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1.
PLoS One ; 13(11): e0206692, 2018.
Article in English | MEDLINE | ID: mdl-30395652

ABSTRACT

As an integral membrane protein, purification and characterization of phospho-N- acetylmuramyl- pentapeptide translocase MraY have proven difficult. Low yield and concerns of retaining stability and activity after detergent solubilization have hampered the structure-function analysis. The recently developed detergent-free styrene-maleic acid (SMA) co-polymer system offers an alternative approach that may overcome these disadvantages. In this study, we used the detergent free system to purify MraY from Bacillus subtilis. This allowed efficient extraction of MraY that was heterologously produced in Escherichia coli membranes into SMA-wrapped nanodiscs. The purified MraY embedded in these nanodiscs (SMA-MraY) was comparable to the micellar MraY extracted with a conventional detergent (DDM) with regard to the yield and the purity of the recombinant protein but required significantly less time. The predominantly alpha-helical secondary structure of the protein in SMA-wrapped nanodiscs was also more stable against heat denaturation compared to the micellar protein. Thus, this detergent-free system is amenable to extract MraY efficiently and effectively while maintaining the biophysical properties of the protein. However, the apparent activity of the SMA-MraY was reduced compared to that of the detergent-solubilized protein. The present data indicates that this is caused by a lower accessibility of the enzyme in SMA-wrapped nanodiscs towards its polyisoprenoid substrate.


Subject(s)
Bacillus subtilis/enzymology , Bacterial Proteins/isolation & purification , Transferases/isolation & purification , Bacillus subtilis/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Biophysical Phenomena , Detergents , Enzyme Stability , Escherichia coli/genetics , Kinetics , Maleates , Micelles , Nanostructures , Polystyrenes , Protein Conformation, alpha-Helical , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Transferases/chemistry , Transferases/genetics , Transferases (Other Substituted Phosphate Groups)
2.
Biochim Biophys Acta ; 1840(9): 2851-61, 2014 Sep.
Article in English | MEDLINE | ID: mdl-24821014

ABSTRACT

BACKGROUND: Gram-positive bacteria in the phylum Firmicutes synthesize the low molecular weight thiol bacillithiol rather than glutathione or mycothiol. The bacillithiol transferase YfiT from Bacillus subtilis was identified as a new member of the recently discovered DinB/YfiT-like Superfamily. Based on structural similarity using the Superfamily program, we have determined 30 of 31 Staphylococcus aureus strains encode a single bacillithiol transferase from the DinB/YfiT-like Superfamily, while the remaining strain encodes two proteins. METHODS: We have cloned, purified, and confirmed the activity of a recombinant bacillithiol transferase (henceforth called BstA) encoded by the S. aureus Newman ORF NWMN_2591. Moreover, we have studied the saturation kinetics and substrate specificity of this enzyme using in vitro biochemical assays. RESULTS: BstA was found to be active with the co-substrate bacillithiol, but not with other low molecular weight thiols tested. BstA catalyzed bacillithiol conjugation to the model substrates monochlorobimane, 1-chloro-2,4-dinitrobenzene, and the antibiotic cerulenin. Several other molecules, including the antibiotic rifamycin S, were found to react directly with bacillithiol, but the addition of BstA did not enhance the rate of reaction. Furthermore, cells growing in nutrient rich medium exhibited low BstA activity. CONCLUSIONS: BstA is a bacillithiol transferase from S. aureus that catalyzes the detoxification of cerulenin. Additionally, we have determined that bacillithiol itself might be capable of directly detoxifying electrophilic molecules. GENERAL SIGNIFICANCE: BstA is an active bacillithiol transferase from S. aureus Newman and is the first DinB/YfiT-like Superfamily member identified from this organism. Interestingly, BstA is highly divergent from B. subtilis YfiT.


Subject(s)
Bacterial Proteins , Cerulenin/chemistry , Dinitrochlorobenzene/chemistry , Pyrazoles/chemistry , Staphylococcus aureus/enzymology , Transferases , Bacillus subtilis/enzymology , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Catalysis , Substrate Specificity , Transferases/chemistry , Transferases/isolation & purification
3.
Methods Mol Biol ; 1153: 9-20, 2014.
Article in English | MEDLINE | ID: mdl-24777787

ABSTRACT

The first enzyme in the methylerythritol phosphate (MEP) pathway is 1-deoxy-D-xylulose 5-phosphate (DXP) synthase (DXS). As such this enzyme is considered to be important in the control of plastidial isoprenoid production. Measuring the activity of DXS in plant extracts is therefore crucial to understanding the regulation of the MEP pathway. Due to the relatively low amounts of DXS, the activity of this enzyme can only be measured using highly sensitive analytical equipment. Here, a method is described to determine the DXS enzyme activity in a crude plant extract, by measuring DXP production directly using high performance liquid chromatography linked to a tandem triple quadrupole mass spectrometry detector (LC-MS/MS).


