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1.
Bioorg Med Chem Lett ; 29(16): 2076-2078, 2019 08 15.
Article in English | MEDLINE | ID: mdl-31300341

ABSTRACT

Mitomycins, produced by several Streptomyces strains, are potent anticancer antibiotics that comprise an aziridine ring fused to a tricyclic mitosane core. Mitomycins have remarkable ability to crosslink DNA with high efficiency. Despite long clinical history of mitomycin C, the biosynthesis of mitomycins, especially mitosane core formation, remains unknown. Here, we report in vitro characterization of three proteins, MmcB (acyl carrier protein), MitE (acyl AMP ligase), and MitB (glycosyltransferase) involved in mitosane core formation. We show that 3-amino-5-hydroxybenzoic acid (AHBA) is first loaded onto MmcB by MitE at the expense of ATP. MitB then catalyzes glycosylation of AHBA-MmcB with uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) to generate a key intermediate, GlcNAc-AHBA-MmcB, which contains all carbon and nitrogen atoms of the mitosane core. These results provide important insight into mitomycin biosynthesis.


Subject(s)
Acyl Carrier Protein/chemistry , Antibiotics, Antineoplastic/chemistry , Bacterial Proteins/chemistry , Carbon-Sulfur Ligases/chemistry , Glycosyltransferases/chemistry , Mitomycins/biosynthesis , Aminobenzoates/chemistry , Biocatalysis , Hydroxybenzoates/chemistry , Mitomycins/chemistry , Streptomyces/enzymology
2.
Biochemistry ; 58(25): 2804-2808, 2019 06 25.
Article in English | MEDLINE | ID: mdl-31188570

ABSTRACT

Mitomycins make up a group of antitumor natural products that are biosynthesized from aminohydroxybenzoic acid (AHBA) and N-acetylglucosamine (GlcNAc). While the biosynthetic gene cluster was reported two decades ago, the mechanism by which the two building blocks, AHBA and GlcNAc, are coupled during biosynthesis remained uncharacterized. Here we report evidence that AHBA is first loaded onto an MmcB acyl carrier protein (ACP) by a MitE acyl ACP synthetase, followed by a transfer of GlcNAc from UDP-GlcNAc by MitB. The results suggest that the early steps of mitomycin biosynthesis proceed via intermediates linked to MmcB.


Subject(s)
Acyl Carrier Protein/chemistry , Carbon-Sulfur Ligases/chemistry , Mitomycin/biosynthesis , N-Acetylglucosaminyltransferases/chemistry , Aminobenzoates/chemistry , Enzyme Assays , Hydroxybenzoates/chemistry , Kinetics , Streptomyces/enzymology
3.
Chembiochem ; 19(13): 1391-1395, 2018 07 04.
Article in English | MEDLINE | ID: mdl-29603548

ABSTRACT

Naturally occurring lactams, such as the polyketide-derived macrolactams, provide a diverse class of natural products that could enhance existing chemically produced lactams. Although ß-amino acid loading in the fluvirucin B2 polyketide pathway was proposed by a previously identified putative biosynthetic gene cluster, biochemical characterization of the complete loading enzymes has not been described. Here we elucidate the complete biosynthetic pathway of the ß-amino acid loading pathway in fluvirucin B2 biosynthesis. We demonstrate the promiscuity of the loading pathway to utilize a range of amino acids and further illustrate the ability to introduce non-native acyl transferases to selectively transfer ß-amino acids onto a polyketide synthase (PKS) loading platform. The results presented here provide a detailed biochemical description of ß-amino acid selection and will further aid in future efforts to develop engineered lactam-producing PKS platforms.


