-limiting enzyme in this pathway.

Protoporphyrinogen IX oxidase from Plasmodium falciparum is anaerobic and is localized to the mitochondrion

Earlier studies in this laboratory had shown that the malarial parasite can synthesize heme de novo and inhibition of the pathway leads to death of the parasite. It has been proposed that the pathway for the biosynthesis of heme in Plasmodium falciparum is unique involving three different cellular compartments, namely mitochondrion, apicoplast and cytosol. Experimental evidences are now available for the functionality and localization of all the enzymes of this pathway, except protoporphyrinogen IX oxidase (PfPPO), the penultimate enzyme. In the present study. PfPPO has been cloned, expressed and shown to be localized to the mitochondrion by immunofluorescence microscopy. Interestingly, the enzyme has been found to be active only under anaerobic conditions and is dependent on electron transport chain (ETC) acceptors for its activity. The native enzyme present in the parasite is inhibited by the ETC inhibitors, atovaquone and antimycin. Atovaquone, a well known inhibitor of parasite dihydroorotate dehydrogenase, dependent on the ETC, inhibits synthesis of heme as well in P. falciparum culture. A model is proposed to explain the ETC dependence of both the pyrimidine and heme-biosynthetic pathways in P. falciparum. (C) 2010 Elsevier B.V. All rights reserved.

Cloning, expression, purification and preliminary X-ray crystallographic studies of 2-methylisocitrate lyase from Salmonella typhimurium. Acta Crystallogr

In Salmonella typhimurium, propionate is oxidized to pyruvate via the 2-methylcitric acid cycle. The last step of this cycle, the cleavage of 2-methylisocitrate to succinate and pyruvate, is catalysed by 2-methylisocitrate lyase (EC 4.1.3.30). Methylisocitrate lyase (molecular weight 32 kDa) with a C-terminal polyhistidine affinity tag has been cloned and overexpressed in Escherichia coli and purified and crystallized under different conditions using the hanging-drop vapour-diffusion technique. Crystals belong to the orthogonal space group P2(1)2(1)2(1), with unit-cell parameters a = 63.600, b = 100.670, c = 204.745 Angstrom. A complete data set to 2.5 Angstrom resolution has been collected using an image-plate detector system mounted on a rotating-anode X-ray generator.

Synthesis and Identification of a new class of antileukemic agents containing 2-(arylcarboxamide)-(S)-6-amino-4,5,6,7-tetrahydrobenzod]thiazole

Recently we have reported the effect of (S)-6-aryl urea/thiourea substituted-2-amino-4,5,6,7-tetrahydrobenzod]thiazole derivatives as potent anti-leukemic agents. To elucidate further the Structure Activity Relationship (SAR) studies on the anti-leukemic activity of (S)-2,6-diamino-4,5,6,7 tetrahydrobenzod]thiazole moiety, a series of 2-arlycarboxamide substituted-(S)-6-amino-4,5,6,7-tetrahydrobenzod]thiazole were designed, synthesized and evaluated for their anti-leukemic activity by trypan blue exclusion, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH) assays and cell cycle analysis. Results suggest that the position, number and bulkiness of the substituent on the phenyl ring of aryl carboxamide moiety at 2nd position of 6-amino-4,5,6,7-tetrhydrobenzod]thiazole play a key role in inhibiting the proliferation of leukemia cells. Compounds with ortho substitution showed poor activity and with meta and para substitution showed good activity. (C) 2010 Elsevier Masson SAS. All rights reserved.

X-ray studies on crystalline complexes involving amino acids and peptides XIX. Effects of change in chirality in the complexes of succinic acid with dl- and l-arginine

Crystals of dl-arginine hemisuccinate dihydrate (I)(monoclinic; P21/c; a = 5.292, b = 16.296, c = 15.203 Å; α= 92.89°; Z = 4) and l-arginine hemisuccinate hemisuccinic acid monohydrate (II) (triclinic; P1; a = 5.099; b = 10.222, c = 14.626 Å; α= 77.31, β= 89.46, γ= 78.42°; Z = 2) were grown under identical conditions from aqueous solutions of the components in molar proportions. The structures were solved by direct methods and refined to R = 0.068 for 2585 observed reflections in the case of (I) and R = 0.036 for 2154 observed reflections in the case of (11). Two of the three crystallographically independent arginine molecules in the complexes have conformations different from those observed so far in the crystal structures containing arginine. The succinic acid molecules and the succinate ions in the structures are centrosymmetric and planar. The crystal structure of (II) is highly pseudosymmetric. Arginine-succinate interactions in both the complexes involve specific guanidyl-carboxylate interactions. The basic elements of aggregation in both the structures are ribbons made up of alternating arginine dimers and succinate ions. However, the ribbons pack in different ways in the two structures. (II) presents an interesting case in which two ionisation states of the same molecule coexist in a crystal. The two complexes provide a good example of the effect of change in chirality on stoichiometry, conformation, aggregation, and ionisation state in the solid state.