An NAD(+)-dependent glutamate dehydrogenase cloned from the ruminal ciliate protozoan, Entodinium caudatum.
An NAD(+)-dependent glutamate dehydrogenase (GDH; EC 1.4.1.24) was cloned from the ruminal ciliate protozoan, Entodinium caudatum. The gene had high sequence similarity to GDH genes from the Bacteroides (class)--a class of bacteria which is highly represented in the rumen. When expressed in Escherichia coli the enzyme had a high affinity for ammonia and alpha-ketoglutarate (apparent K(m) of 2.33 and 0.71 mM, respectively) and a low affinity for glutamate (apparent K(m) of 98 mM). GDH activity and GDH mRNA concentration were increased by incubating washed E. caudatum cells with ammonia and antibiotics. These results suggest that the GDH is an anabolic enzyme catalysing the assimilation of ammonia by E. caudatum in the rumen and that the gene was probably acquired by lateral gene transfer from a ruminal bacterium.
Newbold
C James
CJ
Rowett Research Institute, Greenburn Road, Aberdeen AB21 9SB, Scotland, United Kingdom.
McEwan
Neil R
NR
Calza
Roger E
RE
Chareyron
Emilie N
EN
Duval
Stéphane M
SM
Eschenlauer
Sylvain C P
SC
McIntosh
Freda M
FM
Nelson
Nancy
N
Travis
Anthony J
AJ
Wallace
R John
RJ
D000595:Amino Acid Sequence; D000641:Ammonia / Q000378:metabolism; D000818:Animals; D001439:Bacteroides / Q000235:genetics; D001483:Base Sequence; D016798:Ciliophora / Q000201:enzymology*; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D022761:Gene Transfer, Horizontal; D017125:Genes, Protozoan; D005969:Glutamate Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D012333:RNA, Messenger / Q000032:analysis; D016053:RNA, Protozoan / Q000032:analysis; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D016054:DNA, Protozoan; D007656:Ketoglutaric Acids; D012333:RNA, Messenger; D016053:RNA, Protozoan; D011994:Recombinant Proteins; D018698:Glutamic Acid; D000641:Ammonia; D005969:Glutamate Dehydrogenase