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Single arginine mutation in two yeast isocitrate dehydrogenases: biochemical characterization and functional
Ping Song1, Huanhuan Wei1, Zhengyu Cao1
1Institute of Molecular Biology and Biotechnology, Anhui Normal University, No. 1 Beijing East Road, Wuhu, 241000, Anhui, China.
Plos One
|December 16, 2014
Summary
Mutating key arginine residues in yeast isocitrate dehydrogenase (IDH) enzymes conferred neomorphic activity, producing 2-hydroxyglutarate (2-HG). This study explores the conserved function of IDH mutations, relevant to cancer research.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Isocitrate dehydrogenase (IDH) mutations are implicated in tumorigenesis.
- A specific mutation in human IDH1 (HcIDH) alters enzyme activity, producing 2-hydroxyglutarate (2-HG).
Purpose of the Study:
- To investigate if analogous mutations in yeast IDH enzymes (Saccharomyces cerevisiae NADP+-IDH1 and Yarrowia lipolytica NADP+-IDH) yield similar functional alterations.
- To characterize the enzymatic properties of mutated yeast IDH enzymes.
Main Methods:
- Site-directed mutagenesis to create Arg-to-His mutations (ScIDH1 R148H, YlIDH R141H).
- Enzyme kinetics assays to determine kinetic parameters (Km, kcat/Km) for isocitrate oxidation and alpha-ketoglutarate (α-KG) reduction.
- Gas chromatography/time of flight-mass spectrometry (GC/TOF-MS) to confirm 2-HG production.
Main Results:
- Mutated yeast IDH enzymes (ScIDH1 R148H, YlIDH R141H) showed significantly reduced catalytic efficiency for isocitrate oxidation.
- Both mutants gained neomorphic activity, converting α-KG to 2-HG.
- The yeast mutants exhibited higher affinity and catalytic efficiency for α-KG reduction compared to the HcIDH R132H mutant.
Conclusions:
- The Arg-to-His mutation in yeast IDH enzymes mimics the neomorphic activity observed in human IDH mutations.
- This study provides a yeast model for investigating the in vivo physiological roles of IDH mutations relevant to cancer.
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