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1.
N Biotechnol ; 72: 114-121, 2022 Dec 25.
Article in English | MEDLINE | ID: mdl-36307012

ABSTRACT

In Escherichia coli, acyl carrier protein (ACP) is posttranslationally converted into its active holo-ACP form via covalent linkage of 4'-phosphopantetheine (4'-PP) to residue serine-36. We found that the long flexible 4'-PP arm could react chemoselectively with the iodoacetyl group introduced on solid supports with high efficiency under mild conditions. Based on this finding, we developed site-selective immobilisation of proteins via the active holo-ACP fusion tag, independently of the physicochemical properties of the protein of interest. Furthermore, the molecular ratios of co-immobilised proteins can be manipulated because the tethering process is predominantly directed by the molar concentrations of diverse holo-ACP fusions during co-immobilisation. Conveniently tuning the molecular ratios of co-immobilised proteins allows their cooperation, leading to a highly productive multi-protein co-immobilisation system. Kinetic studies of enzymes demonstrated that α-amylase (Amy) and methyl parathion hydrolase (MPH) immobilised via active tag holo-ACP had higher catalytic efficiency (kcat/Km) in comparison with their corresponding counterparts immobilised via the sulfhydryl groups (-SH) of these proteins. The immobilised holo-ACP-Amy also presented higher thermostability compared with free Amy. The enhanced α-amylase thermostability upon immobilisation via holo-ACP renders it more suitable for industrial application.


Subject(s)
Acyl Carrier Protein , Pantetheine , Kinetics , Pantetheine/chemistry , Pantetheine/metabolism , Acyl Carrier Protein/chemistry , Acyl Carrier Protein/metabolism , Escherichia coli/metabolism , alpha-Amylases/metabolism , Immobilized Proteins/metabolism
2.
Proc Natl Acad Sci U S A ; 119(34): e2206494119, 2022 08 23.
Article in English | MEDLINE | ID: mdl-35969793

ABSTRACT

Complex carbapenems are important clinical antibiotics used to treat recalcitrant infections. Their biosynthetic gene clusters contain three essential B12-dependent radical S-adenosylmethionine (rSAM) enzymes. The majority of characterized enzymes in this subfamily catalyze methyl transfer, but only one is required to sequentially install all methionine-derived carbons in complex carbapenems. Therefore, it is probable that the other two rSAM enzymes have noncanonical functions. Through a series of fermentation and in vitro experiments, we show that ThnL uses radical SAM chemistry to catalyze thioether bond formation between C2 of a carbapenam precursor and pantetheine, uniting initial bicycle assembly common to all carbapenems with later tailoring events unique to complex carbapenems. ThnL also catalyzes reversible thiol/disulfide redox on pantetheine. Neither of these functions has been observed previously in a B12-dependent radical SAM enzyme. ThnL expands the known activity of this subclass of enzymes beyond carbon-carbon bond formation or rearrangement. It is also the only radical SAM enzyme currently known to catalyze carbon-sulfur bond formation with only an rSAM Fe-S cluster and no additional auxiliary clusters.


Subject(s)
Carbapenems , Iron-Sulfur Proteins , S-Adenosylmethionine , Vitamin B 12 , Carbapenems/biosynthesis , Carbapenems/chemistry , Carbon , Iron-Sulfur Proteins/chemistry , Pantetheine/chemistry , S-Adenosylmethionine/chemistry , Sulfides , Vitamin B 12/chemistry
3.
Acta Crystallogr F Struct Biol Commun ; 77(Pt 9): 294-302, 2021 Sep 01.
Article in English | MEDLINE | ID: mdl-34473106

ABSTRACT

Acyltransferases are responsible for the selection and loading of acyl units onto carrier proteins in polyketide and fatty-acid biosynthesis. Despite the importance of protein-protein interactions between the acyltransferase and the carrier protein, structural information on acyltransferase-carrier protein interactions is limited because of the transient interactions between them. In the biosynthesis of the polyketide vicenistatin, the acyltransferase VinK recognizes the carrier protein VinL for the transfer of a dipeptidyl unit. The crystal structure of a VinK-VinL covalent complex formed with a 1,2-bismaleimidoethane cross-linking reagent has been determined previously. Here, the crystal structure of a VinK-VinL covalent complex formed with a pantetheine cross-linking probe is reported at 1.95 Šresolution. In the structure of the VinK-VinL-probe complex, the pantetheine probe that is attached to VinL is covalently connected to the side chain of the mutated Cys106 of VinK. The interaction interface between VinK and VinL is essentially the same in the two VinK-VinL complex structures, although the position of the pantetheine linker slightly differs. This structural observation suggests that interface interactions are not affected by the cross-linking strategy used.


