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Farnesyl pyrophosphate synthase (FPPS) is the major molecular target of nitrogen-containing bisphosphonates (N-BPs), used clinically as bone resorption inhibitors. We investigated the role of threonine 201 (Thr201) and tyrosine 204 (Tyr204) residues in substrate binding, catalysis and inhibition by N-BPs, employing kinetic and crystallographic studies of mutated FPPS proteins. Mutants of Thr201 illustrated the importance of the methyl group in aiding the formation of the Isopentenyl pyrophosphate (IPP) binding site, while Tyr204 mutations revealed the unknown role of this residue in both catalysis and IPP binding. The interaction between Thr201 and the side chain nitrogen of N-BP was shown to be important for tight binding inhibition by zoledronate (ZOL) and risedronate (RIS), although RIS was also still capable of interacting with the main-chain carbonyl of Lys200. The interaction of RIS with the phenyl ring of Tyr204 proved essential for the maintenance of the isomerized enzyme-inhibitor complex. Studies with conformationally restricted analogues of RIS reaffirmed the importance of Thr201 in the formation of hydrogen bonds with N-BPs. In conclusion we have identified new features of FPPS inhibition by N-BPs and revealed unknown roles of the active site residues in catalysis and substrate binding.

Original publication

DOI

10.1016/j.bone.2015.08.020

Type

Journal article

Journal

Bone

Publication Date

12/2015

Volume

81

Pages

478 - 486

Addresses

Botnar Research Centre, Nuffield Department of Orthopaedics Rheumatology and Musculoskeletal Science, University of Oxford, Oxford OX3 7LD, UK; Structural Genomics Consortium, University of Oxford, Oxford OX3 7DQ, UK.

Keywords

Humans, Nitrogen, Diphosphonates, Imidazoles, Tyrosine, Threonine, Recombinant Proteins, Oligonucleotides, Crystallization, Drug Evaluation, Preclinical, Inhibitory Concentration 50, Catalytic Domain, Molecular Conformation, Protein Binding, Mutation, Catalysis, Hydrogen Bonding, Hydrogen-Ion Concentration, Bone Density Conservation Agents, Geranyltranstransferase