Isoprenoid biosynthetic pathways as anti-infective drug targets

2005 ◽  
Vol 33 (4) ◽  
pp. 785-791 ◽  
Author(s):  
F. Rohdich ◽  
A. Bacher ◽  
W. Eisenreich
Keyword(s):  
Helicobacter Pylori ◽  
Plasmodium Falciparum ◽  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Biosynthetic Pathway ◽  
Mevalonate Pathway ◽  
Biosynthetic Pathways ◽  
Isopentenyl Diphosphate ◽  
Human Pathogens ◽  
Dimethylallyl Diphosphate

IPP (isopentenyl diphosphate) and DMAPP (dimethylallyl diphosphate) serve as the universal precursors for the biosynthesis of isoprenoids. Besides the well-known mevalonate pathway, the existence of a second biosynthetic pathway conducive to IPP and DMAPP formation through 1-deoxy-D-xylulose 5-phosphate and 2C-methyl-D-erythritol 4-phosphate was discovered approx. 10 years ago in plants and certain eubacteria. It is now known that this pathway is widely distributed in the bacterial kingdom including major human pathogens, such as Mycobacterium tuberculosis and Helicobacter pylori. The pathway is also essential in the malaria vector Plasmodium falciparum. During the last few years, the genes, enzymes, intermediates and mechanisms of the biosynthetic route have been elucidated by a combination of comparative genomics, enzymology, advanced NMR technology and crystallography. The results provide the basis for the development of novel anti-infective drugs.

The deoxyxylulose phosphate pathway of isoprenoid biosynthesis. Discovery and function of the ispDEFGH genes and their cognate enzymes

2003 ◽  
Vol 75 (2-3) ◽  
pp. 393-405 ◽  
Author(s):  
F. Rohdich ◽  
Stefan Hecht ◽  
Adelbert Bacher ◽  
Wolfgang Eisenreich
Keyword(s):  
Biosynthetic Pathway ◽  
Mevalonate Pathway ◽  
Isoprenoid Biosynthesis ◽  
Catalytic Action ◽  
Isopentenyl Diphosphate ◽  
Dimethylallyl Diphosphate ◽  
And Function

Isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) serve as the universal precursors for the biosynthesis of terpenes. Besides the well-known mevalonate pathway, a second biosynthetic pathway conducive to IPP and DMAPP via 1-deoxy-d-xylulose-5-phosphate and 2C-methyl-d-erythritol-4-phosphate has been discovered recently in plants and certain eubacteria. 2C-Methyl-d-erythritol-4-phosphate, the first committed intermediate of the deoxyxylulose phosphate pathway, is converted into 2C-methyl-d-erythritol 2,4-cyclodiphosphate by the catalytic action of three enzymes specified by the ispDEF genes. The cyclic diphosphate is reductively opened by the IspG protein affording 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate. This compound can be converted into IPP as well as DMAPP by the catalytic action of IspH protein. The enzymes of this pathway are potential targets for novel antibacterial, antimalarial, and herbicide agents.

scholarly journals Characterization of the Mycobacterium tuberculosis 4-Diphosphocytidyl-2-C-Methyl-d-Erythritol Synthase: Potential for Drug Development

2007 ◽  
Vol 189 (24) ◽  
pp. 8922-8927 ◽  
Author(s):  
Hyungjin Eoh ◽  
Amanda C. Brown ◽  
Lori Buetow ◽  
William N. Hunter ◽  
Tanya Parish ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Divalent Cations ◽  
Mep Pathway ◽  
Isopentenyl Diphosphate ◽  
Dimethylallyl Diphosphate ◽  
Methylerythritol Phosphate ◽  
Ph Range ◽  
Support Activity

ABSTRACT Mycobacterium tuberculosis utilizes the methylerythritol phosphate (MEP) pathway for biosynthesis of isopentenyl diphosphate and its isomer, dimethylallyl diphosphate, precursors of all isoprenoid compounds. This pathway is of interest as a source of new drug targets, as it is absent from humans and disruption of the responsible genes has shown a lethal phenotype for Escherichia coli. In the MEP pathway, 4-diphosphocytidyl-2-C-methyl-d-erythritol is formed from 2-C-methyl-d-erythritol 4-phosphate (MEP) and CTP in a reaction catalyzed by a 4-diphosphocytidyl-2-C-methyl-d-erythritol synthase (IspD). In the present work, we demonstrate that Rv3582c is essential for M. tuberculosis: Rv3582c has been cloned and expressed, and the encoded protein has been purified. The purified M. tuberculosis IspD protein was capable of catalyzing the formation of 4-diphosphocytidyl-2-C-methyl-d-erythritol in the presence of MEP and CTP. The enzyme was active over a broad pH range (pH 6.0 to 9.0), with peak activity at pH 8.0. The activity was absolutely dependent upon divalent cations, with 20 mM Mg2+ being optimal, and replacement of CTP with other nucleotide 5′-triphosphates did not support activity. Under the conditions tested, M. tuberculosis IspD had Km values of 58.5 μM for MEP and 53.2 μM for CTP. Calculated k cat and k cat/Km values were 0.72 min−1 and 12.3 mM−1 min−1 for MEP and 1.0 min−1 and 18.8 mM−1 min−1 for CTP, respectively.