Subject(s)
Arabidopsis/enzymology , Enzyme Assays/methods , Erythritol/metabolism , Plant Extracts/metabolism , Transferases/metabolism , Arabidopsis/metabolism , Chromatography, Liquid , Tandem Mass Spectrometry , Transferases/isolation & purification
4.
Plant Physiol Biochem ; 66: 77-83, 2013 May.
Article in English | MEDLINE | ID: mdl-23500709

ABSTRACT

When Glomus intraradices-colonised tomato roots were extracted in methanol at 6 °C, chlorogenic acid (5-caffeoylquinic acid), naturally present in the extract, was slowly converted by transesterification into methyl caffeate. The progress of the reaction could be monitored by HPLC. The reaction only occurred when the ground roots were left in contact with the hydro-alcoholic extract and required the presence of 15-35% water in the mixture. When the roots were extracted in ethanol, chlorogenic acid was transformed to ethyl caffeate in the same conditions. The reaction was also detected in Glomus mosseae-colonised tomato root extracts. It was also detectable in non-mycorrhizal root extracts but was 10-25 times slower. By contrast it was undetectable in extracts of the aerial parts of tomato plants, which also contain high amounts of chlorogenic acid, whether or not these plants were inoculated by the arbuscular mycorrhizal fungus. We found that this transesterification reaction is catalysed by a tomato enzyme, which remains active in hydro-alcoholic mixtures and exhibits chlorogenate-dependant caffeoyltransferase activity in the presence of methanol or ethanol. This transferase activity is inhibited by phenylmethanesulfonyl fluoride. The 4- and 3-caffeoylquinic acid isomers were also used as substrates but were less active than chlorogenic acid. Highest activity was detected in mycorrhizal roots of nutrient-deprived tomato plants. Surprisingly this caffeoyltransferase activity could also be detected in hydro-alcoholic extracts of G. intraradices-colonised roots of leek, sorghum or barrel medic.


Subject(s)
Chlorogenic Acid/metabolism , Mycorrhizae/growth & development , Plant Proteins/isolation & purification , Plant Roots/enzymology , Solanum lycopersicum/enzymology , Transferases/isolation & purification , Caffeic Acids/metabolism , Chromatography, High Pressure Liquid , Enzyme Activation , Enzyme Assays , Enzyme Inhibitors/metabolism , Esterification , Solanum lycopersicum/microbiology , Mycorrhizae/metabolism , Phenylmethylsulfonyl Fluoride/metabolism , Plant Components, Aerial/metabolism , Plant Extracts/chemistry , Plant Proteins/metabolism , Plant Roots/microbiology , Substrate Specificity , Temperature , Transferases/metabolism
5.
Protein Expr Purif ; 88(1): 80-4, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23266652

ABSTRACT

Selenocysteine Synthase (SELA, E.C. 2.9.1.1) from Escherichia coli is a homodecamer pyridoxal-5'-phosphate containing enzyme responsible for the conversion of seryl-tRNA(sec) into selenocysteyl-tRNA(sec) in the biosynthesis of the 21th amino acid, selenocysteine (Sec or U). This paper describes the cloning of the E. coli selA gene into a modified pET29a(+) vector and its expression in E. coli strain WL81460, a crucial modification allowing SELA expression without bound endogenous tRNA(sec). This expression strategy enabled the purification and additional biochemical and biophysical characterization of the SELA decamer. The homogeneous SELA protein was obtained using three chromatographic steps. Size Exclusion Chromatography and Native Gel Electrophoresis showed that SELA maintains a decameric state with molecular mass of approximately 500 kDa with an isoelectric point of 6,03. A predominance of α-helix structures was detected by circular dichroism with thermal stability up to 45 °C. The oligomeric assemblage of SELA was investigated by glutaraldehyde crosslinking experiments indicate that SELA homodecameric structure is the result of a stepwise addition of intermediate oligomeric states and not a direct monomer to homodecamer transition. Our results have contributed to the establishment of a robust expression model for the enzyme free of bound RNA and are of general interest to be taken into consideration in all cases of heterologous/homologous expressions of RNA-binding proteins avoiding the carryover of endogenous RNAs, which may interfere with further biochemical characterizations.


Subject(s)
Escherichia coli/enzymology , Recombinant Proteins/isolation & purification , Transferases/chemistry , Transferases/isolation & purification , Biophysics , Molecular Weight , Protein Structure, Secondary , Pyridoxal Phosphate/chemistry , RNA, Transfer, Amino Acid-Specific/chemistry , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Selenocysteine/biosynthesis , Selenocysteine/chemistry
6.
Protein Expr Purif ; 87(2): 120-8, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23165241

ABSTRACT

The unique bacterial enzyme phosphatidylglycerol: prolipoprotein diacylglyceryl transferase (Lgt) is the least studied enzyme of the ubiquitous bacterial lipoprotein synthetic pathway, mostly due to the low abundance of the enzyme. So far, Lgt has been studied to a limited extent in gram-negative bacteria, mainly in Escherichia coli. We, for the first time, report the isolation of an adequate amount of Lgt from the gram-positive lactic acid bacteria, Lactococcus lactis and compare this wild-type bacterial enzyme with the E. coli enzyme. The L. lactis Lgt, when purified by cationic-exchange chromatography, showed a 20-fold increase in the specific activity compared to that of the load, and 75% of the total Lgt activity loaded was recovered. Kinetically, L. lactis Lgt was found to be similar to the E. coli enzyme with matching K(m) and V(max), whereas the specific activity of the L. lactis enzyme was about 20 times less than that of the E. coli enzyme. Comparative bioinformatic analysis of L. lactis, E. coli and Staphylococcus aureus Lgt revealed that the conserved and catalytically important His-103 residue in E. coli Lgt, was altered to Tyr in L. lactis. Investigations showed that other bacteria where this alteration is visible, form a diversion within the gram-positive bacteria in evolution. Further analysis revealed Mycobacterium smegmatis to be the species which evolved with the alteration of His to Tyr.


