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Crystal structures of IspF from Plasmodium falciparum and Burkholderia cenocepacia: comparisons inform antimicrobial drug target assessment.

O'Rourke PE, Kalinowska-Tłuścik J, Fyfe PK, Dawson A, Hunter WN - BMC Struct. Biol. (2014)

Bottom Line: High-resolution crystal structures of IspF from two important human pathogens have been obtained and compared to orthologues.Ligand binding appears to order a part of the active site involved in substrate recognition.The high degree of structural conservation in and around the IspF active site suggests that any structural model might be suitable to support a program of structure-based drug discovery.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK. w.n.hunter@dundee.ac.uk.

ABSTRACT

Background: 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (IspF) catalyzes the conversion of 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate to 2C-methyl-D-erythritol-2,4-cyclodiphosphate and cytidine monophosphate in production of isoprenoid-precursors via the methylerythritol phosphate biosynthetic pathway. IspF is found in the protozoan Plasmodium falciparum, a parasite that causes cerebral malaria, as well as in many Gram-negative bacteria such as Burkholderia cenocepacia. IspF represents a potential target for development of broad-spectrum antimicrobial drugs since it is proven or inferred as essential in these pathogens and absent from mammals. Structural studies of IspF from these two important yet distinct pathogens, and comparisons with orthologues have been carried out to generate reagents, to support and inform a structure-based approach to early stage drug discovery.

Results: Efficient recombinant protein production and crystallization protocols were developed, and high-resolution crystal structures of IspF from P. falciparum (Emphasis/Emphasis>IspF) and B. cenocepacia (BcIspF) in complex with cytidine nucleotides determined. Comparisons with orthologues, indicate a high degree of order and conservation in parts of the active site where Zn2+ is bound and where recognition of the cytidine moiety of substrate occurs. However, conformational flexibility is noted in that area of the active site responsible for binding the methylerythritol component of substrate. Unexpectedly, one structure of BcIspF revealed two molecules of cytidine monophosphate in the active site, and another identified citrate coordinating to the catalytic Zn2+. In both cases interactions with ligands appear to help order a flexible loop at one side of the active site. Difficulties were encountered when attempting to derive complex structures with other ligands.

Conclusions: High-resolution crystal structures of IspF from two important human pathogens have been obtained and compared to orthologues. The studies reveal new data on ligand binding, with citrate coordinating to the active site Zn2+ and when present in high concentrations cytidine monophosphate displays two binding modes in the active site. Ligand binding appears to order a part of the active site involved in substrate recognition. The high degree of structural conservation in and around the IspF active site suggests that any structural model might be suitable to support a program of structure-based drug discovery.

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Superposition of the active site in BcIspF and PfIspF. Residues that bind CMP or CDP are displayed as sticks. Sequence numbers for BcIspF are followed by those for PfIspF, and residues contributed from the adjacent subunit are marked with’. BcIspF and PfIspF are colored cyan and green respectively. Key interactions are detailed in the text.
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Figure 4: Superposition of the active site in BcIspF and PfIspF. Residues that bind CMP or CDP are displayed as sticks. Sequence numbers for BcIspF are followed by those for PfIspF, and residues contributed from the adjacent subunit are marked with’. BcIspF and PfIspF are colored cyan and green respectively. Key interactions are detailed in the text.

Mentions: The enzyme active site is located in a cleft at the interface of two subunits (Figure 2a) and here Zn2+ is coordinated by the side chains of two histidines and an aspartate [19]. The coordinating residues are Asp10, His12 and His44 in BcIspF, Asp71, His73 and His123 in PfIspF (Figures 3 and 4). These residues are strictly conserved in IspF. In the E. coli enzyme (EcIspF) Glu135, helps coordinate either a Mg2+ or Mn2+ together with the bridging phosphates of the substrate or CDP [19,20]. Although the residue is conserved as Glu137/216 (Figure 4) we do not observe any cation binding here. In 81% of IspF sequences a glutamate is observed at this position, and this is conservatively replaced by aspartate in a further 18%.


