Hetero-oligomeric glutamate dehydrogenase from Thermus thermophilus.
An extremely thermophilic bacterium, Thermus thermophilus, possesses two glutamate dehydrogenase (GDH) genes, gdhA and gdhB, putatively forming an operon on the genome. To elucidate the functions of these genes, the gene products were purified and characterized. GdhA showed no GDH activity, while GdhB showed GDH activity for reductive amination 1.3-fold higher than that for oxidative deamination. When GdhA was co-expressed with His-tag-fused GdhB, GdhA was co-purified with His-tagged GdhB. Compared with GdhB alone, co-purified GdhA-GdhB had decreased reductive amination activity and increased oxidative deamination activity, resulting in a 3.1-fold preference for oxidative deamination over reductive amination. Addition of hydrophobic amino acids affected the GDH activity of the co-purified GdhA-GdhB hetero-complex. Among the amino acids, leucine had the largest effect on activity: addition of 1&emsp14;mM leucine elevated the GDH activity of the co-purified GdhA-GdhB by 974 and 245 % for reductive amination and oxidative deamination, respectively, while GdhB alone did not show such marked activation by leucine. Kinetic analysis revealed that the elevation of GDH activity by leucine is attributable to the enhanced turnover number of GDH. In this hetero-oligomeric GDH system, GdhA and GdhB act as regulatory and catalytic subunits, respectively, and GdhA can modulate the activity of GdhB through hetero-complex formation, depending on the availability of hydrophobic amino acids. This study provides the first finding, to our knowledge, of a hetero-oligomeric GDH that can be regulated allosterically.
Tomita
Takeo
T
Biotechnology Research Center, Department of Biotechnology, The University of Tokyo, Tokyo 113-8657, Japan.
Miyazaki
Takashi
T
Biotechnology Research Center, Department of Biotechnology, The University of Tokyo, Tokyo 113-8657, Japan.
Miyazaki
Junichi
J
Biotechnology Research Center, Department of Biotechnology, The University of Tokyo, Tokyo 113-8657, Japan.
Kuzuyama
Tomohisa
T
Biotechnology Research Center, Department of Biotechnology, The University of Tokyo, Tokyo 113-8657, Japan.
Nishiyama
Makoto
M
RIKEN SPring-8 Center, Hyogo 679-5148, Japan.|Biotechnology Research Center, Department of Biotechnology, The University of Tokyo, Tokyo 113-8657, Japan.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019281:Dimerization; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005969:Glutamate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D017386:Sequence Homology, Amino Acid; D016963:Thermus thermophilus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D005969:Glutamate Dehydrogenase