Subject(s)
Bacterial Proteins/biosynthesis , Bacterial Proteins/isolation & purification , Lactococcus lactis/enzymology , Transferases/biosynthesis , Transferases/isolation & purification , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Chromatography, Ion Exchange , Computational Biology , Escherichia coli/enzymology , Escherichia coli/genetics , Escherichia coli/metabolism , Glucosides/chemistry , Glucosides/metabolism , Kinetics , Lactococcus lactis/genetics , Lactococcus lactis/metabolism , Molecular Sequence Data , Phylogeny , Sequence Alignment , Transferases/chemistry , Transferases/genetics
7.
J Biol Chem ; 287(53): 44406-17, 2012 Dec 28.
Article in English | MEDLINE | ID: mdl-23132863

ABSTRACT

Catechins (flavan-3-ols), the most important secondary metabolites in the tea plant, have positive effects on human health and are crucial in defense against pathogens of the tea plant. The aim of this study was to elucidate the biosynthetic pathway of galloylated catechins in the tea plant. The results suggested that galloylated catechins were biosynthesized via 1-O-glucose ester-dependent two-step reactions by acyltransferases, which involved two enzymes, UDP-glucose:galloyl-1-O-ß-D-glucosyltransferase (UGGT) and a newly discovered enzyme, epicatechin:1-O-galloyl-ß-D-glucose O-galloyltransferase (ECGT). In the first reaction, the galloylated acyl donor ß-glucogallin was biosynthesized by UGGT from gallic acid and uridine diphosphate glucose. In the second reaction, galloylated catechins were produced by ECGT catalysis from ß-glucogallin and 2,3-cis-flavan-3-ol. 2,3-cis-Flavan-3-ol and 1-O-galloyl-ß-D-glucose were appropriate substrates of ECGT rather than 2,3-trans-flavan-3-ol and 1,2,3,4,6-pentagalloylglucose. Purification by more than 1641-fold to apparent homogeneity yielded ECGT with an estimated molecular mass of 241 to 121 kDa by gel filtration. Enzyme activity and SDS-PAGE analysis indicated that the native ECGT might be a dimer, trimer, or tetramer of 60- and/or 58-kDa monomers, and these monomers represent a heterodimer consisting of pairs of 36- or 34- of and 28-kDa subunits. MALDI-TOF-TOF MS showed that the protein SCPL1199 was identified. Epigallocatechin and epicatechin exhibited higher substrate affinities than ß-glucogallin. ECGT had an optimum temperature of 30 °C and maximal reaction rates between pH 4.0 and 6.0. The enzyme reaction was inhibited dramatically by phenylmethylsulfonyl fluoride, HgCl(2), and sodium deoxycholate.


Subject(s)
Camellia sinensis/enzymology , Catechin/metabolism , Gallic Acid/metabolism , Plant Proteins/isolation & purification , Plant Proteins/metabolism , Transferases/isolation & purification , Transferases/metabolism , Camellia sinensis/chemistry , Camellia sinensis/genetics , Enzyme Stability , Kinetics , Plant Proteins/chemistry , Plant Proteins/genetics , Transferases/chemistry , Transferases/genetics
8.
Plant Sci ; 185-186: 309-20, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22325894

ABSTRACT

Green algae exclusively use the methylerythritol 4-phosphate (MEP) pathway for the biosynthesis of isoprenoids. The first enzyme of this pathway is 1-deoxy-D-xylulose 5-phosphate synthase (DXS, EC 2.2.1.7). Green algae have been thought to possess only a single DXS, in contrast to land plants, which have at least two isoforms that serve different roles in metabolism. The green microalga Botryococcus braunii has an extraordinary isoprenoid metabolism, as it produces large amounts of triterpene hydrocarbons. Here, we did cDNA cloning of DXSs from B. braunii and examined enzyme activities of the heterologously expressed proteins. Three distinct DXS isoforms were identified, all of which were functional and had similar kinetic properties, whereas the temperature dependence of enzyme activity showed considerable differences. Transcription of the genes was examined by real time quantitative RT-PCR. The three DXS genes were simultaneously expressed, and the expression levels were highest on day six after subculturing. B. braunii is the first green microalga demonstrated to have multiple DXS isoforms like land plants. This difference to other microalgae seems to mirror its special needs for extensive triterpene production by increasing the metabolic flow through the MEP pathway.


