A preliminary crystallographic analysis of the putative mevalonate diphosphate decarboxylase fromTrypanosoma brucei

Abstract

Mevalonate diphosphate decarboxylase catalyses the last and least well characterized step in the mevalonate pathway for the biosynthesis of isopentenyl pyrophosphate, an isoprenoid precursor. A gene predicted to encode the enzyme from Trypanosoma brucei has been cloned, a highly efficient expression system established and a purification protocol determined. The enzyme gives monoclinic crystals in space group P2 1 , with unit-cell parameters a = 51.5, b = 168.7, c = 54.9 Å , = 118.8 . A Matthews coefficient V M of 2.5 Å 3 Da À1 corresponds to two monomers, each approximately 42 kDa (385 residues), in the asymmetric unit with 50% solvent content. These crystals are well ordered and data to high resolution have been recorded using synchrotron radiation.

Crystal Structures of Trypanosoma brucei and Staphylococcus aureus Mevalonate Diphosphate Decarboxylase Inform on the Determinants of Specificity and Reactivity

“…15 This recombinant enzyme is susceptible to degradation in a matter of days even when stored at low temperatures, as revealed by SDS-PAGE and mass spectrometry analysis (data not shown). It was necessary to use fresh protein for our studies.…”

“…We note that crystals were grown at pH 6.0. 15 SaMDD also assembles as a symmetric dimer, with approximately 70% of atoms at the interface of the two molecules being non-polar. Approximately 15% of the total surface area (2130 Å 2 ) is buried upon dimer formation.…”

“…14 Inspection of genomic data available for a number of Trypanosoma and related Leishmania species reveals putative homologues for the other MVA pathway enzymes, although there are only limited data on these enzymes. 13,15 The enzyme acetoacetyl-CoA transferase initiates the MVA pathway with the condensation of two acetyl coenzyme A molecules to acetoacetyl-CoA. HMG-CoA synthase subsequently catalyzes the addition of another acetyl-CoA to form HMG-CoA, which is then reduced to the central intermediate, MVA, by the NADPH-dependent HMG-CoA reductase.…”