Subject(s)
Acyltransferases/chemistry , Acyltransferases/metabolism , Cross-Linking Reagents/chemistry , Pantetheine/chemistry , Pantetheine/metabolism , Protein Interaction Domains and Motifs , Acyltransferases/genetics , Crystallography, X-Ray , Mutagenesis, Site-Directed , Mutation , Protein Conformation , Substrate Specificity
4.
Int J Biol Macromol ; 179: 144-153, 2021 May 15.
Article in English | MEDLINE | ID: mdl-33667556

ABSTRACT

L. major acyl carrier protein (ACP) is a mitochondrial protein, involved in fatty acid biosynthesis. The protein is expressed as an apo-protein, and post-translationally modified at Ser 37 by a 4'-Phosphopantetheinyl transferase. Crystal structure of the apo-form of the protein at pH 5.5 suggests a four helix bundle fold, typical of ACP's. However, upon lowering the pH to 5.0, it undergoes a conformational transition from α-helix to ß-sheet, and displays amyloid like properties. When left for a few days at room temperature at this pH, the protein forms fibrils, visible under Transmission electron microscopy (TEM). Using an approach combining NMR, biophysical techniques, and mutagenesis, we have identified a Phe residue present on helix II of ACP, liable for this change. Phosphopantetheinylation of LmACP, or mutation of Phe 45 to the corresponding residue in E. coli ACP (methionine), slows down the conformational change. Conversely, substitution of methionine 44 of E. coli ACP with a phenylalanine, causes enhanced ThT binding. Thus, we demonstrate the unique property of an exposed Phe in inducing, and phophopantetheine in inhibiting amyloidogenesis. Taken together, our study adds L. major acyl carrier protein to the list of ACPs that act as pH sensors.


Subject(s)
Acyl Carrier Protein/chemistry , Leishmania major/chemistry , Pantetheine/analogs & derivatives , Phenylalanine/chemistry , Protein Aggregates , Protozoan Proteins/chemistry , Pantetheine/chemistry
5.
Chembiochem ; 22(8): 1357-1367, 2021 04 16.
Article in English | MEDLINE | ID: mdl-33289264

ABSTRACT

Nature uses a diverse array of protein post-translational modifications (PTMs) to regulate protein structure, activity, localization, and function. Among them, protein 4'-phosphopantetheinylation derived from coenzyme A (CoA) is an essential PTM for the biosynthesis of fatty acids, polyketides, and nonribosomal peptides in prokaryotes and eukaryotes. To explore its functions, various chemical probes mimicking the natural structure of 4'-phosphopantetheinylation have been developed. In this minireview, we summarize these chemical probes and describe their applications in direct and metabolic labeling of proteins in bacterial and mammalian cells.


Subject(s)
Coenzyme A/chemistry , Pantetheine/analogs & derivatives , Coenzyme A/metabolism , Models, Molecular , Molecular Structure , Pantetheine/chemistry , Pantetheine/metabolism , Protein Processing, Post-Translational
6.
Bioorg Med Chem ; 28(22): 115740, 2020 11 15.
Article in English | MEDLINE | ID: mdl-33007553

ABSTRACT

Coenzyme A (CoA) is a highly selective inhibitor of the mitotic regulatory enzyme Aurora A kinase, with a novel mode of action. Herein we report the design and synthesis of analogues of CoA as inhibitors of Aurora A kinase. We have designed and synthesised modified CoA structures as potential inhibitors, combining dicarbonyl mimics of the pyrophosphate group with a conserved adenosine headgroup and different length pantetheine-based tail groups. An analogue with a -SH group at the end of the pantotheinate tail showed the best IC50, probably due to the formation of a covalent bond with Aurora A kinase Cys290.