Subject(s)
Chlorophyta/enzymology , Microalgae/enzymology , Plant Leaves/enzymology , Terpenes/metabolism , Transferases/genetics , Amino Acid Sequence , Chlorophyta/genetics , Chlorophyta/metabolism , Conserved Sequence , DNA, Complementary/genetics , Gene Expression/genetics , Gene Expression Regulation, Plant/genetics , Gene Library , Kinetics , Microalgae/genetics , Microalgae/metabolism , Molecular Sequence Data , Phylogeny , Plant Leaves/genetics , Plant Leaves/metabolism , Plant Proteins/genetics , Plant Proteins/isolation & purification , Plant Proteins/metabolism , Protein Isoforms , Recombinant Proteins , Sequence Alignment , Sequence Analysis, DNA , Temperature , Transferases/isolation & purification , Transferases/metabolism
9.
Biochem Cell Biol ; 89(3): 299-307, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21539457

ABSTRACT

The human H-protein is one of four essential components (H-, L-, P-, and T-proteins) of the mammalian glycine cleavage enzyme complex and its function is involved in the pathogenesis and diagnosis of glycine encephalopathy. A transcript corresponding to the glycine cleavage H-protein functional gene was isolated from cultured human skin fibroblasts along with a transcript for a putative processed pseudogene on chromosome 2q33.3. Sequence analysis of the fibroblast H-protein functional gene transcript showed complete identity to that reported from human liver. The H-protein cDNA was subsequently cloned with a hexahistidine affinity tag in the Pichia pastoris plasmid vector pPICZαA and recombined into the yeast genome downstream of the alcohol oxidase promoter for methanol-induced expression. The recombinant H-protein was secreted into the culture medium and purified to homogeneity using a one-step nickel-nitrilotriacetic acid resin column. Approximately 4 mg of homogeneous H-protein was obtained from 1 L of culture medium. Since the attachment of a lipoic acid prosthetic group is required for H-protein function, we have expressed and purified E. coli lipoate protein ligase and succeeded in lipoylating H-protein, converting the apo-H-protein to the functional holo-H-protein. A lipoamide dehydrogenase assay was performed to confirm that the apo-H-protein was inactive, whereas the holo-H-protein was approximately 2.3-fold more active than free lipoic acid as a hydrogen donor in driving the reaction. The availability of copious amounts of human recombinant H-protein by using Pichia pastoris expression and affinity purification will facilitate the elucidation of the structure and function of the H-protein and its relationship to the P-, T-, and L-proteins in the glycine cleavage enzyme complex. In view of the fact that there is no detectable glycine cleavage enzyme activity in human skin fibroblasts, we speculate that a plausible function of the H-protein is to interact with the L-protein, which is also part of the l-ketoglutarate dehydrogenase complex present in fibroblasts.


Subject(s)
Amino Acid Oxidoreductases/isolation & purification , Apoproteins/isolation & purification , Bacterial Proteins/isolation & purification , Carrier Proteins/isolation & purification , Dihydrolipoamide Dehydrogenase/isolation & purification , Escherichia coli/metabolism , Multienzyme Complexes/isolation & purification , Peptide Synthases/isolation & purification , Pichia/metabolism , Recombinant Proteins/isolation & purification , Transferases/isolation & purification , Amino Acid Oxidoreductases/biosynthesis , Amino Acid Oxidoreductases/genetics , Amino Acid Sequence , Apoproteins/biosynthesis , Apoproteins/genetics , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Carrier Proteins/biosynthesis , Carrier Proteins/genetics , Chromatography, Affinity , Cloning, Molecular , DNA, Complementary/analysis , DNA, Complementary/genetics , Dihydrolipoamide Dehydrogenase/biosynthesis , Dihydrolipoamide Dehydrogenase/genetics , Escherichia coli/genetics , Fibroblasts/cytology , Fibroblasts/enzymology , Histidine/metabolism , Humans , Hyperglycinemia, Nonketotic/enzymology , Hyperglycinemia, Nonketotic/pathology , Molecular Sequence Data , Multienzyme Complexes/biosynthesis , Multienzyme Complexes/genetics , Oligopeptides/metabolism , Peptide Synthases/biosynthesis , Peptide Synthases/genetics , Pichia/genetics , Primary Cell Culture , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Sequence Alignment , Sequence Analysis , Skin/cytology , Skin/enzymology , Transferases/biosynthesis , Transferases/genetics
10.
Arch Biochem Biophys ; 505(2): 202-12, 2011 Jan 15.
Article in English | MEDLINE | ID: mdl-21035424

ABSTRACT

The pyrH-encoded uridine 5'-monophosphate kinase (UMPK) is involved in both de novo and salvage synthesis of DNA and RNA precursors. Here we describe Mycobacterium tuberculosis UMPK (MtUMPK) cloning and expression in Escherichia coli. N-terminal amino acid sequencing and electrospray ionization mass spectrometry analyses confirmed the identity of homogeneous MtUMPK. MtUMPK catalyzed the phosphorylation of UMP to UDP, using ATP-Mg²(+) as phosphate donor. Size exclusion chromatography showed that the protein is a homotetramer. Kinetic studies revealed that MtUMPK exhibits cooperative kinetics towards ATP and undergoes allosteric regulation. GTP and UTP are, respectively, positive and negative effectors, maintaining the balance of purine versus pyrimidine synthesis. Initial velocity studies and substrate(s) binding measured by isothermal titration calorimetry suggested that catalysis proceeds by a sequential ordered mechanism, in which ATP binds first followed by UMP binding, and release of products is random. As MtUMPK does not resemble its eukaryotic counterparts, specific inhibitors could be designed to be tested as antitubercular agents.