scholarly journals Deciphering the sugar biosynthetic pathway and tailoring steps of nucleoside antibiotic A201A unveils a GDP-l-galactose mutase

2017 ◽  
Vol 114 (19) ◽  
pp. 4948-4953 ◽  
Author(s):  
Qinghua Zhu ◽  
Qi Chen ◽  
Yongxiang Song ◽  
Hongbo Huang ◽  
Jun Li ◽  
...  
Keyword(s):  
Drug Targeting ◽  
Drug Target ◽  
Biosynthetic Pathway ◽  
Membered Ring ◽  
Antimicrobial Drug ◽  
Biosynthetic Pathways ◽  
Human Pathogens ◽  
Substrate Specificities ◽  
Insight Into

Galactose, a monosaccharide capable of assuming two possible configurational isomers (d-/l-), can exist as a six-membered ring, galactopyranose (Galp), or as a five-membered ring, galactofuranose (Galf). UDP-galactopyranose mutase (UGM) mediates the conversion of pyranose to furanose thereby providing a precursor for d-Galf. Moreover, UGM is critical to the virulence of numerous eukaryotic and prokaryotic human pathogens and thus represents an excellent antimicrobial drug target. However, the biosynthetic mechanism and relevant enzymes that drive l-Galf production have not yet been characterized. Herein we report that efforts to decipher the sugar biosynthetic pathway and tailoring steps en route to nucleoside antibiotic A201A led to the discovery of a GDP-l-galactose mutase, MtdL. Systematic inactivation of 18 of the 33 biosynthetic genes in the A201A cluster and elucidation of 10 congeners, coupled with feeding and in vitro biochemical experiments, enabled us to: (i) decipher the unique enzyme, GDP-l-galactose mutase associated with production of two unique d-mannose-derived sugars, and (ii) assign two glycosyltransferases, four methyltransferases, and one desaturase that regiospecifically tailor the A201A scaffold and display relaxed substrate specificities. Taken together, these data provide important insight into the origin of l-Galf-containing natural product biosynthetic pathways with likely ramifications in other organisms and possible antimicrobial drug targeting strategies.

scholarly journals 1-Deoxy-d-Xylulose 5-Phosphate Reductoisomerase (IspC) from Mycobacterium tuberculosis: towards Understanding Mycobacterial Resistance to Fosmidomycin

2005 ◽  
Vol 187 (24) ◽  
pp. 8395-8402 ◽  
Author(s):  
Rakesh K. Dhiman ◽  
Merrill L. Schaeffer ◽  
Ann Marie Bailey ◽  
Charles A. Testa ◽  
Hataichanok Scherman ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Target ◽  
Pathogenic Bacteria ◽  
Biosynthetic Pathway ◽  
Inhibitory Concentration ◽  
Forward Direction ◽  
Gram Negative Bacteria ◽  
Isopentenyl Diphosphate ◽  
Intrinsic Properties ◽  
Specificity Constant

ABSTRACT 1-Deoxy-d-xylulose 5-phosphate reductoisomerase (IspC) catalyzes the first committed step in the mevalonate-independent isopentenyl diphosphate biosynthetic pathway and is a potential drug target in some pathogenic bacteria. The antibiotic fosmidomycin has been shown to inhibit IspC in a number of organisms and is active against most gram-negative bacteria but not gram positives, including Mycobacterium tuberculosis, even though the mevalonate-independent pathway is the sole isopentenyl diphosphate biosynthetic pathway in this organism. Therefore, the enzymatic properties of recombinant IspC from M. tuberculosis were characterized. Rv2870c from M. tuberculosis converts 1-deoxy-d-xylulose 5-phosphate to 2-C-methyl-d-erythritol 4-phosphate in the presence of NADPH. The enzymatic activity is dependent on the presence of Mg2+ ions and exhibits optimal activity between pH 7.5 and 7.9; the Km for 1-deoxyxylulose 5-phosphate was calculated to be 47.1 μM, and the Km for NADPH was 29.7 μM. The specificity constant of Rv2780c in the forward direction is 1.5 × 106 M−1 min−1, and the reaction is inhibited by fosmidomycin, with a 50% inhibitory concentration of 310 nM. In addition, Rv2870c complements an inactivated chromosomal copy of IspC in Salmonella enterica, and the complemented strain is sensitive to fosmidomycin. Thus, M. tuberculosis resistance to fosmidomycin is not due to intrinsic properties of Rv2870c, and the enzyme appears to be a valid drug target in this pathogen.