Subject(s)
Aurora Kinase A/antagonists & inhibitors , Coenzyme A/pharmacology , Diphosphates/pharmacology , Drug Design , Pantetheine/pharmacology , Protein Kinase Inhibitors/pharmacology , Aurora Kinase A/metabolism , Coenzyme A/chemical synthesis , Coenzyme A/chemistry , Diphosphates/chemistry , Dose-Response Relationship, Drug , Humans , Models, Molecular , Molecular Structure , Pantetheine/chemistry , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Structure-Activity Relationship
7.
FEBS Lett ; 593(6): 622-633, 2019 03.
Article in English | MEDLINE | ID: mdl-30847903

ABSTRACT

In Mycobacterium tuberculosis, acyl carrier protein (AcpM)-mediated fatty acid synthase type II is integral for the synthesis of mycolic acids. AcpM, designated as an atypical ACP, comprises of a putative 33 amino acid long C-terminal extension which is distinctive in nature. Here, we aimed at devising an 'easy-to-go' method for the generation of crypto-AcpM loaded with a solvatochromic probe 7-Nitrobenz-2-oxa-1,3-diazol-4-yl, which is linked to the 4'-phosphopantetheine (Ppant) prosthetic group of AcpM. The crypto-AcpM, coupled with fluorescence spectroscopy and molecular dynamics simulation studies, was employed to explore the elusive dynamics of Ppant arm in AcpM. This investigation establishes the role of the flexible C-terminal extension of AcpM in regulating the prosthetic group sequestration ability by modulating the 'Asp-Ser-Leu' motif.


Subject(s)
Bacterial Proteins/chemistry , Carrier Proteins/chemistry , Coenzyme A/chemistry , Mycobacterium tuberculosis/chemistry , Pantetheine/analogs & derivatives , Amino Acid Motifs , Azoles/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cloning, Molecular , Coenzyme A/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Fluorescent Dyes/chemistry , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Molecular Docking Simulation , Molecular Dynamics Simulation , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Mycolic Acids/metabolism , Nitrobenzenes/chemistry , Pantetheine/chemistry , Pantetheine/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Substrate Specificity
8.
Nat Chem Biol ; 14(5): 474-479, 2018 05.
Article in English | MEDLINE | ID: mdl-29610486

ABSTRACT

Polyketide synthases (PKSs) are microbial multienzymes for the biosynthesis of biologically potent secondary metabolites. Polyketide production is initiated by the loading of a starter unit onto an integral acyl carrier protein (ACP) and its subsequent transfer to the ketosynthase (KS). Initial substrate loading is achieved either by multidomain loading modules or by the integration of designated loading domains, such as starter unit acyltransferases (SAT), whose structural integration into PKS remains unresolved. A crystal structure of the loading/condensing region of the nonreducing PKS CTB1 demonstrates the ordered insertion of a pseudodimeric SAT into the condensing region, which is aided by the SAT-KS linker. Cryo-electron microscopy of the post-loading state trapped by mechanism-based crosslinking of ACP to KS reveals asymmetry across the CTB1 loading/-condensing region, in accord with preferential 1:2 binding stoichiometry. These results are critical for re-engineering the loading step in polyketide biosynthesis and support functional relevance of asymmetric conformations of PKSs.


Subject(s)
Acyl Carrier Protein/chemistry , Polyketide Synthases/chemistry , Ascomycota/metabolism , Catalytic Domain , Cross-Linking Reagents/chemistry , Cryoelectron Microscopy , Crystallography, X-Ray , Escherichia coli/metabolism , Pantetheine/chemistry , Phosphorylation , Propionates/chemistry , Protein Conformation , Protein Domains , Protein Multimerization , Substrate Specificity
9.
Bioorg Chem ; 76: 23-27, 2018 02.
Article in English | MEDLINE | ID: mdl-29107839

