Isolation, cloning, sequence analysis and X-ray structure of dimethyl sulfoxide/trimethylamine N-oxide reductase from Rhodobacter capsulatus.
The periplasmic enzyme dimethyl sulfoxide/trimethylamine N-oxide reductase (DMSOR/TMAOR) from the photosynthetic purple bacterium Rhodobacter capsulatus functions as the terminal electron acceptor in its respiratory chain. The enzyme catalyzes the reduction of highly oxidized substrates like dimethyl sulfoxide (DMSO) or trimethylamine N-oxide (TMAO). At a molybdenum redox centre, two single electrons are transferred from cytochrome c556 to the substrate, e.g. DMSO, generating dimethyl sulfide (DMS) and water. The operon encoding this enzyme 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 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. By an optimized protein purification high yields (5 mg protein/l culture) with a specific activity of 30 U/mg were obtained. The molecular mass was experimentally determined by electrospray mass spectroscopy (MS) to be 85034 Da and from the deduced amino acid sequence of the apoenzyme to be 85033 Da. The enzyme was crystallized in space group P4(1)2(1)2 with unit cell dimensions of a = b = 80.7 A and c = 229.2 A diffracting beyond 1.8 A. The three-dimensional structure was solved by a combination of multiple isomorphous replacement (MIR) and molecular replacement techniques. The atomic model was refined to an R-factor of 0.169 for 57394 independent reflections. The spherical protein consists of four domains with a funnel-like cavity that leads to the freely accessible metal-ion redox center. The sole bis(molybdopterin guanine dinucleotide)molybdenum cofactor (1541 Da) of the single chain protein has the molybdenum ion bound to the cis-dithiolene group of only one molybdopterin guanine dinucleotide (MGD) molecule. In addition, two oxo ligands and the oxygen of a serine side chain are bound to the molybdenum ion.
Knäblein
J
J
Max-Planck-Institut für Biochemie, Martinsried, Germany.
Dobbek
H
H
Ehlert
S
S
Schneider
F
F
D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004269:DNA, Bacterial / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007506:Iron-Sulfur Proteins; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D009876:Operon; D010088:Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D000587:Oxidoreductases Acting on CH-NH Group Donors; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D016354:Rhodobacter capsulatus / Q000201:enzymology*
D004269:DNA, Bacterial; D007506:Iron-Sulfur Proteins; D010088:Oxidoreductases; C021567:methylamine dehydrogenase; D000587:Oxidoreductases Acting on CH-NH Group Donors; D009247:NADH, NADPH Oxidoreductases; C045664:dimethyl sulfoxide reductase