Escherichia coli engineered to synthesize isopentenyl diphosphate and dimethylallyl diphosphate from mevalonate: a novel system for the genetic analysis of the 2-C-methyl-d-erythritol 4-phosphate pathway for isoprenoid biosynthesis

2000 ◽  
Vol 353 (1) ◽  
pp. 59-67 ◽  
Author(s):  
Narciso CAMPOS ◽  
Manuel RODRÍGUEZ-CONCEPCIÓN ◽  
Susanna SAURET-GÜETO ◽  
Francesca GALLEGO ◽  
Luisa-María LOIS ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genetic Analysis ◽  
Mevalonate Pathway ◽  
Mep Pathway ◽  
Isopentenyl Diphosphate ◽  
Isopentenyl Diphosphate Isomerase ◽  
E Coli ◽  
Dimethylallyl Diphosphate ◽  
And Function ◽  
Synthetic Operon

Isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) constitute the basic building block of isoprenoids, a family of compounds that is extraordinarily diverse in structure and function. IPP and DMAPP can be synthesized by two independent pathways: the mevalonate pathway and the recently discovered 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. Although the MEP pathway is essential in most eubacteria, algae and plants and has enormous biotechnological interest, only some of its steps have been determined. We devised a system suitable for the genetic analysis of the MEP pathway in Escherichia coli. A synthetic operon coding for yeast 5-diphosphomevalonate decarboxylase, human 5-phosphomevalonate kinase, yeast mevalonate kinase and E. coli isopentenyl diphosphate isomerase was incorporated in the chromosome of this bacterium. The expression of this operon allowed the synthesis of IPP and DMAPP from mevalonate added exogenously and complementation of lethal mutants of the MEP pathway. We used this system to show that the ygbP, ychB and ygbB genes are essential in E. coli and that the steps catalysed by the products of these genes belong to the trunk line of the MEP pathway.

scholarly journals Isopentenyl diphosphate/dimethylallyl diphosphate-specific Nudix hydrolase from the methanogenic archaeon Methanosarcina mazei

2021 ◽  
Author(s):  
Yumi Ishibashi ◽  
Natsumi Matsushima ◽  
Tomokazu Ito ◽  
Hisashi Hemmi
Keyword(s):  
Mevalonate Pathway ◽  
Kinetic Studies ◽  
Flavin Mononucleotide ◽  
Isopentenyl Diphosphate ◽  
Nudix Hydrolase ◽  
Dimethylallyl Diphosphate ◽  
Methanosarcina Mazei ◽  
Family Protein ◽  
Nudix Hydrolases ◽  
Hydrolysis Of

Abstract Nudix hydrolases typically catalyze the hydrolysis of nucleoside diphosphate linked to moiety X and yield nucleoside monophosphate and X-phosphate, while some of them hydrolyze a terminal diphosphate group of non-nucleosidic compounds and convert it into a phosphate group. Although the number of Nudix hydrolases is usually limited in archaea comparing with those in bacteria and eukaryotes, the physiological functions of most archaeal Nudix hydrolases remain unknown. In this study, a Nudix hydrolase family protein, MM_2582, from the methanogenic archaeon Methanosarcina mazei was recombinantly expressed in Escherichia coli, purified, and characterized. This recombinant protein shows higher hydrolase activity toward isopentenyl diphosphate and short-chain prenyl diphosphates than that toward nucleosidic compounds. Kinetic studies demonstrated that the archaeal enzyme prefers isopentenyl diphosphate and dimethylallyl diphosphate, which suggests its role in the biosynthesis of prenylated flavin mononucleotide, a recently discovered coenzyme that is required, for example, in the archaea-specific modified mevalonate pathway.

scholarly journals Identification of Isopentenol Biosynthetic Genes from Bacillus subtilis by a Screening Method Based on Isoprenoid Precursor Toxicity

2007 ◽  
Vol 73 (19) ◽  
pp. 6277-6283 ◽  
Author(s):  
Sydnor T. Withers ◽  
Shayin S. Gottlieb ◽  
Bonny Lieu ◽  
Jack D. Newman ◽  
Jay D. Keasling
Keyword(s):  
Bacillus Subtilis ◽  
Biosynthetic Pathway ◽  
Screening Method ◽  
Normal Growth ◽  
Terpene Synthase ◽  
Isopentenyl Diphosphate ◽  
E Coli ◽  
Dimethylallyl Diphosphate ◽  
Novel Method ◽  
Prenyl Diphosphate

ABSTRACT We have developed a novel method to clone terpene synthase genes. This method relies on the inherent toxicity of the prenyl diphosphate precursors to terpenes, which resulted in a reduced-growth phenotype. When these precursors were consumed by a terpene synthase, normal growth was restored. We have demonstrated that this method is capable of enriching a population of engineered Escherichia coli for those clones that express the sesquiterpene-producing amorphadiene synthase. In addition, we enriched a library of genomic DNA from the isoprene-producing bacterium Bacillus subtilis strain 6051 in E. coli engineered to produce elevated levels of isopentenyl diphosphate and dimethylallyl diphosphate. The selection resulted in the discovery of two genes (yhfR and nudF) whose protein products acted directly on the prenyl diphosphate precursors and produced isopentenol. Expression of nudF in E. coli engineered with the mevalonate-based isopentenyl pyrophosphate biosynthetic pathway resulted in the production of isopentenol.