ABSTRACT

Dephospho coenzyme A (depCoA) is the last intermediate for CoA biosynthesis, and it can be used as a transcription initiator to prepare CoA-linked RNA by in vitro transcription. However, commercially available depCoA is expensive. We hereby describe a simple and efficient enzymatic synthesis of depCoA in a single-step from commercially available and inexpensive oxidized pantethine (Ox-Pan) and ATP. A plasmid (pCoaDAa) was constructed to co-express and co-purify two enzymes pantothenate kinase (PanK/coaA) and phosphopantetheine adenylyltransferase (PPAT/coaD). Starting from Ox-Pan and ATP, two different synthetic routes of one-pot reaction catalyzed by PanK and PPAT, followed by a simple column purification step, afforded depCoA and its oxidized dimer (Ox-depCoA) with high yields and purity. The simplicity and low cost of our method should make depCoA easily accessible to a broad scientific community, and promote research on CoA-related areas in biology and biomedicine.


Subject(s)
Coenzyme A/chemical synthesis , Nucleotidyltransferases/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Adenosine Triphosphate/chemistry , Amino Acid Sequence , Base Sequence , Chemistry Techniques, Synthetic/methods , Cloning, Molecular/methods , Escherichia coli/enzymology , Nucleotidyltransferases/genetics , Oxidation-Reduction , Pantetheine/analogs & derivatives , Pantetheine/chemistry , Phosphotransferases (Alcohol Group Acceptor)/genetics , Plasmids/genetics
10.
Chembiochem ; 16(1): 156-166, 2015 Jan 02.
Article in English | MEDLINE | ID: mdl-25394180

ABSTRACT

Polyhydroxyalkanoate (PHA) synthases (PhaCs) catalyze the formation of biodegradable PHAs that are considered to be ideal alternatives to non-biodegradable synthetic plastics. However, study of PhaCs has been challenging because the rate of PHA chain elongation is much faster than that of initiation. This difficulty, along with lack of a crystal structure, has become the main hurdle to understanding and engineering PhaCs for economical PHA production. Here we report the synthesis of two carbadethia CoA analogues--sT-CH2-CoA (26 a) and sTet-CH2-CoA (26 b)--as well as sT-aldehyde (saturated trimer aldehyde, 29), as new PhaC inhibitors. Study of these analogues with PhaECAv revealed that 26 a/b and 29 are competitive and mixed inhibitors, respectively. Both the CoA moiety and extension of PHA chain will increase binding affinity; this is consistent with our docking study. Estimation of the Kic values of 26 a and 26 b predicts that a CoA analogue incorporating an octameric hydroxybutanoate (HB) chain might facilitate the formation of a kinetically well-behaved synthase.


Subject(s)
Acyltransferases/chemistry , Aldehydes/chemistry , Bacterial Proteins/chemistry , Coenzyme A/chemistry , Enzyme Inhibitors/chemistry , Pantetheine/analogs & derivatives , Polyhydroxyalkanoates/chemistry , Acyltransferases/antagonists & inhibitors , Acyltransferases/metabolism , Aldehydes/chemical synthesis , Animals , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/metabolism , Biocatalysis , Biodegradation, Environmental , Coenzyme A/chemical synthesis , Cupriavidus necator/chemistry , Cupriavidus necator/enzymology , Dogs , Enzyme Assays , Enzyme Inhibitors/chemical synthesis , Esterases/chemistry , Kinetics , Lipase/chemistry , Molecular Docking Simulation , Pantetheine/chemical synthesis , Pantetheine/chemistry , Polyhydroxyalkanoates/metabolism , Structural Homology, Protein , Substrate Specificity , Sulfolobus solfataricus/chemistry , Sulfolobus solfataricus/enzymology
11.
J Am Chem Soc ; 136(50): 17378-81, 2014 Dec 17.
Article in English | MEDLINE | ID: mdl-25468257

ABSTRACT

We present a spherical micelle generated in a three-step sequence in which a farnesyl-pantetheine conjugate is phosphorylated, adenylated, and phosphorylated once more to generate a farnesyl-CoA amphiphile that self-assembles into spherical micelles. A sphere-to-fibril morphological switch is achieved by enzymatically transferring the farnesyl group of the farnesyl-CoA micelle onto a peptide via phosphopantetheinyl transferase to generate a peptide amphiphile. Each step in the sequence is followed with characterization by HPLC, MS, TEM, and DLS. This system offers an entry into cofactor-mediated peptide decoration by extending the principles of bioresponsive polymeric materials to sequential enzyme cascades.