Crystal structures of IspF from Plasmodium falciparum and Burkholderia cenocepacia: comparisons inform antimicrobial drug target assessment.

O'Rourke PE, Kalinowska-Tłuścik J, Fyfe PK, Dawson A, Hunter WN - BMC Struct. Biol. (2014)

Superposition of the active site in BcIspF and PfIspF. Residues that bind CMP or CDP are displayed as sticks. Sequence numbers for BcIspF are followed by those for PfIspF, and residues contributed from the adjacent subunit are marked with’. BcIspF and PfIspF are colored cyan and green respectively. Key interactions are detailed in the text.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3927217&req=5

Figure 4: Superposition of the active site in BcIspF and PfIspF. Residues that bind CMP or CDP are displayed as sticks. Sequence numbers for BcIspF are followed by those for PfIspF, and residues contributed from the adjacent subunit are marked with’. BcIspF and PfIspF are colored cyan and green respectively. Key interactions are detailed in the text.
Mentions: The enzyme active site is located in a cleft at the interface of two subunits (Figure 2a) and here Zn2+ is coordinated by the side chains of two histidines and an aspartate [19]. The coordinating residues are Asp10, His12 and His44 in BcIspF, Asp71, His73 and His123 in PfIspF (Figures 3 and 4). These residues are strictly conserved in IspF. In the E. coli enzyme (EcIspF) Glu135, helps coordinate either a Mg2+ or Mn2+ together with the bridging phosphates of the substrate or CDP [19,20]. Although the residue is conserved as Glu137/216 (Figure 4) we do not observe any cation binding here. In 81% of IspF sequences a glutamate is observed at this position, and this is conservatively replaced by aspartate in a further 18%.

Bottom Line: High-resolution crystal structures of IspF from two important human pathogens have been obtained and compared to orthologues.Ligand binding appears to order a part of the active site involved in substrate recognition.The high degree of structural conservation in and around the IspF active site suggests that any structural model might be suitable to support a program of structure-based drug discovery.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK. w.n.hunter@dundee.ac.uk.

ABSTRACT

Background: 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (IspF) catalyzes the conversion of 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate to 2C-methyl-D-erythritol-2,4-cyclodiphosphate and cytidine monophosphate in production of isoprenoid-precursors via the methylerythritol phosphate biosynthetic pathway. IspF is found in the protozoan Plasmodium falciparum, a parasite that causes cerebral malaria, as well as in many Gram-negative bacteria such as Burkholderia cenocepacia. IspF represents a potential target for development of broad-spectrum antimicrobial drugs since it is proven or inferred as essential in these pathogens and absent from mammals. Structural studies of IspF from these two important yet distinct pathogens, and comparisons with orthologues have been carried out to generate reagents, to support and inform a structure-based approach to early stage drug discovery.

Results: Efficient recombinant protein production and crystallization protocols were developed, and high-resolution crystal structures of IspF from P. falciparum (Emphasis/Emphasis>IspF) and B. cenocepacia (BcIspF) in complex with cytidine nucleotides determined. Comparisons with orthologues, indicate a high degree of order and conservation in parts of the active site where Zn2+ is bound and where recognition of the cytidine moiety of substrate occurs. However, conformational flexibility is noted in that area of the active site responsible for binding the methylerythritol component of substrate. Unexpectedly, one structure of BcIspF revealed two molecules of cytidine monophosphate in the active site, and another identified citrate coordinating to the catalytic Zn2+. In both cases interactions with ligands appear to help order a flexible loop at one side of the active site. Difficulties were encountered when attempting to derive complex structures with other ligands.

Conclusions: High-resolution crystal structures of IspF from two important human pathogens have been obtained and compared to orthologues. The studies reveal new data on ligand binding, with citrate coordinating to the active site Zn2+ and when present in high concentrations cytidine monophosphate displays two binding modes in the active site. Ligand binding appears to order a part of the active site involved in substrate recognition. The high degree of structural conservation in and around the IspF active site suggests that any structural model might be suitable to support a program of structure-based drug discovery.

Show MeSH
Related in: MedlinePlus