Subject(s)
Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Mycobacterium tuberculosis/enzymology , Pyrimidines/metabolism , Transferases/genetics , Transferases/metabolism , Adenosine Triphosphate/metabolism , Allosteric Regulation , Amino Acid Sequence , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/isolation & purification , Genes, Suppressor , Guanosine Triphosphate/metabolism , Kinetics , Ligands , Molecular Sequence Data , Molecular Weight , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Polymerase Chain Reaction , Sequence Alignment , Sequence Analysis, DNA , Spectrometry, Mass, Electrospray Ionization , Transferases/chemistry , Transferases/isolation & purification , Uridine Triphosphate/metabolism
11.
Glycobiology ; 20(5): 617-28, 2010 May.
Article in English | MEDLINE | ID: mdl-20124190

ABSTRACT

The structures of the pectic polysaccharide rhamnogalacturonan II (RG-II) pectin constituent are remarkably evolutionary conserved in all plant species. At least 12 different glycosyl residues are present in RG-II. Among them is the seldom eight-carbon sugar 3-deoxy-d-manno-octulosonic acid (Kdo) whose biosynthetic pathway has been shown to be conserved between plants and Gram-negative bacteria. Kdo is formed in the cytosol by the condensation of phosphoenol pyruvate with d-arabinose-5-P and then activated by coupling to cytidine monophosphate (CMP) prior to its incorporation in the Golgi apparatus by a Kdo transferase (KDTA) into the nascent polysaccharide RG-II. To gain new insight into RG-II biosynthesis and function, we isolated and characterized null mutants for the unique putative KDTA (AtKDTA) encoded in the Arabidopsis genome. We provide evidence that, in contrast to mutants affecting the RG-II biosynthesis, the extinction of the AtKDTA gene expression does not result in any developmental phenotype in the AtkdtA plants. Furthermore, the structure of RG-II from the null mutants was not altered and contained wild-type amount of Rha-alpha(1-5)Kdo side chain. The cellular localization of AtKDTA was investigated by using laser scanning confocal imaging of the protein fused to green fluorescent protein. In agreement with its cellular prediction, the fusion protein was demonstrated to be targeted to the mitochondria. These data, together with data deduced from sequence analyses of higher plant genomes, suggest that AtKDTA encodes a putative KDTA involved in the synthesis of a mitochondrial not yet identified lipid A-like molecule rather than in the synthesis of the cell wall RG-II.


Subject(s)
Arabidopsis/enzymology , Arabidopsis/genetics , Gene Expression Regulation, Plant/genetics , Transferases/genetics , Amino Acid Sequence , Molecular Sequence Data , Mutation , Pectins/biosynthesis , Pectins/chemistry , Phylogeny , Reverse Transcriptase Polymerase Chain Reaction , Transferases/chemistry , Transferases/isolation & purification
12.
Mol Microbiol ; 73(5): 913-23, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19656295

ABSTRACT

Cell division and cell wall biosynthesis in prokaryotes are driven by partially overlapping multiprotein machineries whose activities are tightly controlled and co-ordinated. So far, a number of protein components have been identified and acknowledged as essential for both fundamental cellular processes. Genes for enzymes of both machineries have been found in the genomes of the cell wall-less genera Chlamydia and Wolbachia, raising questions as to the functionality of the lipid II biosynthesis pathway and reasons for its conservation. We provide evidence on three levels that the lipid II biosynthesis pathway is indeed functional and essential in both genera: (i) fosfomycin, an inhibitor of MurA, catalysing the initial reaction in lipid II biosynthesis, has a detrimental effect on growth of Wolbachia cells; (ii) isolated cytoplasmic membranes from Wolbachia synthesize lipid II ex vivo; and (iii) recombinant MraY and MurG from Chlamydia and Wolbachia exhibit in vitro activity, synthesizing lipid I and lipid II respectively. We discuss the hypothesis that the necessity for maintaining lipid II biosynthesis in cell wall-lacking bacteria reflects an essential role of the precursor in prokaryotic cell division. Our results also indicate that the lipid II pathway may be exploited as an antibacterial target for chlamydial and filarial infections.