Subject(s)
Amino Acids/metabolism , Deoxy Sugars/biosynthesis , Actinobacteria/chemistry , Actinobacteria/enzymology , Acyltransferases/chemistry , Acyltransferases/metabolism , Aminoacylation , Carbon-Sulfur Ligases/chemistry , Carbon-Sulfur Ligases/metabolism , Carboxy-Lyases/chemistry , Carboxy-Lyases/metabolism , Catalysis , Lactams , Molecular Structure , Peptide Synthases/chemistry , Peptide Synthases/metabolism , Protein Domains , Substrate Specificity
4.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1862(11): 1404-1413, 2017 Nov.
Article in English | MEDLINE | ID: mdl-27956138

ABSTRACT

Lysophospholipids (LPLs) are metabolic intermediates in bacterial phospholipid turnover. Distinct from their diacyl counterparts, these inverted cone-shaped molecules share physical characteristics of detergents, enabling modification of local membrane properties such as curvature. The functions of LPLs as cellular growth factors or potent lipid mediators have been extensively demonstrated in eukaryotic cells but are still undefined in bacteria. In the envelope of Gram-negative bacteria, LPLs are derived from multiple endogenous and exogenous sources. Although several flippases that move non-glycerophospholipids across the bacterial inner membrane were characterized, lysophospholipid transporter LplT appears to be the first example of a bacterial protein capable of facilitating rapid retrograde translocation of lyso forms of glycerophospholipids across the cytoplasmic membrane in Gram-negative bacteria. LplT transports lyso forms of the three bacterial membrane phospholipids with comparable efficiency, but excludes other lysolipid species. Once a LPL is flipped by LplT to the cytoplasmic side of the inner membrane, its diacyl form is effectively regenerated by the action of a peripheral enzyme, acyl-ACP synthetase/LPL acyltransferase (Aas). LplT-Aas also mediates a novel cardiolipin remodeling by converting its two lyso derivatives, diacyl or deacylated cardiolipin, to a triacyl form. This coupled remodeling system provides a unique bacterial membrane phospholipid repair mechanism. Strict selectivity of LplT for lyso lipids allows this system to fulfill efficient lipid repair in an environment containing mostly diacyl phospholipids. A rocker-switch model engaged by a pair of symmetric ion-locks may facilitate alternating substrate access to drive LPL flipping into bacterial cells. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.


Subject(s)
Cell Wall/metabolism , Gram-Negative Bacteria/metabolism , Lipogenesis , Lysophospholipids/biosynthesis , Phospholipid Transfer Proteins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Biological Transport , Carbon-Sulfur Ligases/chemistry , Carbon-Sulfur Ligases/metabolism , Lysophospholipids/chemistry , Phospholipid Transfer Proteins/chemistry , Signal Transduction , Substrate Specificity
5.
PLoS One ; 10(12): e0145085, 2015.
Article in English | MEDLINE | ID: mdl-26675168

ABSTRACT

Formaldehyde is a toxin and carcinogen that is both an environmental pollutant and an endogenous metabolite. Formaldehyde metabolism, which is probably essential for all aerobic cells, likely proceeds via multiple mechanisms, including via a glutathione-dependent pathway that is widely conserved in bacteria, plants and animals. However, it is unclear whether the first step in the glutathione-dependent pathway (i.e. formation of S-hydroxymethylglutathione (HMG)) is enzyme-catalysed. We report studies on glutathione-dependent formaldehyde-activating enzyme (GFA) from Paracoccus denitrificans, which has been proposed to catalyse HMG formation from glutathione and formaldehyde on the basis of studies using NMR exchange spectroscopy (EXSY). Although we were able to replicate the EXSY results, time course experiments unexpectedly imply that GFA does not catalyse HMG formation under standard conditions. However, GFA was observed to bind glutathione using NMR and mass spectrometry. Overall, the results reveal that GFA binds glutathione but does not directly catalyse HMG formation under standard conditions. Thus, it is possible that GFA acts as a glutathione carrier that acts to co-localise glutathione and formaldehyde in a cellular context.


