Cloning, sequence analysis, and functional expression of the acetyl coenzyme A synthetase gene from Methanothrix soehngenii in Escherichia coli.
In the acetoclastic methanogen Methanothrix soehngenii, acetate is activated to acetyl coenzyme A by acetyl coenzyme A synthetase (Acs). The acs gene, coding for the single Acs subunit, was isolated from a genomic library of M. soehngenii DNA in Escherichia coli by using antiserum raised against the purified Acs. After introduction in E. coli, the acs gene was expressed, resulting in the production of an immunoreactive protein of 68 kDa, which is approximately 5 kDa smaller than the known size of purified Acs. In spite of this difference in size, the Acs enzymes are produced in similar quantities in E. coli and M. soehngenii and show comparable specific activities. Upstream from the acs gene, consensus archaeal expression signals were identified. Immediately downstream from the acs gene there was a putative transcriptional stop signal. The amino acid sequence deduced from the nucleotide sequence of the acs gene showed homology with those of functionally related proteins, i.e., proteins involved in the binding of coenzyme A, ATP, or both.
Eggen
R I
RI
Department of Microbiology, Wageningen Agricultural University, The Netherlands.
Geerling
A C
AC
Boshoven
A B
AB
de Vos
W M
WM
D000084:Acetate Kinase / Q000378:metabolism; D000106:Acetate-CoA Ligase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000502:physiology; D005798:Genes, Bacterial / Q000235:genetics*; D015151:Immunoblotting; D017019:Methanosarcinaceae / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D016415:Sequence Alignment
D000084:Acetate Kinase; D000106:Acetate-CoA Ligase