Re-design of Rhodobacter sphaeroides dimethyl sulfoxide reductase - Enhancement of adenosine N-1-oxide reductase activity

The periplasmic DMSO reductase from Rhodobacter sphaeroides f. sp. denitrif icans has been expressed in Escherichia coli BL21(DE3) cells in its mature form and with the R. sphaeroides or E. coli N-terminal signal sequence. Whe reas the R. sphaeroides signal sequence prevents formation of active enzyme , addition of a 6x Histag at the N terminus of the mature peptide maximizes production of active enzyme and allows for affinity purification. The reco mbinant protein contains 1.7-1.9 guanines and greater than 0.7 molybdenum a toms per molecule and has a DMSO reductase activity of 3.4-3.7 units/nmol m olybdenum, compared with 3.7 units/nmol molybdenum for enzyme purified from R. sphaeroides. The recombinant enzyme differs from the native enzyme in i ts color and spectrum but is indistinguishable from the native protein afte r redox cycling with reduced methyl viologen and Me,SO. Substitution of Cys for the molybdenum-ligating Ser-147 produced a protein with DMSO reductase activity of 1.4-1.5 units/nmol molybdenum. The mutant protein differs from wild type in its color and absorption spectrum in both the oxidized and re duced states. This substitution leads to losses of 61-99% of activity towar d five substrates, but the adenosine N-1-oxide reductase activity increases by over 400%.