Isolation, cloning, sequence analysis and localization of the operon encoding dimethyl sulfoxide/trimethylamine N-oxide reductase from Rhodobacter capsulatus.
The operon encoding the periplasmic enzymes dimethyl sulfoxide reductase (DMSOR) and trimethylamine N-oxide reductase (TMAOR) from the purple, non-sulfur, photosynthetic bacterium Rhodobacter capsulatus was isolated, cloned and sequenced, and its chromosomal location determined. It was shown by analytical and crystallographic data that DMSOR and TMAOR are identical enzymes. Degenerate primers were derived from short peptide sequences generated by automated Edman degradation and a 700 bp fragment was amplified by nested PCR, subsequently cloned and radioactively labeled to screen a prepared lambda DASH library. Positive lambda clones were subcloned into pBluescript and subsequently transformed into Escherichia coli to sequence the DMSOR/ TMAOR operon. The promoter consisted of an A + T-rich region followed by a -35 region, a putative ribosome binding site, and a leader sequence of 13 amino acid residues. The transcription terminator was a G + C-rich dyad sequence capable of forming a hairpin structure, which may act rho-independently. An optimized protein purification of the wild-type enzyme is also described, giving high yields (5 mg protein per liter of culture) and a specific activity of 30 units/mg. The molecular mass was determined by electrospray mass spectrometry to be 85,034 Da; from the deduced amino acid sequence the molecular mass of the apoenzyme was 85,033 Da.
Knäblein
J
J
Max-Planck-Institut für Biochemie, Martinsried FRG.
Mann
K
K
Ehlert
S
S
Fonstein
M
M
Huber
R
R
Schneider
F
F
D000595:Amino Acid Sequence; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D005798:Genes, Bacterial / Q000235:genetics*; D007506:Iron-Sulfur Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D009876:Operon / Q000235:genetics*; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D000587:Oxidoreductases Acting on CH-NH Group Donors; D011401:Promoter Regions, Genetic / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D016354:Rhodobacter capsulatus / Q000201:enzymology / Q000235:genetics*; D012270:Ribosomes / Q000378:metabolism; D016415:Sequence Alignment; D017421:Sequence Analysis; D017422:Sequence Analysis, DNA; D013728:Terminator Regions, Genetic / Q000235:genetics
D007506:Iron-Sulfur Proteins; D021382:Protein Sorting Signals; D010088:Oxidoreductases; C021567:methylamine dehydrogenase; D000587:Oxidoreductases Acting on CH-NH Group Donors; D009247:NADH, NADPH Oxidoreductases; C045664:dimethyl sulfoxide reductase