Subject(s)
Biosynthetic Pathways/genetics , Chlamydia/genetics , Chlamydia/metabolism , Uridine Diphosphate N-Acetylmuramic Acid/analogs & derivatives , Wolbachia/genetics , Wolbachia/metabolism , Alkyl and Aryl Transferases/antagonists & inhibitors , Bacterial Outer Membrane Proteins/isolation & purification , Bacterial Outer Membrane Proteins/metabolism , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Cell Membrane/metabolism , Chlamydia/drug effects , Enzyme Inhibitors/pharmacology , Fosfomycin/pharmacology , Genes, Bacterial , Genes, Essential , Models, Biological , Monosaccharides/metabolism , N-Acetylglucosaminyltransferases/isolation & purification , N-Acetylglucosaminyltransferases/metabolism , Oligopeptides/metabolism , Transferases/isolation & purification , Transferases/metabolism , Transferases (Other Substituted Phosphate Groups) , Uridine Diphosphate N-Acetylmuramic Acid/biosynthesis , Wolbachia/drug effects
13.
J Biosci Bioeng ; 107(6): 620-2, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19447338

ABSTRACT

cis-Prenyltransferase catalyzes the synthesis of Z,E-mixed prenyl diphosphates by a condensation of isopentenyl diphosphate to an allylic diphosphate. A novel gene encoding a cis-prenyltransferase is cloned from Thermobifida fusca. It showed a unique substrate specificity accepting dimethylallyl diphosphate as a shortest allylic substrate, and synthesizes polyprenyl products up to C(70).


Subject(s)
Actinomycetales/enzymology , Bacterial Proteins/metabolism , Transferases/metabolism , Bacterial Proteins/isolation & purification , Cloning, Molecular , Substrate Specificity , Transferases/isolation & purification
14.
Biochemistry ; 48(22): 4999-5006, 2009 Jun 09.
Article in English | MEDLINE | ID: mdl-19379010

ABSTRACT

Two classes of bacteriophages, the single-stranded DNA Microviridae and the single-stranded RNA Alloleviviridae, accomplish lysis by expressing "protein antibiotics", or polypeptides that inhibit cell wall biosynthesis. Previously, we have provided genetic and physiological evidence that E, a 91-amino acid membrane protein encoded by the prototype microvirus, varphiX174, is a specific inhibitor of the translocase MraY, an essential membrane-embedded enzyme that catalyzes the formation of the murein precursor, Lipid I, from UDP-N-acetylmuramic acid-pentapeptide and the lipid carrier, undecaprenol phosphate. Here we report the first purification of E, which has been refractory to overexpression because of its lethality to Escherichia coli. Moreover, using a fluorescently labeled analogue of the sugar-nucleotide substrate, we demonstrate that E acts as a noncompetitive inhibitor of detergent-solubilized MraY, with respect to both soluble and lipid substrates. In addition, we show that the E sensitivity of five MraY mutant proteins, produced from alleles selected for resistance to E, can be correlated to the apparent affinities determined by in vivo multicopy suppression experiments. These results are inconsistent with previous reports that E inhibited membrane-embedded MraY but not the detergent-solubilized enzyme, which led to a model in which E functions by binding MraY and blocking the formation of an essential heteromultimeric complex involving MraY and other murein biosynthesis enzymes. We discuss a new model in which E binds to MraY at a site composed of the two transmembrane domains within which the E resistance mutations map and the fact that the result of this binding is a conformational change that inactivates the enzyme.


Subject(s)
Bacteriolysis/physiology , Bacteriophage phi X 174/chemistry , Bacteriophage phi X 174/physiology , Viral Proteins/isolation & purification , Viral Proteins/physiology , Alleles , Amino Acid Sequence , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Bacteriolysis/genetics , Drug Resistance, Bacterial/genetics , Fluorescent Dyes/metabolism , Molecular Sequence Data , Substrate Specificity/genetics , Transferases/antagonists & inhibitors , Transferases/genetics , Transferases/isolation & purification , Transferases/metabolism , Transferases (Other Substituted Phosphate Groups) , Viral Proteins/chemistry , Viral Proteins/genetics
15.
Appl Environ Microbiol ; 74(21): 6703-8, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18791012

ABSTRACT

To investigate potential complementary activities of multiple enzymes belonging to the same family within a single microorganism, we chose a set of Old Yellow Enzyme (OYE) homologs of Pseudomonas putida. The physiological function of these enzymes is not well established; however, an activity associated with OYE family members from different microorganisms is their ability to reduce nitroaromatic compounds. Using an in silico approach, we identified six OYE homologs in P. putida KT2440. Each gene was subcloned into an expression vector, and each corresponding gene product was purified to homogeneity prior to in vitro analysis for its catalytic activity against 2,4,6-trinitrotoluene (TNT). One of the enzymes, called XenD, lacked in vitro activity, whereas the other five enzymes demonstrated type I hydride transferase activity and reduced the nitro groups of TNT to hydroxylaminodinitrotoluene derivatives. XenB has the additional ability to reduce the aromatic ring of TNT to produce Meisenheimer complexes, defined as type II hydride transferase activity. The condensations of the primary products of type I and type II hydride transferases react with each other to yield diarylamines and nitrite; the latter can be further reduced to ammonium and serves as a nitrogen source for microorganisms in vivo.


