PMID- 17956607
OWN - NLM
STAT- MEDLINE
DCOM- 20080108
LR  - 20211020
IS  - 1472-6807 (Electronic)
IS  - 1472-6807 (Linking)
VI  - 7
DP  - 2007 Oct 23
TI  - The structure of Mycobacteria 2C-methyl-D-erythritol-2,4-cyclodiphosphate 
      synthase, an essential enzyme, provides a platform for drug discovery.
PG  - 68
AB  - 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.
FAU - Buetow, Lori
AU  - Buetow L
AD  - Division of Biological Chemistry and Molecular Microbiology, College of Life 
      Sciences, University of Dundee, Dundee DD1 5EH, UK. l.buetow@dundee.ac.uk
FAU - Brown, Amanda C
AU  - Brown AC
FAU - Parish, Tanya
AU  - Parish T
FAU - Hunter, William N
AU  - Hunter WN
LA  - eng
GR  - Wellcome Trust/United Kingdom
PT  - Comparative Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20071023
PL  - England
TA  - BMC Struct Biol
JT  - BMC structural biology
JID - 101088689
RN  - 0 (Bacterial Proteins)
RN  - 0 (DNA, Bacterial)
RN  - 0 (Ligands)
RN  - 0 (Protein Subunits)
RN  - 059QF0KO0R (Water)
RN  - I38ZP9992A (Magnesium)
RN  - J41CSQ7QDS (Zinc)
SB  - IM
MH  - Amino Acid Sequence
MH  - Bacterial Proteins/*chemistry/genetics/isolation & purification/metabolism
MH  - Binding Sites
MH  - Cloning, Molecular
MH  - Conserved Sequence
MH  - Crystallography, X-Ray
MH  - DNA, Bacterial/genetics/isolation & purification
MH  - *Drug Design
MH  - Electroporation
MH  - Gene Deletion
MH  - Gene Expression
MH  - Genes, Bacterial
MH  - Hydrogen Bonding
MH  - Hydrophobic and Hydrophilic Interactions
MH  - Ligands
MH  - Magnesium/metabolism
MH  - Models, Biological
MH  - Models, Chemical
MH  - Models, Molecular
MH  - Molecular Sequence Data
MH  - Molecular Structure
MH  - Mycobacterium smegmatis/chemistry/genetics/metabolism
MH  - Mycobacterium tuberculosis/*enzymology/genetics/metabolism
MH  - Plasmids
MH  - Pliability
MH  - Protein Binding
MH  - Protein Structure, Secondary
MH  - Protein Subunits/chemistry/metabolism
MH  - Sequence Analysis, DNA
MH  - Sequence Homology, Amino Acid
MH  - Substrate Specificity
MH  - Water/chemistry
MH  - Zinc/metabolism
PMC - PMC2151065
EDAT- 2007/10/25 09:00
MHDA- 2008/01/09 09:00
PMCR- 2007/10/23
CRDT- 2007/10/25 09:00
PHST- 2007/05/08 00:00 [received]
PHST- 2007/10/23 00:00 [accepted]
PHST- 2007/10/25 09:00 [pubmed]
PHST- 2008/01/09 09:00 [medline]
PHST- 2007/10/25 09:00 [entrez]
PHST- 2007/10/23 00:00 [pmc-release]
AID - 1472-6807-7-68 [pii]
AID - 10.1186/1472-6807-7-68 [doi]
PST - epublish
SO  - BMC Struct Biol. 2007 Oct 23;7:68. doi: 10.1186/1472-6807-7-68.