Subject(s)
Bacteria/enzymology , Nanostructures/chemistry , Farnesol/chemistry , Micelles , Molecular Structure , Pantetheine/chemistry
12.
J Pharm Biomed Anal ; 97: 141-50, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24863372

ABSTRACT

Pantethine (d-bis-(N-pantothenyl-ß-aminoethyl)-disulfide, PAN), the stable disulfide form of pantetheine, has beneficial effects in vascular diseases being able to decrease the hyperlipidaemia, moderate the platelet function and prevent the lipid peroxidation. Furthermore, recent studies suggested that PAN may be an effective therapeutic agent for cerebral malaria and, possibly, for neurodegenerative processes. Interestingly, in the literature, there were no data dealing with the chemical stability and the analytical aspects of PAN. Hence, in the present work the chemical stability of PAN was for the first time established through a forced degradation study followed by liquid chromatography tandem mass spectrometry investigation showing the formation of three degradation products of PAN (PD1, PD2 and POx) arising from hydrolytic, thermal and oxidative stresses. Based on these data a stability-indicating LC-UV method for simultaneous estimation of PAN, and its most relevant degradation product (PD1) was developed and validated; moreover the method allowed also the separation and the quantification of the preservative system, constituted by a paraben mixture. The method showed linearity for PAN (0.4-1.2mgmL(-1)), MHB, PHB (0.4-1.2µgmL(-1)) and PD1 (2.5-100µgmL(-1)); the precision, determined in terms of intra-day and inter-day precision, expressed as RSDs, were in the ranges 0.4-1.2 and 0.7-1.4, respectively. The method demonstrated to be accurate and robust; indeed the average recoveries were 100.2, 99.9, and 100.0% for PAN, MHB and PHB, respectively, and 99.9% for PD1. By applying small variations of the mobile phase composition, counter-ion concentration and pH the separation of analytes was not affected. Finally, the applicability of this method was evaluated analyzing the available commercial forms at release as well as during stability studies.


Subject(s)
Drug Stability , Pantetheine/analogs & derivatives , Capsules , Chromatography, Liquid/methods , Limit of Detection , Molecular Structure , Pantetheine/analysis , Pantetheine/chemistry , Parabens/analysis , Tandem Mass Spectrometry/methods , Ultraviolet Rays
13.
Proteins ; 82(9): 2067-77, 2014 Sep.
Article in English | MEDLINE | ID: mdl-24634061

ABSTRACT

While the cis-acyltransferase modular polyketide synthase assembly lines have largely been structurally dissected, enzymes from within the recently discovered trans-acyltransferase polyketide synthase assembly lines are just starting to be observed crystallographically. Here we examine the ketoreductase (KR) from the first polyketide synthase module of the bacillaene nonribosomal peptide synthetase/polyketide synthase at 2.35-Å resolution. This KR naturally reduces both α- and ß-keto groups and is the only KR known to do so during the biosynthesis of a polyketide. The isolated KR not only reduced an N-acetylcysteamine-bound ß-keto substrate to a D-ß-hydroxy product, but also an N-acetylcysteamine-bound α-keto substrate to an L-α-hydroxy product. That the substrates must enter the active site from opposite directions to generate these stereochemistries suggests that the acyl-phosphopantetheine moiety is capable of accessing very different conformations despite being anchored to a serine residue of a docked acyl carrier protein. The features enabling stereocontrolled α-ketoreduction may not be extensive since a KR that naturally reduces a ß-keto group within a cis-acyltransferase polyketide synthase was identified that performs a completely stereoselective reduction of the same α-keto substrate to generate the D-α-hydroxy product. A sequence analysis of trans-acyltransferase KRs reveals that a single residue, rather than a three-residue motif found in cis-acyltransferase KRs, is predictive of the orientation of the resulting ß-hydroxyl group.


