The inducible trimethylamine-N-oxide reductase of Escherichia coli K12: biochemical and immunological studies.

The inducible trimethylamine-N-oxide reductase which migrates on non-denaturing polyacrylamide gels with an RF of 0.22, has been purified from the soluble fraction of wild-type E. coli K12. The molecular weight of the purified enzyme estimated by molecular-sieve chromatography is about 230,000. It is composed of two subunits of molecular weight 110,000. Antiserum specific for the enzyme has been produced. Gel filtration on Sephadex G-200 of the soluble fraction gave two peaks of trimethylamine-N-oxide reductase, one with an Mr of 230,000 and an RF of 0.22, and another with an Mr of 120,000 and an RF of 0.36. Since the anti-trimethylamine-N-oxide reductase serum recognises the two forms and shows a single subunit with an Mr of 110,000, we conclude that in E. coli there is a single inducible trimethylamine-N-oxide reductase which can exist as a dimer or a monomer. Other immunological studies with anti-trimethylamine-N-oxide reductase serum on crude extracts prepared from cells grown in the absence of inducer showed that the constitutive trimethylamine-N-oxide reductase was not recognised by the antiserum. The same analyses carried out on a tor mutant (defective in the structural gene of the inducible enzyme) confirmed without ambiguity that the constitutive enzyme is immunologically distinct from the inducible enzyme. In the same way, using the anti-trimethylamine-N-oxide reductase serum, rocket immunoelectrophoresis analyses were able to show that the inducible apoenzyme is not regulated by the fnr gene product and that molybdate does not seem necessary for the synthesis or stabilisation of this enzyme.