Role of conserved glycine in zinc-dependent medium chain dehydrogenase/reductase superfamily

Manish Kumar Tiwari, Raushan Kumar Singh, Ranjitha Singh, Marimuthu Jeya, Huimin Zhao, Jung Kul Lee

Research output: Contribution to journalArticlepeer-review

Abstract

The medium-chain dehydrogenase/reductase (MDR) superfamily consists of a large group of enzymes with a broad range of activities. Members of this superfamily are currently the subject of intensive investigation, but many aspects, including the zinc dependence of MDR superfamily proteins, have not yet have been adequately investigated. Using a density functional theorybased screening strategy, we have identified a strictly conserved glycine residue (Gly) in the zinc-dependent MDR superfamily. To elucidate the role of this conserved Gly in MDR, we carried out a comprehensive structural, functional, and computational analysis of fourMDRenzymes through a series of studies including site-directed mutagenesis, isothermal titration calorimetry, electron paramagnetic resonance (EPR), quantum mechanics, and molecular mechanics analysis. Gly substitution by other amino acids posed a significant threat to the metal binding affinity and activity of MDR superfamily enzymes. Mutagenesis at the conserved Gly resulted in alterations in the coordination of the catalytic zinc ion, with concomitant changes in metal-ligand bond length, bond angle, and the affinity (Kd) toward the zinc ion. The Gly mutants also showed different spectroscopic properties in EPR compared with those of the wild type, indicating that the binding geometries of the zinc to the zinc binding ligands were changed by the mutation. The present results demonstrate that the conserved Gly in the GHE motif plays a role in maintaining the metal binding affinity and the electronic state of the catalytic zinc ion during catalysis of theMDRsuperfamily enzymes.

Original languageEnglish (US)
Pages (from-to)19429-19439
Number of pages11
JournalJournal of Biological Chemistry
Volume287
Issue number23
DOIs
StatePublished - Jun 1 2012

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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