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1.  Biosynthesis of A Water-Soluble Lipid I Analogue and A Convenient Assay for Translocase I 
Analytical biochemistry  2014;461:36-45.
Translocase I (MraY/MurX) is an essential enzyme in growth of the vast majority of bacteria that catalyzes the transformation from UDP-MurNAc-pentapeptide (Park’s nucleotide) to prenyl-MurNAc-pentapeptide (lipid I), the first membrane-anchored peptidoglycan precursor. MurX has been received considerable attentions to the development of new TB drugs due to the fact that the MurX inhibitors kill exponentially growing Mycobacterium tuberculosis (Mtb) much faster than clinically used TB drugs. Lipid I isolated from Mtb contains the C50-prenyl unit that shows very poor water-solubility, and thus, this chemical characteristic of lipid I renders MurX enzyme assays impractical for screening and lacks reproducibility of the enzyme assays. We have established a scalable chemical synthesis of Park’s nucleotide-Nε-dansylthiourea 2 that can be used as a MurX enzymatic substrate to form lipid I analogues. In our investigation of minimum structure requirement of the prenyl phosphate in the MraY/MurX-catalyzed lipid I analogue synthesis with 2, we found that neryl phosphate (C10-phosphate) can be recognized by MraY/MurX to generate the water-soluble lipid I analogue in quantitative yield under the optimized conditions. Herein, we report a rapid and robust analytical method for quantifying MraY/MurX inhibitory activity of library molecules.
PMCID: PMC4296562  PMID: 24939461
Mur X; Mra Y; Translocase I; Mycobacterium tuberculosis; Water-soluble lipid I; Park’s nucleotide; MraY assay; HTS; MraY inhibitors
2.  Genetic defects in dolichol metabolism 
Congenital disorders of glycosylation (CDG) comprise a group of inborn errors of metabolism with abnormal glycosylation of proteins and lipids. Patients with defective protein N-glycosylation are identified in routine metabolic screening via analysis of serum transferrin glycosylation. Defects in the assembly of the dolichol linked Glc3Man9GlcNAc2 glycan and its transfer to proteins lead to the (partial) absence of complete glycans on proteins. These defects are called CDG-I and are located in the endoplasmic reticulum (ER) or cytoplasm. Defects in the subsequent processing of protein bound glycans result in the presence of truncated glycans on proteins. These defects are called CDG-II and the enzymes involved are located mainly in the Golgi apparatus. In recent years, human defects have been identified in dolichol biosynthesis genes within the group of CDG-I patients. This has increased interest in dolichol metabolism, has resulted in specific recognizable clinical symptoms in CDG-I and has offered new mechanistic insights in dolichol biosynthesis. We here review its biosynthetic pathways, the clinical and biochemical phenotypes in dolichol-related CDG defects, up to the formation of dolichyl-P-mannose (Dol-P-Man), and discuss existing evidence of regulatory networks in dolichol metabolism to provide an outlook on therapeutic strategies.
PMCID: PMC4281381  PMID: 25270028
3.  The effects of statins on the mevalonic acid pathway in recombinant yeast strains expressing human HMG-CoA reductase 
BMC Biotechnology  2013;13:68.
The yeast Saccharomyces cerevisiae can be a useful model for studying cellular mechanisms related to sterol synthesis in humans due to the high similarity of the mevalonate pathway between these organisms. This metabolic pathway plays a key role in multiple cellular processes by synthesizing sterol and nonsterol isoprenoids. Statins are well-known inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the key enzyme of the cholesterol synthesis pathway. However, the effects of statins extend beyond their cholesterol-lowering action, since inhibition of HMGR decreases the synthesis of all products downstream in the mevalonate pathway. Using transgenic yeast expressing human HMGR or either yeast HMGR isoenzyme we studied the effects of simvastatin, atorvastatin, fluvastatin and rosuvastatin on the cell metabolism.
Statins decreased sterol pools, prominently reducing sterol precursors content while only moderately lowering ergosterol level. Expression of genes encoding enzymes involved in sterol biosynthesis was induced, while genes from nonsterol isoprenoid pathways, such as coenzyme Q and dolichol biosynthesis or protein prenylation, were diversely affected by statin treatment. Statins increased the level of human HMGR protein substantially and only slightly affected the levels of Rer2 and Coq3 proteins involved in non-sterol isoprenoid biosynthesis.
