Enzymes depending on the pterin molybdenum cofactor: sequence families, spectroscopic properties of molybdenum and possible cofactor-binding domains

Re-design of Rhodobacter sphaeroides Dimethyl Sulfoxide Reductase

“…The expression results suggest that the 42-residue periplasmic signal sequence found on the precursor enzyme (16, 35) may actually interfere with formation of the active R. sphaeroides enzyme in the cytoplasm of E. coli. Further optimization attempts revealed that the cultures required molybdate supplementation in the LB medium to produce active enzyme 2 and that expression of the active enzyme was much more effective at 26 than at 37°C (Fig. 2B).…”

“…BL21(DE3) cells expressing recombinant DMSO reductase fused with the His-tag appeared to produce more active enzyme than cells expressing the mature enzyme alone. 2 It has been demonstrated that the proteins involved in synthesis of the molybdenum cofactor are present at higher levels under anaerobic conditions, since many of the prokaryotic molybdenum-containing enzymes are anaerobic respiratory enzymes (36,37). Expression of recombinant E. coli DMSO reductase was shown to be more effective under anaerobic conditions in a glycerol/fumarate medium (24).…”

“…The majority of molybdoenzymes contain one or more strongly chromophoric prosthetic groups in addition to molybdenum (1,2). Although the properties of their molybdenum centers have been studied by EPR and EXAFS, 1 other techniques such as MCD, resonance Raman spectroscopy (RR), and UV-visible absorption spectroscopy have not been applicable to these enzymes because the weakly absorbing molybdenum centers are masked by the other stronger chromophores.…”

Identification by mutational analysis of four critical residues in the molybdenum cofactor domain of eukaryotic nitrate reductase

“…Alignment of the NR amino acid sequences has already allowed to pinpoint several conserved residues in plant, but also fungal and algal MoCo domain [3,5]. For some of these residues, the conservation extends also to the MoCo domain sequences of animal sulfite oxidases [30].…”

“…All molybdoenzymes, except nitrogenase, contain a molybdenum cofactor (MoCo) which consists of the metal ion bound to a pterin moiety called molybdopterin [2]. So far X-ray structural informations are not available for MoCo-containing enzymes and, although these enzymes have been the subject of many biochemical and spectroscopic studies [3], little is known of their structure/function relationships.…”