Subject(s)
Flavoproteins/metabolism , NADPH Dehydrogenase/metabolism , Pseudomonas putida/enzymology , Transferases/metabolism , Cloning, Molecular , Flavoproteins/genetics , Flavoproteins/isolation & purification , Gene Expression , Metabolic Networks and Pathways , NADPH Dehydrogenase/genetics , NADPH Dehydrogenase/isolation & purification , Nitrites/metabolism , Phylogeny , Quaternary Ammonium Compounds/metabolism , Sequence Homology, Amino Acid , Transferases/genetics , Transferases/isolation & purification , Trinitrotoluene/metabolism
16.
Bioorg Med Chem Lett ; 18(19): 5320-3, 2008 Oct 01.
Article in English | MEDLINE | ID: mdl-18783951

ABSTRACT

We report on a target-based approach to identify possible Mycobacterium tuberculosis DXS inhibitors from the structure of a known transketolase inhibitor. A small focused library of analogs was assembled in order to begin elucidating some meaningful structure-activity relationships of 3-(4-chloro-phenyl)-5-benzyl-4H-pyrazolo[1,5-a]pyrimidin-7-one. Ultimately we found that 2-methyl-3-(4-fluorophenyl)-5-(4-methoxy-phenyl)-4H-pyrazolo[1,5-a]pyrimidin-7-one, although still weak, was able to inhibit M. tuberculosis DXS with an IC(50) of 10.6 microM.


Subject(s)
Antitubercular Agents/chemical synthesis , Antitubercular Agents/pharmacology , Drug Design , Mycobacterium tuberculosis/enzymology , Pyrazoles/chemical synthesis , Pyrazoles/pharmacology , Pyrimidines/chemical synthesis , Pyrimidines/pharmacology , Transferases/genetics , Transferases/metabolism , Antitubercular Agents/chemistry , Combinatorial Chemistry Techniques , Inhibitory Concentration 50 , Molecular Structure , Mycobacterium tuberculosis/genetics , Pyrazoles/chemistry , Pyrimidines/chemistry , Structure-Activity Relationship , Transferases/antagonists & inhibitors , Transferases/chemistry , Transferases/isolation & purification
17.
J Cell Biol ; 182(3): 531-42, 2008 Aug 11.
Article in English | MEDLINE | ID: mdl-18695044

ABSTRACT

Regulatory pathways for protein glycosylation are poorly understood, but expression of branchpoint enzymes is critical. A key branchpoint enzyme is the T-synthase, which directs synthesis of the common core 1 O-glycan structure (T-antigen), the precursor structure for most mucin-type O-glycans in a wide variety of glycoproteins. Formation of active T-synthase, which resides in the Golgi apparatus, requires a unique molecular chaperone, Cosmc, encoded on Xq24. Cosmc is the only molecular chaperone known to be lost through somatic acquired mutations in cells. We show that Cosmc is an endoplasmic reticulum (ER)-localized adenosine triphosphate binding chaperone that binds directly to human T-synthase. Cosmc prevents the aggregation and ubiquitin-mediated degradation of the T-synthase. These results demonstrate that Cosmc is a molecular chaperone in the ER required for this branchpoint glycosyltransferase function and show that expression of the disease-related Tn antigen can result from deregulation or loss of Cosmc function.


Subject(s)
Endoplasmic Reticulum/metabolism , Molecular Chaperones/metabolism , Adenosine Triphosphate/metabolism , Animals , CHO Cells , Cell Line, Tumor , Conserved Sequence , Cricetinae , Cricetulus , Disulfides/metabolism , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/enzymology , Endoplasmic Reticulum Chaperone BiP , Glycosylation/drug effects , Golgi Apparatus/drug effects , Golgi Apparatus/enzymology , Heat-Shock Proteins/metabolism , Humans , Intracellular Membranes/drug effects , Intracellular Membranes/enzymology , Leupeptins/pharmacology , Mutant Proteins/metabolism , Organ Specificity/drug effects , Proteasome Inhibitors , Protein Sorting Signals , Protein Transport/drug effects , Solubility/drug effects , Transferases/isolation & purification , Transferases/metabolism , Ubiquitination/drug effects , Vertebrates/metabolism
18.
J Microbiol Biotechnol ; 18(3): 457-64, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18388462

ABSTRACT

An extracellular enzyme (RMEBE) possessing alpha- (1-->4)-(1-->6)-transferring activity was purified to homogeneity from Rhodothermus marinus by combination of ammonium sulfate precipitation, Q-Sepharose ion-exchange, and Superdex- 200 gel filtration chromatographies, and preparative native polyacrylamide gel electrophoresis. The purified enzyme had an optimum pH of 6.0 and was highly thermostable with a maximal activity at 80 degrees . Its half-life was determined to be 73.7 and 16.7 min at 80 and 85 degrees , respectively. The enzyme was also halophilic and highly halotolerant up to about 2 M NaCl, with a maximal activity at 0.5M. The substrate specificity of RMEBE suggested that it possesses partial characteristics of both glucan branching enzyme and neopullulanase. RMEBE clearly produced branched glucans from amylose, with partial alpha-(1-->4)-hydrolysis of amylose and starch. At the same time, it hydrolyzed pullulan partly to panose, and exhibited alpha-(1-->4)-(1-->6)-transferase activity for small maltooligosaccharides, producing disproportionated alpha-(1-->6)-branched maltooligosaccharides. The enzyme preferred maltopentaose and maltohexaose to smaller maltooligosaccharides for production of longer branched products. Thus, the results suggest that RMEBE might be applied for production of branched oligosaccharides from small maltodextrins at high temperature or even at high salinity.


