is a unicellular parasitic protozoan with a complex life cycle involving both insects and mammals. The parasites are transmitted into the bloodstream of mammals by the bite of the tsetse fly, causing African sleeping sickness in humans. No vaccines are available, the drugs in current use have severe side-effects and drug resistance is also rapidly increasing. The enzymes of the mevalonate (MVA) pathway of isoprenoid precursor biosynthesis represent potential drug targets in trypanosomes since active sterol biosynthesis has been shown to be essential for growth and survival in T. cruzi
). For example, sterol-synthesis inhibitors such as terbinafine (an inhibitor of squalene epoxidase, downstream of the MVA pathway) have been shown to retard growth and lead to parasite death (Buckner et al.
) and inhibitors of T. cruzi
HMG-CoA reductase (a central enzyme of the MVA pathway) have been shown to potentiate the anti-proliferative effects of terbinafine (Urbina et al.
). This would suggest that inhibition of the MVA pathway offers hope for future drug development against trypanosomatid parasites. Furthermore, several MVA-pathway enzymes, including MDD, have been shown to be essential in yeast (Oulmouden & Karst, 1990
; Tsay & Robinson, 1991
; Bergès et al.
) which, like trypanosomatids, is dependent on the MVA pathway for isoprenoid precursor biosynthesis.
Isoprenoids, which represent one of the largest families of compounds in nature, contribute to many important and diverse biochemical roles: for example, quinones participate in electron-transport processes, sterols such as cholesterol or ergosterol are components of membrane structures, dolichol is required for glycoprotein synthesis and isoprenyl moieties are involved in membrane anchorage and also signalling processes (Sacchettini & Poulter, 1997
). The essential building block for all isoprenoids is the five-carbon isopentenyl diphosphate (IPP). Two pathways lead to IPP biosynthesis, those being either the mevalonate (MVA) pathway or the non-mevalonate (also called the 1-deoxy-d
-xylulose-5-phosphate or DOXP) pathway (Eisenreich et al.
; Rohmer, 1999
). Trypanosomes, as is the case for all eukaryotes, archaebacteria and a few eubacteria, use the MVA pathway for IPP biosynthesis. This biosynthetic pathway provides new potential drug targets for exploitation in the search for improved therapies against parasitic diseases caused by trypanosomatid infection and it is important to study the enzyme components further.
Mevalonate diphosphate decarboxylase (MDD) catalyses the last step in the pathway, using mevalonate diphosphate in an ATP-dependent decarboxylation step to make IPP. MDD is a GHMP kinase superfamily member, so-called because the family originally included galacto-, homoserine, mevalonate and phosphomevalonate kinases. There is interest in this family from a structural perspective since members all share the characteristic α/β-fold of the GHMP kinase family and similar sequence homology (in the range 10–20% identity), yet they catalyze different reactions and utilize very different substrates. Only three ternary complex structures are available from this superfamily, including homoserine kinase (Krishna et al.
), galactokinase (Hartley et al.
) and 4-diphosphocytidyl-2C
-erythritol kinase (Miallau et al.
), and thus there is limited detailed structural information as to how individual members achieve substrate specificity. Only a single structure of MDD, that of the Saccharomyces cerevisiae
enzyme, is available to date and this is in the apo form (Bonanno et al.
). Detailed kinetic and mutagenesis studies of this enzyme have suggested a possible mechanism (Dhe-Paganon et al.
) and which residues might contribute to substrate binding (Krepkiy & Miziorko, 2004
), but ligand–complex structures remain essential to achieve a detailed understanding of the catalytic mechanism and ligand specificity of MDD necessary to support a programme of structure-based inhibitor design.
Here, we describe the cloning of the putative mevalonate diphosphate decarboxylase gene from T. brucei, the construction of a highly efficient protein-expression system and purification of the recombinant enzyme (of approximate molecular weight 42.4 kDa, 385 residues), along with crystallization and preliminary diffraction experiments.