Cofactor Biosynthetic Pathways in Mycobacterium tuberculosis as Potential Drug Targets

2014 ◽  
Vol 10 (2) ◽  
pp. 97-108
Author(s):  
Shilpika Pandey ◽  
Sarthak Gaur ◽  
Neha Topno ◽  
Sidharth Chopra ◽  
Arunava Dasgupta
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Biosynthetic Pathways ◽  
Potential Drug ◽  
Potential Drug Targets

scholarly journals The Sphingolipid Biosynthetic Pathway Is a Potential Target for Chemotherapy against Chagas Disease

2011 ◽  
Vol 2011 ◽  
pp. 1-13 ◽  
Author(s):  
Carolina Macedo Koeller ◽  
Norton Heise
Keyword(s):  
Chagas Disease ◽  
Drug Targets ◽  
Biosynthetic Pathway ◽  
Protozoan Parasite ◽  
Surface Expression ◽  
Sequence Information ◽  
Insect Vector ◽  
Biosynthetic Pathways ◽  
Genome Sequence Information ◽  
Intracellular Stage

The protozoan parasite Trypanosoma cruzi is the causative agent of human Chagas disease, for which there currently is no cure. The life cycle of T. cruzi is complex, including an extracellular phase in the triatomine insect vector and an obligatory intracellular stage inside the vertebrate host. These phases depend on a variety of surface glycosylphosphatidylinositol-(GPI-) anchored glycoconjugates that are synthesized by the parasite. Therefore, the surface expression of GPI-anchored components and the biosynthetic pathways of GPI anchors are attractive targets for new therapies for Chagas disease. We identified new drug targets for chemotherapy by taking the available genome sequence information and searching for differences in the sphingolipid biosynthetic pathways (SBPs) of mammals and T. cruzi. In this paper, we discuss the major steps of the SBP in mammals, yeast and T. cruzi, focusing on the IPC synthase and ceramide remodeling of T. cruzi as potential therapeutic targets for Chagas disease.

Molecular cloning, expression profiling and functional analyses of a cDNA encoding isopentenyl diphosphate isomerase from Gossypium barbadense

2009 ◽  
Vol 29 (2) ◽  
pp. 111-119 ◽  
Author(s):  
Yechun Wang ◽  
Chengxiang Qiu ◽  
Fei Zhang ◽  
Binhui Guo ◽  
Zhiqi Miao ◽  
...  
Keyword(s):  
Mevalonate Pathway ◽  
Plant Defence ◽  
Gossypium Barbadense ◽  
Full Length ◽  
Isopentenyl Diphosphate ◽  
Reading Frame ◽  
Full Length Cdna ◽  
Dimethylallyl Diphosphate ◽  
Rapid Amplification ◽  
High Level

Gossypol, a type of plant defence sesquiterpenoid phytoalexin, is synthesized from the MEP (2C-methyl-D-erythritol 4-phosphate) and MVA (mevalonate) pathway in the isoprenoid biosynthetic system. The key step is the isomerization of IPP (isopentenyl diphosphate) to DMAPP (dimethylallyl diphosphate), which is catalysed by IPI (IPP isomerase; EC 5.3.3.2). A full-length cDNA encoding IPI (designated GbIPI) was cloned from Gossypium barbadense by RACE (rapid amplification of cDNA ends). The full-length cDNA of GbIPI was 1205 bp and contained a 906 bp ORF (open reading frame) encoding a protein of 302 amino acids, with a predicted molecular mass of 34.39 kDa and an isoelectric point of 6.07. Amino acid sequence analysis revealed that the GbIPI has a high level of similarity to other IPIs. Southern-blot analysis revealed that GbIPI belongs to a small gene family. Expression analysis indicated that GbIPI expression is highest in stems, followed by leaves, and is lowest in roots, and that the expression of GbIPI could be induced by Verticillium dahliae Kleb, MeJA (methyl jasmonate) and SA (salicylic acid). The functional colour assay indicated that GbIPI could accelerate the accumulation of β-carotene in Escherichia coli transformants. The cloning and functional analysis of GbIPI will be useful in increasing understanding of the role of IPI in isoprenoid biosynthesis at the molecular level.

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