Genes and enzymes of the acetyl cycle of arginine biosynthesis in the extreme thermophilic bacterium Thermus thermophilus HB27.
An arginine biosynthetic gene cluster, argC-argJ, of the extreme thermophilic bacterium Thermus thermophilus HB27 was isolated by heterologous complementation of an Escherichia coli acetylornithinase mutant. The recombinant plasmid (pTHM1) conferred ornithine acetyltransferase activity to the E. coli host, implying that T. thermophilus uses the energetically more economic pathway for the deacetylation of acetylornithine. pTHM1 was, however, unable to complement an E. coli argA mutant and no acetylglutamate synthase activity could be detected in E. coli argA cells containing pTHM1. The T. thermophilus argJ-encoded enzyme is thus monofunctional and is unable to use acetyl-CoA to acetylate glutamate (contrary to the Bacillus stearothermophilus homologue). Alignment of several ornithine acetyltransferase amino acid sequences showed no obvious pattern that could account for this difference; however, the monofunctional enzymes proved to have shorter N-termini. Sequence analysis of the pTHM1 3.2 kb insert revealed the presence of the argC gene (encoding N-acetylglutamate-5-semialdehyde dehydrogenase) upstream of the argJ gene. Alignment of several N-acetylglutamate-5-semialdehyde dehydrogenase amino acid sequences allowed identification of two strongly conserved putative motifs for cofactor binding: a putative FAD-binding site and a motif reminiscent of the NADPH-binding fingerprint. The relationship between the amino acid content of both enzymes and thermostability is discussed and an effect of the GC content bias is indicated. Transcription of both the argC and argJ genes appeared to be vector-dependent. The argJ-encoded enzyme activity was twofold repressed by arginine in the native host and was inhibited by ornithine. Both upstream of the argC gene and downstream of the argJ gene an ORF with unknown function was found, indicating that the organization of the arginine biosynthetic genes in T. thermophilus is new.
Baetens
Margot
M
Vlaams Interuniversitair Instituut voor Biotechnologie, Emile Grysonlaan 1, B-1070 Brussel, Belgium.|Microbiologie, Vrije Universiteit Brussel, Emile Grysonlaan 1, B-1070 Brussel, Belgium.
Legrain
Christianne
C
Research Institute CERIA-COOVI, Emile Grysonlaan 1, B-1070 Brussel, Belgium.
Boyen
Anne
A
Vlaams Interuniversitair Instituut voor Biotechnologie, Emile Grysonlaan 1, B-1070 Brussel, Belgium.|Microbiologie, Vrije Universiteit Brussel, Emile Grysonlaan 1, B-1070 Brussel, Belgium.
Glansdorff
Nicolas
N
Research Institute CERIA-COOVI, Emile Grysonlaan 1, B-1070 Brussel, Belgium.|Vlaams Interuniversitair Instituut voor Biotechnologie, Emile Grysonlaan 1, B-1070 Brussel, Belgium.|Microbiologie, Vrije Universiteit Brussel, Emile Grysonlaan 1, B-1070 Brussel, Belgium.
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000123:Acetyltransferases / Q000235:genetics / Q000378:metabolism; D000445:Aldehyde Oxidoreductases; D000581:Amidohydrolases / Q000235:genetics; D000595:Amino Acid Sequence; D051046:Amino-Acid N-Acetyltransferase; D001120:Arginine / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001482:Base Composition; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D005971:Glutamates / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family; D016254:Mutagenesis, Insertional; D009249:NADP / Q000378:metabolism; D016366:Open Reading Frames; D009952:Ornithine / Q000378:metabolism; D010957:Plasmids; D011995:Recombination, Genetic; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D016963:Thermus thermophilus / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic; D014170:Transformation, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D005971:Glutamates; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D000105:Acetyl Coenzyme A; D001120:Arginine; D009952:Ornithine; D000445:Aldehyde Oxidoreductases; C089846:argC protein, Bacteria; D000123:Acetyltransferases; D051046:Amino-Acid N-Acetyltransferase; C020646:glutamate acetyltransferase; D000581:Amidohydrolases; C018732:N-acetylornithine deacetylase; C016195:N-acetylglutamic acid