Subject(s)
Acyltransferases/chemistry , Alcohol Oxidoreductases/chemistry , Alcohol Oxidoreductases/ultrastructure , Bacterial Proteins/chemistry , Bacterial Proteins/ultrastructure , Polyketide Synthases/chemistry , Polyketides/chemistry , Amino Acid Sequence , Bacillus subtilis/enzymology , Crystallography, X-Ray , Models, Molecular , Molecular Sequence Data , Pantetheine/analogs & derivatives , Pantetheine/chemistry , Peptide Synthases , Polyenes
14.
Bioorg Khim ; 40(2): 170-7, 2014.
Article in Russian | MEDLINE | ID: mdl-25895336

ABSTRACT

Convenient two-step synthesis of conjugates of HS-CoA and D-pantetheine with aminooxy analogues of Spm, Spd and Put was suggested. The use of acetone linker provided target conjugates with quantitative yields. The activity of CoA-derived "bisubstrate" inhibitors being active at microM concentrations was at least 100 times better than that of corresponding derivatives of D-pantetheine.


Subject(s)
Acetyltransferases/chemical synthesis , Polyamines/metabolism , Spermidine/chemical synthesis , Spermine/chemical synthesis , Acetyl Coenzyme A/chemistry , Acetyltransferases/chemistry , Bacteria/chemistry , Bacteria/metabolism , Bacteria/pathogenicity , Kinetics , Pantetheine/chemistry , Polyamines/chemistry , Spermidine/chemistry , Spermine/chemistry
15.
Antimicrob Agents Chemother ; 57(12): 6005-15, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24041904

ABSTRACT

Inhibitors of 4'-phosphopantetheine adenylyltransferase (PPAT) were identified through high-throughput screening of the AstraZeneca compound library. One series, cycloalkyl pyrimidines, showed inhibition of PPAT isozymes from several species, with the most potent inhibition of enzymes from Gram-positive species. Mode-of-inhibition studies with Streptococcus pneumoniae and Staphylococcus aureus PPAT demonstrated representatives of this series to be reversible inhibitors competitive with phosphopantetheine and uncompetitive with ATP, binding to the enzyme-ATP complex. The potency of this series was optimized using structure-based design, and inhibition of cell growth of Gram-positive species was achieved. Mode-of-action studies, using generation of resistant mutants with targeted sequencing as well as constructs that overexpress PPAT, demonstrated that growth suppression was due to inhibition of PPAT. An effect on bacterial burden was demonstrated in mouse lung and thigh infection models, but further optimization of dosing requirements and compound properties is needed before these compounds can be considered for progress into clinical development. These studies validated PPAT as a novel target for antibacterial therapy.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Nucleotidyltransferases/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Staphylococcus aureus/drug effects , Streptococcus pneumoniae/drug effects , Animals , Anti-Bacterial Agents/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Binding, Competitive , Crystallography, X-Ray , Drug Discovery , Enzyme Inhibitors/chemistry , Female , Lung/drug effects , Lung/microbiology , Mice , Models, Molecular , Nucleotidyltransferases/chemistry , Nucleotidyltransferases/metabolism , Pantetheine/analogs & derivatives , Pantetheine/chemistry , Pneumococcal Infections/drug therapy , Pneumococcal Infections/microbiology , Pneumonia, Bacterial/drug therapy , Pneumonia, Bacterial/microbiology , Small Molecule Libraries/chemistry , Staphylococcus aureus/enzymology , Staphylococcus aureus/growth & development , Streptococcus pneumoniae/enzymology , Streptococcus pneumoniae/growth & development , Thigh/microbiology
16.
PLoS One ; 8(9): e74271, 2013.
Article in English | MEDLINE | ID: mdl-24040220

