Structural insights into domain movement and cofactor specificity of glutamate dehydrogenase from Corynebacterium glutamicum.
Glutamate dehydrogenase (GDH) is an enzyme involved in the synthesis of amino acids by converting glutamate to α-ketoglutarate, and vice versa. To investigate the molecular mechanism of GDH, we determined a crystal structure of the Corynebacterium glutamicum-derived GDH (CgGDH) in complex with its NADP cofactor and α-ketoglutarate substrate. CgGDH functions as a hexamer, and each CgGDH monomer comprises 2 separate domains; a Rossmann fold cofactor-binding domain and a substrate-binding domain. The structural comparison between the apo- and cofactor/substrate-binding forms revealed that the CgGDH enzyme undergoes a domain movement during catalysis. In the apo-form, CgGDH exists as an open state, and upon binding of the substrate and cofactor the protein undergoes a conformation change to a closed state. Our structural study also revealed that CgGDH has cofactor specificity for NADP, but not NAD, and this was confirmed by GDH activity measurements. Residues involved in the stabilization of the NADP cofactor and the α-ketoglutarate substrate were identified, and their roles in substrate/cofactor binding were confirmed by site-directed mutagenesis experiments.
School of Life Sciences, KNU Creative BioResearch Group, Kyungpook National University, Daehak-ro 80, Buk-ku, Daegu 702-701, Republic of Korea.
Hyeoncheol Francis
HF
Son
Paik Kwang Industrial Company Ltd., 57 Oehang-4 Gil, Gunsan, Jellabuk-do 573-879, Republic of Korea.
Il-Kwon
IK
Kim
School of Life Sciences, KNU Creative BioResearch Group, Kyungpook National University, Daehak-ro 80, Buk-ku, Daegu 702-701, Republic of Korea. Electronic address: kkim@knu.ac.kr.
Kyung-Jin
KJ
Kim
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D048230:Corynebacterium glutamicum / Q000201:enzymology* / Q000235:genetics; D018360:Crystallography, X-Ray; D005969:Glutamate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D001426:Bacterial Proteins; D007656:Ketoglutaric Acids; D009249:NADP; D005969:Glutamate Dehydrogenase
Cofactor specificity; Corynebacterium glutamicum; Glutamate dehydrogenase; Structure