Isolation, cloning, sequence analysis and X-ray structure of dimethyl sulfoxide/trimethylamine N-oxide reductase from Rhodobacter capsulatus.

摘要: The periplasmic enzyme dimethyl sulfoxide/trimethylamine N-oxide reductase (DMSOR/TMAOR) from the photosynthetic purple bacterium Rhodobacter capsulatus functions as terminal electron acceptor in its respiratory chain. catalyzes reduction of highly oxidized substrates like sulfoxide (DMSO) or trimethylamine (TMAO). At a molybdenum redox centre, two single electrons are transferred cytochrome c556 to substrate, e.g. DMSO, generating sulfide (DMS) and water. operon encoding this was isolated, cloned sequenced, chromosomal location determined. It shown by analytical crystallographic data that DMSOR TMAOR identical enzymes. Degenerate primers were derived short peptide sequences 700 bp fragment amplified nested PCR, subsequently radioactively labeled screen prepared lambda DASH library. Positive clones subcloned into pBluescript transformed Escherichia coli sequence DMSOR/TMAOR operon. By an optimized protein purification high yields (5 mg protein/l culture) with specific activity 30 U/mg obtained. molecular mass experimentally determined electrospray spectroscopy (MS) be 85034 Da deduced amino acid apoenzyme 85033 Da. crystallized space group P4(1)2(1)2 unit cell dimensions = b 80.7 A c 229.2 diffracting beyond 1.8 A. three-dimensional structure solved combination multiple isomorphous replacement (MIR) techniques. atomic model refined R-factor 0.169 for 57394 independent reflections. spherical consists four domains funnel-like cavity leads freely accessible metal-ion center. sole bis(molybdopterin guanine dinucleotide)molybdenum cofactor (1541 Da) chain has ion bound cis-dithiolene only one molybdopterin dinucleotide (MGD) molecule. In addition, oxo ligands oxygen serine side ion.