Statins influence the sterol pool, gene expression and protein levels of enzymes from the sterol and nonsterol isoprenoid biosynthesis branches and this effect depends on the type of statin administered. Our model system is a cheap and convenient tool for characterizing individual statins or screening for novel ones, and could also be helpful in individualized selection of the most efficient HMGR inhibitors leading to the best response and minimizing serious side effects.
PMCID: PMC3765880  PMID: 24128347
HMG-CoA reductase; Statins; Yeast expression system; Heterologous proteins; Mevalonate pathway
4.  Reconstitution of Functional Mycobacterial Arabinosyltransferase AftC Proteoliposome and Assessment of Decaprenylphosphorylarabinose Analogues as Arabinofuranosyl Donors 
ACS chemical biology  2011;6(8):819-828.
Arabinosyltransferases are a family of membrane-bound glycosyltransferases involved in the biosynthesis of the arabinan segment of two key glycoconjugates, arabinogalactan and lipoarabinomannan, in the mycobacterial cell wall. All arabinosyl-transferases identified have been found to be essential for the growth of Mycobcterium tuberculosis and are potential targets for developing new antituberculosis drugs. Technical bottlenecks in designing enzyme assays for screening for inhibitors of these enzymes are (1) the enzymes are membrane proteins and refractory to isolation; and (2) the sole arabinose donor, decaprenylphosphoryl-d-arabinofuranose is sparingly produced and difficult to isolate, and commercial substrates are not available. In this study, we have synthesized several analogues of decaprenylphosphoryl-d-arabinofuranose by varying the chain length and investigated their arabinofuranose (Araf) donating capacity. In parallel, an essential arabinosyltransferase (AftC), an enzyme that introduces α-(1→3) branch points in the internal arabinan domain in both arabinogalactan and lipoarabinomannan synthesis, has been expressed, solubilized, and purified for the first time. More importantly, it has been shown that the AftC is active only when reconstituted in a proteoliposome using mycobacterial phospholipids and has a preference for diacylated phosphatidylinositoldimannoside (Ac2PIM2), a major cell wall associated glycolipid. α-(1→3) branched arabinans were generated when AftC–liposome complex was used in assays with the (Z,Z)-farnesylphosphoryl d-arabinose linear α-d-Araf-(1→5)3–5 oligosaccharide acceptors and not with the acceptor that had a α-(1→3) branch point preintroduced.
PMCID: PMC3158817  PMID: 21595486
5.  SRD5A3 is required for the conversion of polyprenol to dolichol, essential for N-linked protein glycosylation 
Cell  2010;142(2):203-217.
N-linked glycosylation is the most frequent modification of secreted and membrane-bound proteins in eukaryotic cells, disruption of which is the basis of the Congenital Disorders of Glycosylation (CDG). We describe a new type of CDG caused by mutations in the steroid 5α-reductase type 3 (SRD5A3) gene. Patients have mental retardation, ophthalmologic and cerebellar defects. We found that SRD5A3 is necessary for the reduction of the alpha-isoprene unit of polyprenols to form dolichols, required for synthesis of dolichol-linked monosaccharides and the oligosaccharide precursor used for N-glycosylation. The presence of residual dolichol in cells depleted for this enzyme suggests the existence of an unexpected alternative pathway for dolichol de novo biosynthesis. Our results thus suggest that SRD5A3 is likely to be the long-sought polyprenol reductase and reveal the genetic basis of one of the earliest steps in protein N-linked glycosylation.
PMCID: PMC2940322  PMID: 20637498
N-glycosylation; dolichol; polyprenol; SRD5A3
6.  Quantification of dolichol in the human lens with different types of cataracts 
Molecular Vision  2009;15:1573-1579.
To quantify and characterize dolichol species in cataractous and clear human lenses.
Whole lenses were collected from cadaver eyeballs from the C.H. Nagri Eye Bank and Red Cross Society Eye Bank (Ahmedabad, India). Cataractous nuclei were collected after extracapsular cataract extraction (ECCE). Wet weight for all the lenses was taken and were stored at –50 °C until used. Dolichol was extracted using a standard protocol and then analyzed using High Performance Liquid Chromatography (HPLC) on a 4.6 mm×60 mm Hypersil-Octadecylsilane (ODS; 3 μm) reversed phase column using a Waters dual pump apparatus, a Waters gradient programmer, and an ultraviolet (UV) detector set at 210 nm. Dolichol 13 was used as an internal standard, and dolichol mixture from the liver was used as an external qualitative standard.