Subject(s)
Amylases/chemistry , Amylases/isolation & purification , Extracellular Space/enzymology , Rhodothermus/enzymology , Seawater/microbiology , Amylases/genetics , Amylases/metabolism , Amylopectin , Amylose/metabolism , Enzyme Stability , Hydrogen-Ion Concentration , Molecular Weight , Oligosaccharides/metabolism , Starch/metabolism , Substrate Specificity , Temperature , Transferases/chemistry , Transferases/genetics , Transferases/isolation & purification , Transferases/metabolism
19.
FEMS Microbiol Rev ; 32(2): 208-33, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18081839

ABSTRACT

The biosynthesis of bacterial cell wall peptidoglycan is a complex process involving many different steps taking place in the cytoplasm (synthesis of the nucleotide precursors) and on the inner and outer sides of the cytoplasmic membrane (assembly and polymerization of the disaccharide-peptide monomer unit, respectively). This review summarizes the current knowledge on the membrane steps leading to the formation of the lipid II intermediate, i.e. the substrate of the polymerization reactions. It makes the point on past and recent data that have significantly contributed to the understanding of the biosynthesis of undecaprenyl phosphate, the carrier lipid required for the anchoring of the peptidoglycan hydrophilic units in the membrane, and to the characterization of the MraY and MurG enzymes which catalyze the successive transfers of the N-acetylmuramoyl-peptide and N-acetylglucosamine moieties onto the carrier lipid, respectively. Enzyme inhibitors and antibacterial compounds interfering with these essential metabolic steps and interesting targets are presented.


Subject(s)
Bacterial Proteins , Lipids/biosynthesis , Peptidoglycan/biosynthesis , Bacteria/chemistry , Bacteria/metabolism , Bacterial Outer Membrane Proteins/antagonists & inhibitors , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/isolation & purification , Bacterial Outer Membrane Proteins/metabolism , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Kinetics , Lipids/chemistry , Monosaccharides/chemical synthesis , Monosaccharides/chemistry , Monosaccharides/metabolism , N-Acetylglucosaminyltransferases/antagonists & inhibitors , N-Acetylglucosaminyltransferases/chemistry , N-Acetylglucosaminyltransferases/isolation & purification , N-Acetylglucosaminyltransferases/metabolism , Oligopeptides/chemical synthesis , Oligopeptides/chemistry , Oligopeptides/metabolism , Polyisoprenyl Phosphates/chemistry , Polyisoprenyl Phosphates/metabolism , Substrate Specificity , Terpenes/metabolism , Transferases/antagonists & inhibitors , Transferases/isolation & purification , Transferases/metabolism , Transferases (Other Substituted Phosphate Groups) , Uridine Diphosphate N-Acetylmuramic Acid/analogs & derivatives , Uridine Diphosphate N-Acetylmuramic Acid/chemical synthesis , Uridine Diphosphate N-Acetylmuramic Acid/chemistry , Uridine Diphosphate N-Acetylmuramic Acid/metabolism
20.
J Bacteriol ; 189(9): 3471-8, 2007 May.
Article in English | MEDLINE | ID: mdl-17337583

ABSTRACT

P(II) proteins have been shown to be key players in the regulation of nitrogen fixation and ammonia assimilation in bacteria. The mode by which these proteins act as signals is by being in either a form modified by UMP or the unmodified form. The modification, as well as demodification, is catalyzed by a bifunctional enzyme encoded by the glnD gene. The regulation of this enzyme is thus of central importance. In Rhodospirillum rubrum, three P(II) paralogs have been identified. In this study, we have used purified GlnD and P(II) proteins from R. rubrum, and we show that for the uridylylation activity of R. rubrum GlnD, alpha-ketoglutarate is the main signal, whereas glutamine has no effect. This is in contrast to, e.g., the Escherichia coli system. Furthermore, we show that all three P(II) proteins are uridylylated, although the efficiency is dependent on the cation present. This difference may be of importance in understanding the effects of the P(II) proteins on the different target enzymes. Furthermore, we show that the deuridylylation reaction is greatly stimulated by glutamine and that Mn(2+) is required.


Subject(s)
Cations, Divalent/pharmacology , Ketoglutaric Acids/pharmacology , PII Nitrogen Regulatory Proteins/metabolism , Rhodospirillum rubrum/enzymology , Transferases/metabolism , Uridine Monophosphate/metabolism , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Electrophoretic Mobility Shift Assay , Enzyme Activators/pharmacology , Escherichia coli/enzymology , Glutamine/pharmacology , PII Nitrogen Regulatory Proteins/isolation & purification , Transferases/isolation & purification
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