Association of the mcrD gene product with methyl coenzyme M reductase in Methanococcus vannielii.
The mcrD gene, subcloned from the methyl coenzyme M reductase (MR) encoding mcrBDCGA operon in Methanococcus vannielii, has been expressed at a high level in Escherichia coli. Rabbit antibodies, raised against the product of this gene (rgpmcrD, recombinant gene product of mcrD) purified from E. coli, have been used to quantitate gpmcrD in M. vannielii and to follow its fate during MR purification. The molar ratio of gpmcrD to MR was found to be approx. 1:15 in cells of M. vannielii taken from batch cultures at all stages of growth. Sedimentation of lysates of M. vannielii cells through sucrose gradients and analyses of the fractions obtained by Western blotting and immunoprecipitation have demonstrated the presence of a macromolecular complex containing both gpmcrD and MR. Addition of mcrD antibodies or removal of gpmcrD from lysates of M. vannielii cells by immunoprecipitation decreased the rates of methanogenesis in vitro by approx. 20%. Addition of purified rgpmcrD to these lysates did not stimulate methanogenesis.
Stroup
D
D
Department of Microbiology, Ohio State University, Columbus 43210.
Reeve
J N
JN
D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005798:Genes, Bacterial; D017017:Methanococcus / Q000201:enzymology / Q000235:genetics*; D009876:Operon; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism
D010088:Oxidoreductases; C021572:methyl coenzyme M reductase