Subject(s)
Bacterial Proteins/chemistry , Carbon-Sulfur Ligases/chemistry , Paracoccus denitrificans/enzymology , Amino Acid Sequence , Bacterial Proteins/metabolism , Carbon-Sulfur Ligases/metabolism , Glutathione/analogs & derivatives , Glutathione/metabolism , Molecular Sequence Data
6.
Proteins ; 81(7): 1232-44, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23444054

ABSTRACT

In thermophilic bacteria, specific 2-thiolation occurs on the conserved ribothymidine at position 54 (T54) in tRNAs, which is necessary for survival at high temperatures. T54 2-thiolation is achieved by the tRNA thiouridine synthetase TtuA and sulfur-carrier proteins. TtuA has five conserved CXXC/H motifs and the signature PP motif, and belongs to the TtcA family of tRNA 2-thiolation enzymes, for which there is currently no structural information. In this study, we determined the crystal structure of a TtuA homolog from the hyperthermophilic archeon Pyrococcus horikoshii at 2.1 Å resolution. The P. horikoshii TtuA forms a homodimer, and each subunit contains a catalytic domain and unique N- and C-terminal zinc fingers. The catalytic domain has much higher structural similarity to that of another tRNA modification enzyme, TilS (tRNA(Ile)2 lysidine synthetase), than to the other type of tRNA 2-thiolation enzyme, MnmA. Three conserved cysteine residues are clustered in the putative catalytic site, which is not present in TilS. An in vivo mutational analysis in the bacterium Thermus thermophilus demonstrated that the three conserved cysteine residues and the putative ATP-binding residues in the catalytic domain are important for the TtuA activity. A positively charged surface that includes the catalytic site and the two zinc fingers is likely to provide the tRNA-binding site.


Subject(s)
Amino Acyl-tRNA Synthetases/chemistry , Bacterial Proteins/chemistry , Carbon-Sulfur Ligases/chemistry , Protein Structure, Tertiary , Thermus thermophilus/enzymology , Thiouridine/chemistry , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Escherichia coli/enzymology , Models, Molecular , Mutation
7.
Anal Biochem ; 417(2): 264-73, 2011 Oct 15.
Article in English | MEDLINE | ID: mdl-21771578

ABSTRACT

Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), encodes for an astonishing 34 fatty acid adenylating enzymes (FadDs), which play key roles in lipid metabolism. FadDs involved in lipid biosynthesis are functionally nonredundant and serve to link fatty acid and polyketide synthesis to produce some of the most architecturally complex natural lipids including the essential mycolic acids as well as the virulence-conferring phthiocerol dimycocerosates, phenolic glycolipids, and mycobactins. Here we describe the systematic development and optimization of a fluorescence polarization assay to identify small molecule inhibitors as potential antitubercular agents. We fluorescently labeled a bisubstrate inhibitor to generate a fluorescent probe/tracer, which bound with a K(D) of 245 nM to FadD28. Next, we evaluated assay performance by competitive binding experiments with a series of known ligands and assessed the impact of control parameters including incubation time, stability of the signal, temperature, and DMSO concentration. As a final level of validation the LOPAC1280 library was screened in a 384-well plate format and the assay performed with a Z-factor of 0.75, demonstrating its readiness for high-throughput screening.


Subject(s)
Carbon-Sulfur Ligases/chemistry , Fluorescence Polarization/methods , High-Throughput Screening Assays/methods , Mycobacterium tuberculosis/enzymology , Carbon-Sulfur Ligases/antagonists & inhibitors , Carbon-Sulfur Ligases/genetics , Catalytic Domain , Fatty Acids/chemistry , Fatty Acids/metabolism , Fluorescent Dyes/chemical synthesis , Lipid Metabolism
8.
J Am Chem Soc ; 133(6): 1757-9, 2011 Feb 16.
Article in English | MEDLINE | ID: mdl-21247153

ABSTRACT

Ovothiols are histidine-derived thiols that were first isolated from marine invertebrates. We have identified a 5-histidylcysteine sulfoxide synthase (OvoA) as the first ovothiol biosynthetic enzyme and characterized OvoAs from Erwinia tasmaniensis and Trypanosoma cruzi . Homologous enzymes are encoded in more than 80 genomes ranging from proteobacteria to animalia.