The highest dolichol concentration was found in nuclear cataract (2.54±0.6 μg) followed by posterior subcapsular cataract (1.4±0.35 μg), and the lowest levels were observed in cortical cataract (0.37±0.06 μg). The level of dolichol concentration in cataractous lenses was statistically significantly higher than the levels in clear lenses (1.0±04.3 μg; p<0.01).
The dolichol concentration was significantly higher in lenses with nuclear cataract. A significant difference in dolichol concentration was observed between the different types of cataract. It suggests that dolichol and other isoprenoids may be associated with cataractogenesis.
PMCID: PMC2728568  PMID: 19693292
7.  Polyisoprenol Specificity in the Campylobacter jejuni N-linked Glycosylation Pathway 
Biochemistry  2007;46(50):14342-14348.
Campylobacter jejuni contains a general N-linked glycosylation pathway in which a heptasaccharide is sequentially assembled onto a polyisoprenyl-diphosphate carrier and subsequently transferred to the asparagine side chain of an acceptor protein. The enzymes in the pathway function at a membrane interface and have in common amphiphilic membrane-bound polyisoprenyl-linked substrates. Herein we examine the potential role of the polyisoprene component of the substrates by investigating the relative substrate efficiencies of polyisoprene-modified analogs in individual steps in the pathway. Chemically defined substrates for PglC, PglJ and PglB are prepared via semisynthetic approaches. The substrates included polyisoprenols of varying length, double bond geometry, and degree of saturation to probe the role of the hydrophobic polyisoprene in substrate specificity. Kinetic analysis reveals that all three enzymes exhibit distinct preferences for the polyisoprenol carrier whereby cis-double bond geometry and α-unsaturation of the native substrate are important features, while the precise polyisoprene length may be less critical. These finding suggest that the polyisoprenol carrier plays a specific role in the function of these enzymes beyond a purely physical role as a membrane anchor. These studies underscore the potential of the C. jejuni N-linked glycosylation pathway as a system for investigating the biochemical and biophysical roles of polyisoprenol carriers common to prokaryotic and eukaryotic glycosylation.
PMCID: PMC2585822  PMID: 18034500
Campylobacter jejuni; PglB; PglC; PglJ; polyisoprene donor
8.  Farnesyl Phosphates Are Endogenous Ligands of Lysophosphatidic Acid Receptors: Inhibition of LPA GPCR and Activation of PPAR 
Biochimica et biophysica acta  2006;1761(12):1506-1514.
Oligoprenyl phosphates are key metabolic intermediates for the biosynthesis of steroids, the side chain of ubiqinones, and dolichols and the posttranslational isoprenylation of proteins. Farnesyl phosphates are isoprenoid phosphates that resemble polyunsaturated fatty alcohol phosphates, which we have recently shown to be the minimal pharmacophores of lysophosphatidic acid (LPA) receptors. Here we examine whether farnesyl phosphates can interact with the cell surface and nuclear receptors for LPA. Both farnesyl phosphate and farnesyl diphosphate potently and specifically antagonized LPA-elicited intracellular Ca2+-mobilization mediated through the LPA3 receptor, while causing only modest inhibition at the LPA2 receptor and no measurable effect at the LPA1 receptor. Farnesol also inhibited LPA3 but was much less effective. The estimated dissociation constant of LPA3 for farnesyl phosphate is 48 ± 12 nM and 155 ± 30 nM for farnesyl diphosphate. The transcription factor peroxisome proliferator-activated receptor gamma (PPARγ) binds to and is activated by LPA and its analogs including fatty alcohol phosphates. We found that both farnesyl phosphate and diphosphate, but not farnesol, compete with the binding of the synthetic PPARγ agonist [3H]rosiglitazone and activate the PPARγ-mediated gene transcription. Farnesyl monophosphate at 1 μM, but not diphosphate, activated PPARα and PPARβ/δ reporter gene expression. These results indicate new potential roles for the oligoprenyl phosphates as potential endogenous modulators of LPA targets and show that the polyisoprenoid chain is recognized by some LPA receptors.
PMCID: PMC1766556  PMID: 17092771
farnesyl phosphate; isoprenoid; LPA; lysophospholipid; GPCR; PPARγ

Results 1-8 (8)