title: Identification of catalytic residues of a very large NAD-glutamate dehydrogenase from Janthinobacterium lividum by site-directed mutagenesis.
abstract: We previously found a very large NAD-dependent glutamate dehydrogenase with approximately 170 kDa subunit from Janthinobacterium lividum (Jl-GDH) and predicted that GDH reaction occurred in the central domain of the subunit. To gain further insights into the role of the central domain, several single point mutations were introduced. The enzyme activity was completely lost in all single mutants of R784A, K810A, K820A, D885A, and S1142A. Because, in sequence alignment analysis, these residues corresponded to the residues responsible for glutamate binding in well-known small GDH with approximately 50 kDa subunit, very large GDH and well-known small GDH may share the same catalytic mechanism. In addition, we demonstrated that C1141, one of the three cysteine residues in the central domain, was responsible for the inhibition of enzyme activity by HgCl2, and HgCl2 functioned as an activating compound for a C1141T mutant. At low concentrations, moreover, HgCl2 was found to function as an activating compound for a wild-type Jl-GDH. This suggests that the mechanism for the activation is entirely different from that for the inhibition.
authors.affiliation: a Division of Environmental Symbiosis Studies, Graduate School of Integrated Arts and Sciences , The University of Tokushima , Tokushima , Japan.
authors.forename: Ryushi
authors.initials: R
authors.lastname: Kawakami
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Sakuraba
authors.forename: Toshihisa
authors.initials: T
authors.lastname: Ohshima
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D055162:Biocatalysis; D042521:Burkholderiaceae / Q000201:enzymology* / Q000235:genetics; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D057075:Enzyme Assays; D015870:Gene Expression; D005969:Glutamate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008627:Mercuric Chloride / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D021122:Protein Subunits; D018698:Glutamic Acid; D008627:Mercuric Chloride; D005969:Glutamate Dehydrogenase; C061421:glutamate dehydrogenase (NAD(P)+); D003545:Cysteine
keywords: Janthinobacterium lividum; activation by Hg2+; glutamate-binding residues; inhibition by Hg2+; very large glutamate dehydrogenase