Site-specific insertion of selenium into the redox-active disulfide of the flavoprotein augmenter of liver regeneration

Stephanie Ramadan, Colin Thorpe, Sharon Rozovsky

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Augmenter of liver regeneration (sfALR) is a small disulfide-bridged homodimeric flavoprotein with sulfhydryl oxidase activity. Here, we investigate the catalytic and spectroscopic consequences of selectively replacing C145 by a selenocysteine to complement earlier studies in which random substitution of ∼90% of the 6 cysteine residues per sfALR monomer was achieved growing Escherichia coli on selenite. A selenocysteine insertion sequence (SECIS) element was installed within the gene for human sfALR. SecALR2 showed a spectrum comparable to that of wild-type sfALR. The catalytic efficiency of SecALR2 towards dithiothreitol was 6.8-fold lower than a corresponding construct in which position 145 was returned to a cysteine residue while retaining the additional mutations introduced with the SECIS element. This all-cysteine control enzyme formed a mixed disulfide between C142 and β-mercaptoethanol releasing C145 to form a thiolate-flavin charge transfer absorbance band at ∼530 nm. In contrast, SecALR2 showed a prominent long-wavelength absorbance at 585 nm consistent with the expectation that a selenolate would be a better charge-transfer donor to the isoalloxazine ring. These data show the robustness of the ALR protein fold towards the multiple mutations required to insert the SECIS element and provide the first example of a selenolate to flavin charge-transfer complex.

Original languageEnglish
Pages (from-to)60-65
Number of pages6
JournalArchives of Biochemistry and Biophysics
Volume548
DOIs
Publication statusPublished - 15 Apr 2014
Externally publishedYes

Fingerprint

Selenocysteine
Flavoproteins
Liver Regeneration
Selenium
Disulfides
Liver
Oxidation-Reduction
DNA Transposable Elements
Cysteine
Charge transfer
Selenious Acid
Mutation
Mercaptoethanol
Dithiothreitol
Escherichia coli
Substitution reactions
Genes
Monomers
Wavelength
Enzymes

Keywords

  • Augmenter of liver regeneration
  • Charge-transfer
  • Disulfide
  • Flavin
  • Selenium
  • Selenocysteine insertion sequence

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Site-specific insertion of selenium into the redox-active disulfide of the flavoprotein augmenter of liver regeneration. / Ramadan, Stephanie; Thorpe, Colin; Rozovsky, Sharon.

In: Archives of Biochemistry and Biophysics, Vol. 548, 15.04.2014, p. 60-65.

Research output: Contribution to journalArticle

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