Subject(s)
Carbon-Sulfur Ligases/metabolism , Erwinia/enzymology , Methylhistidines/metabolism , Trypanosoma cruzi/enzymology , Animals , Carbon-Sulfur Ligases/chemistry , Carbon-Sulfur Ligases/genetics , Genome/genetics , Protein Structure, Tertiary
9.
Biochemistry ; 45(33): 10008-19, 2006 Aug 22.
Article in English | MEDLINE | ID: mdl-16906759

ABSTRACT

The gene encoding the unique soluble acyl-acyl carrier protein synthetase (AasS) of the bioluminescent Vibrio harveyi strain B392 has been isolated by expression cloning in Escherichia coli. This enzyme catalyzes the ATP-dependent acylation of the thiol of acyl carrier protein (ACP) with fatty acids with chain lengths from C4 to C18. The gene (called aasS) encodes a protein of 60 kDa, a hexahistidine-tagged version of which was readily expressed in E. coli and purified in large quantities. Surprisingly, the sequence of the encoded protein was significantly more similar to that of an acyl-CoA synthetase of the distantly related bacterium, Thermus thermophilus, than to that of the membrane-bound acyl-acyl carrier protein synthetase of E. coli, an enzyme that catalyzes the same reaction from a more closely related organism. Indeed, the AasS sequence can readily be modeled on the known crystal structures of the T. thermophilus acyl-CoA synthetase with remarkably high levels of conservation of the catalytic site residues. To test the possible role of AasS in the fatty aldehyde-dependent bioluminescence pathway of V. harveyi, the chromosomal aasS gene of the organism was disrupted by insertion of a kanamycin cassette by homologous recombination. The resulting aasS::kan strains retained low levels of acyl-acyl carrier protein synthetase consistent with prior indications of a second such activity in this bacterium. The mutant strains grew normally and had normal levels of bioluminescence but were deficient in the incorporation of exogenous octanoic acid into the cellular phospholipids of V. harveyi, particularly at low octanoate concentrations. These data indicate that AasS is responsible for a high-affinity and high-capacity uptake system that efficiently converts exogenous fatty acids into acyl-ACP species competent to enter the fatty acid biosynthetic cycle.


Subject(s)
Carbon-Sulfur Ligases/metabolism , Coenzyme A Ligases/metabolism , Vibrio/enzymology , Aldehydes/metabolism , Base Sequence , Binding Sites , Carbon-Sulfur Ligases/chemistry , Carbon-Sulfur Ligases/isolation & purification , Coenzyme A Ligases/chemistry , Coenzyme A Ligases/isolation & purification , Electrophoresis, Polyacrylamide Gel , Escherichia coli/enzymology , Fatty Acids/metabolism , Luminescent Measurements , Membrane Proteins , Molecular Sequence Data , Molecular Weight , Mutation , Saccharomyces cerevisiae Proteins , Substrate Specificity , Thermus thermophilus/enzymology
10.
Article in English | MEDLINE | ID: mdl-16582482

ABSTRACT

FadD28 from Mycobacterium tuberculosis belongs to the fatty-acyl AMP ligase (FAAL) family of proteins. It is essential for the biosynthesis of a virulent phthiocerol dimycocerosate (PDIM) lipid that is only found in the cell wall of pathogenic mycobacteria. The N-terminal domain, comprising of the first 460 residues, was crystallized by the hanging-drop vapour-diffusion method at 295 K. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 50.97, b = 60.74, c = 136.54 angstroms. The crystal structure of the N-terminal domain of FadD28 at 2.35 angstroms resolution has been solved using the MAD method.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Carbon-Sulfur Ligases/chemistry , Carbon-Sulfur Ligases/metabolism , Mycobacterium tuberculosis/enzymology , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Carbon-Sulfur Ligases/isolation & purification , Crystallization , Crystallography, X-Ray , Peptide Fragments/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism
11.
J Biol Chem ; 280(4): 2826-30, 2005 Jan 28.
Article in English | MEDLINE | ID: mdl-15548539

