Role of Conserved Glycine in Zinc-dependent Medium Chain Dehydrogenase/Reductase Superfamily

• Published in: The Journal of biological chemistry Vol. 287; no. 23; pp. 19429 - 19439
• Main Authors: Tiwari, Manish Kumar, Singh, Raushan Kumar, Singh, Ranjitha, Jeya, Marimuthu, Zhao, Huimin, Lee, Jung-Kul
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.06.2012; American Society for Biochemistry and Molecular Biology
• Subjects: Alcohol Dehydrogenase - chemistry; Alcohol Dehydrogenase - genetics; Alcohol Dehydrogenase - metabolism; Amino Acid Motifs; Coordination Geometry; Density Functional Theory; Electron Spin Resonance Spectroscopy; Enzyme Catalysis; Enzyme Mechanisms; Enzymology; Fungal Proteins - chemistry; Fungal Proteins - genetics; Fungal Proteins - metabolism; Glycine - chemistry; Glycine - genetics; Glycine - metabolism; Metalloenzymes; Molecular Modeling; Mutagenesis; Mutagenesis, Site-Directed; Neurospora crassa - enzymology; Neurospora crassa - genetics; Protein Structure, Tertiary; Zinc - chemistry; Zinc - metabolism; Enzyme Mechanisms; Molecular Modeling; Mutagenesis; Enzyme Catalysis; Density Functional Theory; Coordination Geometry; Metalloenzymes
• DOI: 10.1074/jbc.M111.335752
• ISSN: 0021-9258; 1083-351X

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 (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 the MDR superfamily enzymes. The function of second-shell residues is not well understood in zinc-dependent medium chain dehydrogenase/reductases (MDRs). The strictly conserved second-shell residue Gly-77 was characterized using a wide variety of methods. Gly-77 maintains the metal binding affinity and electronic state of the catalytic zinc ion. This study provides the first insights into the role of a conserved glycine in the MDR superfamily.