title: Role of conserved glycine in zinc-dependent medium chain dehydrogenase/reductase superfamily.
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 theory-based 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 four MDR enzymes 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 (K(d)) 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 the MDR superfamily enzymes.
authors.affiliation: Department of Chemical Engineering, Konkuk University, Seoul 143-701, Korea.
authors.forename: Manish Kumar
authors.initials: MK
authors.lastname: Tiwari
authors.forename: Raushan Kumar
authors.initials: RK
authors.lastname: Singh
authors.forename: Ranjitha
authors.initials: R
authors.lastname: Singh
authors.forename: Marimuthu
authors.initials: M
authors.lastname: Jeya
authors.forename: Huimin
authors.initials: H
authors.lastname: Zhao
authors.forename: Jung-Kul
authors.initials: JK
authors.lastname: Lee
mesh_terms: D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D004578:Electron Spin Resonance Spectroscopy; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005998:Glycine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics; D017434:Protein Structure, Tertiary; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D000426:Alcohol Dehydrogenase; D015032:Zinc; D005998:Glycine