ABSTRACT

Helicobacter pylori is a major etiologic agent associated with the development and maintenance of human gastritis. The goal of this study was to develop novel antibiotics against H. pylori, and we thus targeted H. pylori phosphopantetheine adenylyltransferase (HpPPAT). PPAT catalyzes the penultimate step in coenzyme A biosynthesis. Its inactivation effectively prevents bacterial viability, making it an attractive target for antibacterial drug discovery. We employed virtual high-throughput screening and the HpPPAT crystal structure to identify compounds in the PubChem database that might act as inhibitors of HpPPAT. d-amethopterin is a potential inhibitor for blocking HpPPAT activity and suppressing H. pylori viability. Following treatment with d-amethopterin, H. pylori exhibited morphological characteristics associated with cell death. d-amethopterin is a mixed inhibitor of HpPPAT activity; it simultaneously occupies the HpPPAT 4'-phosphopantetheine- and ATP-binding sites. Its binding affinity is in the micromolar range, implying that it is sufficiently potent to serve as a lead compound in subsequent drug development. Characterization of the d-amethopterin and HpPPAT interaction network in a docked model will allow us to initiate rational drug optimization to improve the inhibitory efficacy of d-amethopterin. We anticipate that novel, potent, and selective HpPPAT inhibitors will emerge for the treatment of H. pylori infection.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Helicobacter pylori/drug effects , Methotrexate/pharmacology , Nucleotidyltransferases/antagonists & inhibitors , Adenosine Triphosphate/chemistry , Anti-Bacterial Agents/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Binding Sites , Coenzyme A/antagonists & inhibitors , Coenzyme A/biosynthesis , Coenzyme A/chemistry , Databases, Chemical , Drug Discovery , Enzyme Inhibitors/chemistry , Helicobacter pylori/chemistry , Helicobacter pylori/enzymology , High-Throughput Screening Assays , Methotrexate/chemistry , Microbial Sensitivity Tests , Molecular Docking Simulation , Nucleotidyltransferases/chemistry , Nucleotidyltransferases/metabolism , Pantetheine/analogs & derivatives , Pantetheine/chemistry , Protein Binding
17.
Chem Biol ; 20(9): 1135-46, 2013 Sep 19.
Article in English | MEDLINE | ID: mdl-23993461

ABSTRACT

Protein·protein interactions, which often involve interactions among an acyl carrier protein (ACP) and ACP partner enzymes, are important for coordinating polyketide biosynthesis. However, the nature of such interactions is not well understood, especially in the fungal nonreducing polyketide synthases (NR-PKSs) that biosynthesize toxic and pharmaceutically important polyketides. Here, we employ mechanism-based crosslinkers to successfully probe ACP and ketosynthase (KS) domain interactions in NR-PKSs. We found that crosslinking efficiency is closely correlated with the strength of ACP·KS interactions and that KS demonstrates strong starter unit selectivity. We further identified positively charged surface residues by KS mutagenesis, which mediates key interactions with the negatively charged ACP surface. Such complementary/matching contact pairs can serve as "adapter surfaces" for future efforts to generate new polyketides using NR-PKSs.


Subject(s)
Polyketide Synthases/metabolism , Acyl Carrier Protein/chemistry , Acyl Carrier Protein/metabolism , Amino Acid Sequence , Cross-Linking Reagents/chemistry , Electrophoresis, Polyacrylamide Gel , Molecular Sequence Data , Mutagenesis , Pantetheine/chemistry , Polyketide Synthases/chemistry , Polyketide Synthases/genetics , Polyketides/chemistry , Polyketides/metabolism , Protein Interaction Domains and Motifs , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Sequence Alignment
18.
ACS Chem Biol ; 8(3): 530-4, 2013 Mar 15.
Article in English | MEDLINE | ID: mdl-23270378

ABSTRACT

Vanins are enzymes with pantetheinase activity and are presumed to play a role in the recycling of pantothenic acid (vitamin B5) from pantetheine. Pantothenic acid is an essential nutrient required to synthesize coenzyme A, a cofactor involved in many biological processes such as fatty acid synthesis and oxidation of pyruvate to fuel the citric acid cycle. Hydrolysis of pantetheine also liberates cysteamine, a known antioxidant. Vanin-1 is highly expressed in liver and is under transcriptional control of PPAR-α and nutritional status, suggesting a role in energy metabolism. The lack of potent and specific inhibitors of vanins has hampered detailed investigation of their function. We hereby report the design, synthesis, and characterization of a novel pantetheine analogue, RR6, that acts as a selective, reversible, and competitive vanin inhibitor at nanomolar concentration. Oral administration of RR6 in rats completely inhibited plasma vanin activity and caused alterations of plasma lipid concentrations upon fasting, thereby illustrating its potential use in chemical biology research.