ABSTRACT

The crystal structures of glutathione-dependent formaldehyde-activating enzyme (Gfa) from Paracoccus denitrificans, which catalyzes the formation of S-hydroxymethylglutathione from formaldehyde and glutathione, and its complex with glutathione (Gfa-GTT) have been determined. Gfa has a new fold with two zinc-sulfur centers, one that is structural (zinc tetracoordinated) and one catalytic (zinc apparently tricoordinated). In Gfa-GTT, the catalytic zinc is displaced due to disulfide bond formation of glutathione with one of the zinc-coordinating cysteines. Soaking crystals of Gfa-GTT with formaldehyde restores the holoenzyme. Accordingly, the displaced zinc forms a complex by scavenging formaldehyde and glutathione. The activation of formaldehyde and of glutathione in this zinc complex favors the final nucleophilic addition, followed by relocation of zinc in the catalytic site. Therefore, the structures of Gfa and Gfa-GTT draw the critical association between a dynamic zinc redox switch and a nucleophilic addition as a new facet of the redox activity of zinc-sulfur sites.


Subject(s)
Carbon-Sulfur Ligases/chemistry , Paracoccus denitrificans/enzymology , Zinc/chemistry , Crystallography, X-Ray , Cysteine/chemistry , Escherichia coli/metabolism , Formaldehyde/pharmacology , Glutathione/chemistry , Glutathione/pharmacology , Models, Molecular , Oxidation-Reduction , Protein Binding , Protein Conformation , Protein Folding
12.
J Biol Chem ; 277(33): 29369-76, 2002 Aug 16.
Article in English | MEDLINE | ID: mdl-12034706

ABSTRACT

Fatty acyl-CoA synthetase (FACS, fatty acid:CoA ligase, AMP forming; EC ) plays a central role in intermediary metabolism by catalyzing the formation of fatty acyl-CoA. In Escherichia coli this enzyme, encoded by the fadD gene, is required for the coupled import and activation of exogenous long-chain fatty acids. The E. coli FACS (FadD) contains two sequence elements, which comprise the ATP/AMP signature motif ((213)YTGGTTGVAKGA(224) and (356)GYGLTE(361)) placing it in the superfamily of adenylate-forming enzymes. A series of site-directed mutations were generated in the fadD gene within the ATP/AMP signature motif site to evaluate the role of this conserved region to enzyme function and to fatty acid transport. This approach revealed two major classes of fadD mutants with depressed enzyme activity: 1) those with 25-45% wild type activity (fadD(G216A), fadD(T217A), fadD(G219A), and fadD(K222A)) and 2) those with 10% or less wild-type activity (fadD(Y213A), fadD(T214A), and fadD(E361A)). Using anti-FadD sera, Western blots demonstrated the different mutant forms of FadD that were present and had localization patterns equivalent to the wild type. The defect in the first class was attributed to a reduced catalytic efficiency although several mutant forms also had a reduced affinity for ATP. The mutations resulting in these biochemical phenotypes reduced or essentially eliminated the transport of exogenous long-chain fatty acids. These data support the hypothesis that the FACS FadD functions in the vectorial movement of exogenous fatty acids across the plasma membrane by acting as a metabolic trap, which results in the formation of acyl-CoA esters.


Subject(s)
Adenosine Monophosphate/metabolism , Adenosine Triphosphate/metabolism , Amino Acids/metabolism , Carbon-Sulfur Ligases/metabolism , Escherichia coli/enzymology , Fatty Acids/metabolism , Amino Acid Sequence , Biological Transport , Carbon-Sulfur Ligases/chemistry , Carbon-Sulfur Ligases/genetics , Cell Membrane/enzymology , Molecular Sequence Data , Mutagenesis, Site-Directed , Sequence Homology, Amino Acid
13.
J Biol Chem ; 277(5): 3069-72, 2002 Feb 01.
Article in English | MEDLINE | ID: mdl-11741920

