title: The inhibition of human farnesyl pyrophosphate synthase by nitrogen-containing bisphosphonates. Elucidating the role of active site threonine 201 and tyrosine 204 residues using enzyme mutants.
abstract: 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.
authors: 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.
authors: Maria K
authors: MK
authors: Tsoumpra
authors: Structural Genomics Consortium, University of Oxford, Oxford OX3 7DQ, UK.
authors: Joao R
authors: JR
authors: Muniz
authors: Chemistry Department, University of Cincinnati, Cincinnati, OH 45221, USA.
authors: Bobby L
authors: BL
authors: Barnett
authors: Botnar Research Centre, Nuffield Department of Orthopaedics Rheumatology and Musculoskeletal Science, University of Oxford, Oxford OX3 7LD, UK.
authors: Aaron A
authors: AA
authors: Kwaasi
authors: Structural Genomics Consortium, University of Oxford, Oxford OX3 7DQ, UK.
authors: Ewa S
authors: ES
authors: Pilka
authors: Structural Genomics Consortium, University of Oxford, Oxford OX3 7DQ, UK.
authors: Kathryn L
authors: KL
authors: Kavanagh
authors: Monsanto, St Louis, MO, USA.
authors: Artem
authors: A
authors: Evdokimov
authors: Shamrock Structures, Woodridge, Illinois, USA.
authors: Richard L
authors: RL
authors: Walter
authors: Structural Genomics Consortium, University of Oxford, Oxford OX3 7DQ, UK.
authors: Frank
authors: F
authors: Von Delft
authors: Structural Genomics Consortium, University of Oxford, Oxford OX3 7DQ, UK; Department of Chemistry, University of Rochester, Rochester, NY 14627, USA.
authors: Frank H
authors: FH
authors: Ebetino
authors: 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.
authors: Udo
authors: U
authors: Oppermann
authors: Botnar Research Centre, Nuffield Department of Orthopaedics Rheumatology and Musculoskeletal Science, University of Oxford, Oxford OX3 7LD, UK; Mellanby Centre for Bone Research, University of Sheffield Medical School, Sheffield S10 2RX, UK.
authors: R Graham G
authors: RGG
authors: Russell
authors: 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. Electronic address: james.dunford@ndorms.ox.ac.uk.
authors: James E
authors: JE
authors: Dunford
mesh_terms: D050071:Bone Density Conservation Agents / Q000627:therapeutic use; D002384:Catalysis; D020134:Catalytic Domain; D003460:Crystallization; D004164:Diphosphonates / Q000737:chemistry* / Q000627:therapeutic use; D004353:Drug Evaluation, Preclinical; D051228:Geranyltranstransferase / Q000037:antagonists & inhibitors* / Q000737:chemistry; D006801:Humans; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000627:therapeutic use; D020128:Inhibitory Concentration 50; D008968:Molecular Conformation; D009154:Mutation; D009584:Nitrogen / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D013912:Threonine / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry; D000077211:Zoledronic Acid
chemical_list: D050071:Bone Density Conservation Agents; D004164:Diphosphonates; D007093:Imidazoles; D009841:Oligonucleotides; D011994:Recombinant Proteins; D013912:Threonine; D014443:Tyrosine; D000077211:Zoledronic Acid; D051228:Geranyltranstransferase; D009584:Nitrogen
keywords: Active site mutant; Bisphosphonate; Drug binding; Farnesyl pyrophosphate synthase; Inhibition mechanism; Substrate binding