Subject(s)
Amidohydrolases/antagonists & inhibitors , Drug Discovery , Pantetheine/pharmacology , Small Molecule Libraries/pharmacology , Amidohydrolases/metabolism , Animals , Cattle , Disease , Dose-Response Relationship, Drug , Female , GPI-Linked Proteins/antagonists & inhibitors , GPI-Linked Proteins/metabolism , Humans , Male , Molecular Structure , Pantetheine/analogs & derivatives , Pantetheine/chemistry , Rats , Rats, Wistar , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/chemistry , Structure-Activity Relationship
19.
Org Biomol Chem ; 10(10): 1992-2002, 2012 Mar 14.
Article in English | MEDLINE | ID: mdl-22293823

ABSTRACT

Acyl carrier proteins are critical components of fatty acid and polyketide biosynthesis. Their primary function is to shuttle intermediates between active sites via a covalently bound phosphopantetheine arm. Small molecules capable of acylating this prosthetic group will provide a simple and reversible means of introducing novel functionality onto carrier protein domains. A series of N-activated ß-lactams are prepared to examine site-specific acylation of the phosphopantetheine-thiol. In general, ß-lactams are found to be significantly more reactive than our previously studied ß-lactones. Selectivity for the holo over apo-form of acyl carrier proteins is demonstrated indicating that only the phosphopantetheine-thiol is modified. Incorporation of an N-propargyloxycarbonyl group provides an alkyne handle for conjugation to fluorophores and affinity labels. The utility of these groups for mechanistic interrogation of a critical step in polyketide biosynthesis is examined through comparison to traditional probes. In all, we expect the probes described in this study to serve as valuable and versatile tools for mechanistic interrogation.


Subject(s)
Acyl Carrier Protein/analysis , Fluorescent Dyes/chemistry , beta-Lactams/chemistry , Acylation , Electrophoresis, Polyacrylamide Gel , Mass Spectrometry , Pantetheine/analogs & derivatives , Pantetheine/chemistry , Sulfhydryl Compounds/chemistry
20.
ACS Chem Biol ; 7(3): 470-5, 2012 Mar 16.
Article in English | MEDLINE | ID: mdl-22217014

ABSTRACT

The most common mechanism of resistance to aminoglycoside antibiotics entails bacterial expression of drug-metabolizing enzymes, such as the clinically widespread aminoglycoside N-6'-acetyltransferase (AAC(6')). Aminoglycoside-CoA bisubstrates are highly potent AAC(6') inhibitors; however, their inability to penetrate cells precludes in vivo studies. Some truncated bisubstrates are known to cross cell membranes, yet their activities against AAC(6') are in the micromolar range at best. We report here the synthesis and biological activity of aminoglycoside-pantetheine derivatives that, although devoid of AAC(6') inhibitory activity, can potentiate the antibacterial activity of kanamycin A against an aminoglycoside-resistant strain of Enterococcus faecium. Biological studies demonstrate that these molecules are potentially extended to their corresponding full-length bisubstrates by enzymes of the coenzyme A biosynthetic pathway. This work provides a proof-of-concept for the utility of prodrug compounds activated by enzymes of the coenzyme A biosynthetic pathway, to resensitize resistant strains of bacteria to aminoglycoside antibiotics.


Subject(s)
Aminoglycosides/pharmacology , Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial/drug effects , Enterococcus faecium/drug effects , Kanamycin/pharmacology , Prodrugs/metabolism , Prodrugs/pharmacology , Acetyltransferases/antagonists & inhibitors , Acetyltransferases/metabolism , Aminoglycosides/chemical synthesis , Aminoglycosides/chemistry , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Coenzyme A/biosynthesis , Coenzyme A/metabolism , Dose-Response Relationship, Drug , Enterococcus faecium/cytology , Enterococcus faecium/enzymology , Kanamycin/chemical synthesis , Kanamycin/chemistry , Pantetheine/chemical synthesis , Pantetheine/chemistry , Pantetheine/pharmacology , Structure-Activity Relationship
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