title: Virtual mutagenesis of isocitrate dehydrogenase 1 involved in glioblastoma multiforme.
abstract: BACKGROUND
  Site A132Arg mutations potentially impair the affinity of isocitrate dehydrogenase 1 (IDH1) for its substrate isocitrate (ICT), consequently reducing the production of α-ketoglutarate and leading to tumor growth through the induction of the hypoxia-inducible factor-1 (HIF-1) pathway. However, given that the roles of other active sites in IDH1 substrate binding remain unclear, we aimed to investigate IDH1 mutation pattern and its influence on enzyme function.
  
  
  METHODS
  Fifteen IDH1 catalytic active site candidates were selected for in silico mutagenesis and protein homology modeling. Binding free energy of the IDH1/ICT complexes with single-site mutations was compared with that of the wild type. The affinity of 10 IDH1 catalytic active sites for the ICT substrate was further calculated.
  
  
  RESULTS
  The IDH1 active site included seven residues from chain A (A77Thr, A94Ser, A100Arg, A132Arg, A109Arg, A275Asp, and A279Asp) and three residues from chain B (B214Thr, B212Lys, and B252Asp) that constituted the substrate ICT-binding site. These residues were located within 0.5 nm of ICT, indicating a potential interaction with the substrate. IDH1 changes of binding free energy (ΔE) suggested that the A132Arg residue from chain A contributes three hydrogen bonds to the ICT α-carboxyl and β-carboxyl groups, while the other nine residues involved in ICT binding form only one or two hydrogen bonds. Amino acid substitutes at A132Arg, A109Arg, and B212Lys sites, had the greatest effect on enzyme affinity for its substrate.
  
  
  CONCLUSIONS
  Mutations at sites A132Arg, A109Arg, and B212Lys reduced IDH1 affinity for ICT, indicating these active sites may play a central role in substrate binding. Mutations at sites A77Thr, A94Ser, and A275Asp increased the affinity of IDH1 for ICT, which may enhance IDN1 catalytic activity. Mutant IDH1 proteins with higher catalytic activity than the wild-type IDH1 could potentially be used as a novel gene therapy for glioblastoma multiforme.
authors: Department of Neurosurgery, Affiliated Hospital of Hebei University, Baoding, Hebei 071000, China.
authors: Ming-dong
authors: MD
authors: Wang
authors: Yan-fang
authors: YF
authors: Shi
authors: Hong
authors: H
authors: Wang
authors: Jia-liang
authors: JL
authors: Wang
authors: Wen-bin
authors: WB
authors: Ma
authors: Ren-zhi
authors: RZ
authors: Wang
mesh_terms: D020134:Catalytic Domain / Q000235:genetics; D005909:Glioblastoma / Q000235:genetics*; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000235:genetics* / Q000378:metabolism*; D007523:Isocitrates / Q000378:metabolism; D016296:Mutagenesis; D009154:Mutation; D011485:Protein Binding; D013329:Structure-Activity Relationship
chemical_list: D007523:Isocitrates; C034219:isocitric acid; D007521:Isocitrate Dehydrogenase