ABSTRACT

The formation of S-hydroxymethylglutathione from formaldehyde and glutathione is a central reaction in the consumption of the cytotoxin formaldehyde in some methylotrophic bacteria as well as in many other organisms. We describe here the discovery of an enzyme from Paracoccus denitrificans that accelerates this spontaneous condensation reaction. The rates of S-hydroxymethylglutathione formation and cleavage were determined under equilibrium conditions via two-dimensional proton exchange NMR spectroscopy. The pseudo first order rate constants k(1)* were estimated from the temperature dependence of the reaction and the signal to noise ratio of the uncatalyzed reaction. At 303 K and pH 6.0 k(1)* was found to be 0.02 s(-1) for the spontaneous reaction. A 10-fold increase of the rate constant was observed upon addition of cell extract from P. denitrificans grown in the presence of methanol corresponding to a specific activity of 35 units mg(-1). Extracts of cells grown in the presence of succinate revealed a lower specific activity of 11 units mg(-1). The enzyme catalyzing the conversion of formaldehyde and glutathione was purified and named glutathione-dependent formaldehyde-activating enzyme (Gfa). The gene gfa is located directly upstream of the gene for glutathione-dependent formaldehyde dehydrogenase, which catalyzes the subsequent oxidation of S-hydroxymethylglutathione. Putative proteins with sequence identity to Gfa from P. denitrificans are present also in Rhodobacter sphaeroides, Sinorhizobium meliloti, and Mesorhizobium loti.


Subject(s)
Carbon-Sulfur Ligases/metabolism , Glutathione/analogs & derivatives , Glutathione/biosynthesis , Paracoccus denitrificans/enzymology , Carbon-Sulfur Ligases/chemistry , Carbon-Sulfur Ligases/isolation & purification , Culture Media , Formaldehyde/metabolism , Glutathione/metabolism , Kinetics , Magnetic Resonance Spectroscopy/methods , Nitrogen Fixation , Paracoccus denitrificans/growth & development
14.
Biochem J ; 360(Pt 2): 471-9, 2001 Dec 01.
Article in English | MEDLINE | ID: mdl-11716776

ABSTRACT

Fatty acids are activated in an ATP-dependent manner before they are utilized. We describe here how the 10 S triacylglycerol biosynthetic multienzyme complex from Rhodotorula glutinis is capable of activating non-esterified fatty acids for the synthesis of triacylglycerol. The photolabelling of the complex with [(32)P]azido-ATP showed labelling of a 35 kDa polypeptide. The labelled polypeptide was identified as acyl-acyl carrier protein (ACP) synthetase, which catalyses the ATP-dependent ligation of fatty acid with ACP to form acyl-ACP. The enzyme was purified by successive PAGE separations to apparent homogeneity from the soluble fraction of oleaginous yeast and its apparent molecular mass was 35 kDa under denaturing and reducing conditions. Acyl-ACP synthetase was specific for ATP. The K(m) values for palmitic, stearic, oleic and linoleic acids were found to be 42.9, 30.4, 25.1 and 22.7 microM, respectively. The antibodies to acyl-ACP synthetase cross-reacted with Escherichia coli acyl-ACP synthetase. Anti-ACP antibodies showed no cross-reactivity with the purified acyl-ACP synthetase, indicating no bound ACP with the enzyme. Immunoprecipitations with antibodies to acyl-ACP synthetase revealed that this enzyme is a part of the 10 S triacylglycerol biosynthetic complex. These results demonstrate that the soluble acyl-ACP synthetase plays a novel role in activating fatty acids for triacylglycerol biosynthesis in oleaginous yeast.


Subject(s)
Acyl Carrier Protein/metabolism , Carbon-Sulfur Ligases/chemistry , Carbon-Sulfur Ligases/isolation & purification , Rhodotorula/enzymology , Triglycerides/biosynthesis , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Carbon-Sulfur Ligases/metabolism , Cytidine Triphosphate/metabolism , Enzyme Activation , Fatty Acids/metabolism , Guanosine Triphosphate/metabolism , Kinetics , Molecular Sequence Data , Multienzyme Complexes/chemistry , Multienzyme Complexes/isolation & purification , Multienzyme Complexes/metabolism , Uridine Triphosphate/metabolism
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