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Re-design of Rhodobacter sphaeroides dimethyl sulfoxide reductase. Enhancement of adenosine N1-oxide reductase activity.

Publication ,  Journal Article
Hilton, JC; Temple, CA; Rajagopalan, KV
Published in: J Biol Chem
March 26, 1999

The periplasmic DMSO reductase from Rhodobacter sphaeroides f. sp. denitrificans 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. Whereas the R. sphaeroides signal sequence prevents formation of active enzyme, addition of a 6x His-tag at the N terminus of the mature peptide maximizes production of active enzyme and allows for affinity purification. The recombinant protein contains 1.7-1.9 guanines and greater than 0.7 molybdenum atoms per molecule and has a DMSO reductase activity of 3.4-3.7 units/nmol molybdenum, compared with 3.7 units/nmol molybdenum for enzyme purified from R. sphaeroides. The recombinant enzyme differs from the native enzyme in its color and spectrum but is indistinguishable from the native protein after redox cycling with reduced methyl viologen and Me2SO. 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 reduced states. This substitution leads to losses of 61-99% of activity toward five substrates, but the adenosine N1-oxide reductase activity increases by over 400%.

Duke Scholars

Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

March 26, 1999

Volume

274

Issue

13

Start / End Page

8428 / 8436

Location

United States

Related Subject Headings

  • Substrate Specificity
  • Rhodobacter sphaeroides
  • Recombinant Proteins
  • Pteridines
  • Protein Engineering
  • Oxidoreductases
  • Mutagenesis, Site-Directed
  • Molybdenum Cofactors
  • Molybdenum
  • Metalloproteins
 

Citation

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Hilton, J. C., Temple, C. A., & Rajagopalan, K. V. (1999). Re-design of Rhodobacter sphaeroides dimethyl sulfoxide reductase. Enhancement of adenosine N1-oxide reductase activity. J Biol Chem, 274(13), 8428–8436. https://doi.org/10.1074/jbc.274.13.8428
Hilton, J. C., C. A. Temple, and K. V. Rajagopalan. “Re-design of Rhodobacter sphaeroides dimethyl sulfoxide reductase. Enhancement of adenosine N1-oxide reductase activity.J Biol Chem 274, no. 13 (March 26, 1999): 8428–36. https://doi.org/10.1074/jbc.274.13.8428.
Hilton JC, Temple CA, Rajagopalan KV. Re-design of Rhodobacter sphaeroides dimethyl sulfoxide reductase. Enhancement of adenosine N1-oxide reductase activity. J Biol Chem. 1999 Mar 26;274(13):8428–36.
Hilton, J. C., et al. “Re-design of Rhodobacter sphaeroides dimethyl sulfoxide reductase. Enhancement of adenosine N1-oxide reductase activity.J Biol Chem, vol. 274, no. 13, Mar. 1999, pp. 8428–36. Pubmed, doi:10.1074/jbc.274.13.8428.
Hilton JC, Temple CA, Rajagopalan KV. Re-design of Rhodobacter sphaeroides dimethyl sulfoxide reductase. Enhancement of adenosine N1-oxide reductase activity. J Biol Chem. 1999 Mar 26;274(13):8428–8436.

Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

March 26, 1999

Volume

274

Issue

13

Start / End Page

8428 / 8436

Location

United States

Related Subject Headings

  • Substrate Specificity
  • Rhodobacter sphaeroides
  • Recombinant Proteins
  • Pteridines
  • Protein Engineering
  • Oxidoreductases
  • Mutagenesis, Site-Directed
  • Molybdenum Cofactors
  • Molybdenum
  • Metalloproteins