The objective of this work is to investigate the structures of two nucleotide
binding proteins: mevalonate kinase (MVK) and FtsZ.
MVK is the key enzyme involved in terpenoid biosynthesis. In this study, we
solved the crystal structures of the M. jannaschii MVK apoprotein, as well as the protein
in complex with ligands. Its fold was analyzed and firmly established within the GHMP
kinase family, in which homoserine kinase (HSK), phosphomevalonate kinase and
galactokinase also belong. Structural analysis in combination with enzyme kinetics
studies revealed the mechanism of this enzyme upon substrate binding, catalysis and
inhibition. In particular, the phosphate-binding loop was found to be critically involved
in the binding of nucleotides and terpenoids, via the interaction with a di-phosphate
moiety from the ligand. An enzymatic reaction mechanism was constructed based on our
structural data and it is consistent with kinetics studies from the literature. In this
mechanism, the invariant residue Asp 155 functions as a general base that increases the
nucleophilicity of the phosphoryl acceptor. Finally, a virtual screening study has been performed to explore the ligand binding potential of MVK. Compounds predicted to
bind MVK were tested and analyzed.
FtsZ is a prokaryotic homologue of tubulin that forms the apparatus for bacterial
cell division. The structure of a crystal filament of the M. tuberculosis FtsZ complexed
with GDP was described in this study. It shows an anti-parallel, left-handed double
helical architecture. Compared with the straight crystal filament revealed earlier by other
groups, the catalytic T7 loop in our structure is found to be outside the nucleotide
binding site, indicating the GTPase is inactive. Furthermore, the buried surface area in
our crystal filament is less, probably suggesting the helical FtsZ filament is less stable.
We therefore proposed that the hydrolysis of GTP and the releasing of the ?-phosphate
group will trigger the rearrangement of the FtsZ fibler, characterized by the exclusion of
the T7 loop, which might lead to a less stable helical filament and would be the first step
for the disassembly of FtsZ polymer.
- Sacchettini, James Professor