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1.  Characterization of Aquifex aeolicus 4-diphosphocytidyl-2C-methyl-d-erythritol kinase – ligand recognition in a template for antimicrobial drug discovery 
The Febs Journal  2008;275(11):2779-2794.
4-Diphosphocytidyl-2C-methyl-d-erythritol kinase (IspE) catalyses the ATP-dependent conversion of 4-diphosphocytidyl-2C-methyl-d-erythritol (CDPME) to 4-diphosphocytidyl-2C-methyl-d-erythritol 2-phosphate with the release of ADP. This reaction occurs in the non-mevalonate pathway of isoprenoid precursor biosynthesis and because it is essential in important microbial pathogens and absent from mammals it represents a potential target for anti-infective drugs. We set out to characterize the biochemical properties, determinants of molecular recognition and reactivity of IspE and report the cloning and purification of recombinant Aquifex aeolicus IspE (AaIspE), kinetic data, metal ion, temperature and pH dependence, crystallization and structure determination of the enzyme in complex with CDP, CDPME and ADP. In addition, 4-fluoro-3,5-dihydroxy-4-methylpent-1-enylphosphonic acid (compound 1) was designed to mimic a fragment of the substrate, a synthetic route to 1 was elucidated and the complex structure determined. Surprisingly, this ligand occupies the binding site for the ATP α-phosphate not the binding site for the methyl-d-erythritol moiety of CDPME. Gel filtration and analytical ultracentrifugation indicate that AaIspE is a monomer in solution. The enzyme displays the characteristic α/β galacto-homoserine-mevalonate-phosphomevalonate kinase fold, with the catalytic centre positioned in a deep cleft between the ATP- and CDPME-binding domains. Comparisons indicate a high degree of sequence conservation on the IspE active site across bacterial species, similarities in structure, specificity of substrate recognition and mechanism. The biochemical characterization, attainment of well-ordered and reproducible crystals and the models resulting from the analyses provide reagents and templates to support the structure-based design of broad-spectrum antimicrobial agents.
doi:10.1111/j.1742-4658.2008.06418.x
PMCID: PMC2655357  PMID: 18422643
enzyme–ligand complex; GHMP kinase; isoprenoid biosynthesis; molecular recognition; non-mevalonate pathway
2.  Structural and mechanistic insights into type II trypanosomatid tryparedoxin-dependent peroxidases 
Biochemical Journal  2008;414(Pt 3):375-381.
TbTDPX (Trypanosoma brucei tryparedoxin-dependent peroxidase) is a genetically validated drug target in the fight against African sleeping sickness. Despite its similarity to members of the GPX (glutathione peroxidase) family, TbTDPX2 is functional as a monomer, lacks a selenocysteine residue and relies instead on peroxidatic and resolving cysteine residues for catalysis and uses tryparedoxin rather than glutathione as electron donor. Kinetic studies indicate a saturable Ping Pong mechanism, unlike selenium-dependent GPXs, which display infinite Km and Vmax values. The structure of the reduced enzyme at 2.1 Å (0.21 nm) resolution reveals that the catalytic thiol groups are widely separated [19 Å (0.19 nm)] and thus unable to form a disulphide bond without a large conformational change in the secondary-structure architecture, as reported for certain plant GPXs. A model of the oxidized enzyme structure is presented and the implications for small-molecule inhibition are discussed.
doi:10.1042/BJ20080889
PMCID: PMC2552391  PMID: 18522537
dithiol-dependent peroxidase; drug discovery; glutathione peroxidase; Leishmania; Trypanosoma; trypanothione; GPX, glutathione peroxidase; His6, hexahistidine; Lm, Leishmania major; PEG, poly(ethylene glycol); Pt, Populus trichocarpaxdeltoides (hybrid poplar); r.m.s.d., root mean square deviation; Tb, Trypanosoma brucei; TDPX, tryparedoxin-dependent peroxidase; TryX, tryparedoxin
3.  The Synthesis of UDP-N-acetylglucosamine Is Essential for Bloodstream Form Trypanosoma brucei in Vitro and in Vivo and UDP-N-acetylglucosamine Starvation Reveals a Hierarchy in Parasite Protein Glycosylation*S⃞ 
The Journal of Biological Chemistry  2008;283(23):16147-16161.
A gene encoding Trypanosoma brucei UDP-N-acetylglucosamine pyrophosphorylase was identified, and the recombinant protein was shown to have enzymatic activity. The parasite enzyme is unusual in having a strict substrate specificity for N-acetylglucosamine 1-phosphate and in being located inside a peroxisome-like microbody, the glycosome. A bloodstream form T. brucei conditional null mutant was constructed and shown to be unable to sustain growth in vitro or in vivo under nonpermissive conditions, demonstrating that there are no alternative metabolic or nutritional routes to UDP-N-acetylglucosamine and providing a genetic validation for the enzyme as a potential drug target. The conditional null mutant was also used to investigate the effects of N-acetylglucosamine starvation in the parasite. After 48 h under nonpermissive conditions, about 24 h before cell lysis, the status of parasite glycoprotein glycosylation was assessed. Under these conditions, UDP-N-acetylglucosamine levels were less than 5% of wild type. Lectin blotting and fluorescence microscopy with tomato lectin revealed that poly-N-acetyllactosamine structures were greatly reduced in the parasite. The principal parasite surface coat component, the variant surface glycoprotein, was also analyzed. Endoglycosidase digestions and mass spectrometry showed that, under UDP-N-acetylglucosamine starvation, the variant surface glycoprotein was specifically underglycosylated at its C-terminal Asn-428 N-glycosylation site. The significance of this finding, with respect to the hierarchy of site-specific N-glycosylation in T. brucei, is discussed.
doi:10.1074/jbc.M709581200
PMCID: PMC2414269  PMID: 18381290

Results 1-3 (3)