2uzh: Difference between revisions

Revision as of 19:51, 21 February 2008

MYCOBACTERIUM SMEGMATIS 2C-METHYL-D-ERYTHRITOL-2,4-CYCLODIPHOSPHATE SYNTHASE (ISPF)

OverviewOverview

BACKGROUND: The prevalence of tuberculosis, the prolonged and expensive treatment that this disease requires and an increase in drug resistance indicate an urgent need for new treatments. The 1-deoxy-D-xylulose 5-phosphate pathway of isoprenoid precursor biosynthesis is an attractive chemotherapeutic target because it occurs in many pathogens, including Mycobacterium tuberculosis, and is absent from humans. To underpin future drug development it is important to assess which enzymes in this biosynthetic pathway are essential in the actual pathogens and to characterize them. RESULTS: The fifth enzyme of this pathway, encoded by ispF, is 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (IspF). A two-step recombination strategy was used to construct ispF deletion mutants in M. tuberculosis but only wild-type double crossover strains were isolated. The chromosomal copy could be deleted when a second functional copy was provided on an integrating plasmid, demonstrating that ispF is an essential gene under the conditions tested thereby confirming its potential as a drug target. We attempted structure determination of the M. tuberculosis enzyme (MtIspF), but failed to obtain crystals. We instead analyzed the orthologue M. smegmatis IspF (MsIspF), sharing 73% amino acid sequence identity, at 2.2 A resolution. The high level of sequence conservation is particularly pronounced in and around the active site. MsIspF is a trimer with a hydrophobic cavity at its center that contains density consistent with diphosphate-containing isoprenoids. The active site, created by two subunits, comprises a rigid CDP-Zn2+ binding pocket with a flexible loop to position the 2C-methyl-D-erythritol moiety of substrate. Sequence-structure comparisons indicate that the active site and interactions with ligands are highly conserved. CONCLUSION: Our study genetically validates MtIspF as a therapeutic target and provides a model system for structure-based ligand design.

About this StructureAbout this Structure

2UZH is a Single protein structure of sequence from Mycobacterium smegmatis with , , , , , and as ligands. Active as 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, with EC number 4.6.1.12 Known structural/functional Sites: , , , , , , , , , , , , , and . Full crystallographic information is available from OCA.

ReferenceReference

The structure of Mycobacteria 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase, an essential enzyme, provides a platform for drug discovery., Buetow L, Brown AC, Parish T, Hunter WN, BMC Struct Biol. 2007 Oct 23;7:68. PMID:17956607

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 ==Overview==
-ABSTRACT: BACKGROUND: The prevalence of tuberculosis, the prolonged and, expensive treatment that this disease requires and an increase in drug, resistance indicate an urgent need for new treatments. The, 1-deoxy-D-xylulose 5-phosphate pathway of isoprenoid precursor, biosynthesis is an attractive chemotherapeutic target because it occurs in, many pathogens, including Mycobacterium tuberculosis, and is absent from, humans. To underpin future drug development it is important to assess, which enzymes in this biosynthetic pathway are essential in the actual, pathogens and to characterize them. RESULTS: The fifth enzyme of this, pathway, encoded by ispF, is 2C-methyl-D-erythritol-2,4-cyclodiphosphate, synthase (IspF). A two-step recombination strategy was used to construct, ispF deletion mutants in M. tuberculosis but only wild-type double, crossover strains were isolated. The chromosomal copy could be deleted, when a second functional copy was provided on an integrating plasmid, demonstrating that ispF is an essential gene under the conditions tested, thereby confirming its potential as a drug target. We attempted structure, determination of the M. tuberculosis enzyme (MtIspF), but failed to obtain, crystals. We instead analyzed the orthologue M. smegmatis IspF (MsIspF), sharing 73% amino acid sequence identity, at 2.2 A resolution. The high, level of sequence conservation is particularly pronounced in and around, the active site. MsIspF is a trimer with a hydrophobic cavity at its, center that contains density consistent with diphosphate-containing, isoprenoids. The active site, created by two subunits, comprises a rigid, CDP-Zn2+ binding pocket with a flexible loop to position the, 2C-methyl-D-erythritol moiety of substrate. Sequence-structure comparisons, indicate that the active site and interactions with ligands are highly, conserved. CONCLUSIONS: Our study genetically validates MtIspF as a, therapeutic target and provides a model system for structure-based ligand, design.
+BACKGROUND: The prevalence of tuberculosis, the prolonged and expensive treatment that this disease requires and an increase in drug resistance indicate an urgent need for new treatments. The 1-deoxy-D-xylulose 5-phosphate pathway of isoprenoid precursor biosynthesis is an attractive chemotherapeutic target because it occurs in many pathogens, including Mycobacterium tuberculosis, and is absent from humans. To underpin future drug development it is important to assess which enzymes in this biosynthetic pathway are essential in the actual pathogens and to characterize them. RESULTS: The fifth enzyme of this pathway, encoded by ispF, is 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (IspF). A two-step recombination strategy was used to construct ispF deletion mutants in M. tuberculosis but only wild-type double crossover strains were isolated. The chromosomal copy could be deleted when a second functional copy was provided on an integrating plasmid, demonstrating that ispF is an essential gene under the conditions tested thereby confirming its potential as a drug target. We attempted structure determination of the M. tuberculosis enzyme (MtIspF), but failed to obtain crystals. We instead analyzed the orthologue M. smegmatis IspF (MsIspF), sharing 73% amino acid sequence identity, at 2.2 A resolution. The high level of sequence conservation is particularly pronounced in and around the active site. MsIspF is a trimer with a hydrophobic cavity at its center that contains density consistent with diphosphate-containing isoprenoids. The active site, created by two subunits, comprises a rigid CDP-Zn2+ binding pocket with a flexible loop to position the 2C-methyl-D-erythritol moiety of substrate. Sequence-structure comparisons indicate that the active site and interactions with ligands are highly conserved. CONCLUSION: Our study genetically validates MtIspF as a therapeutic target and provides a model system for structure-based ligand design.
 ==About this Structure==
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 ==Reference==
-The structure of Mycobacteria 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase, an essential enzyme, provides a platform for drug discovery., Buetow L, Brown AC, Parish T, Hunter WN, BMC Struct Biol. 2007 Oct 23;7(1):68. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=17956607 17956607]
+The structure of Mycobacteria 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase, an essential enzyme, provides a platform for drug discovery., Buetow L, Brown AC, Parish T, Hunter WN, BMC Struct Biol. 2007 Oct 23;7:68. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=17956607 17956607]
 [[Category: 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase]]
 [[Category: Mycobacterium smegmatis]]
 [[Category: Single protein]]
-[[Category: Brown, A.C.]]
+[[Category: Brown, A C.]]
 [[Category: Buetow, L.]]
-[[Category: Hunter, W.N.]]
+[[Category: Hunter, W N.]]
 [[Category: Parish, T.]]
 [[Category: ACT]]
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 [[Category: non-mevalonate pathway of isoprenoid biosynthesis]]
-''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sun Feb  3 10:48:43 2008''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 18:51:52 2008''