title: Site-specific insertion of selenium into the redox-active disulfide of the flavoprotein augmenter of liver regeneration.
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 ∼530nm. 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, United States.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Schaefer-Ramadan
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, United States.
authors.forename: Colin
authors.initials: C
authors.lastname: Thorpe
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, United States. Electronic address: rozovsky@udel.edu.
authors.forename: Sharon
authors.initials: S
authors.lastname: Rozovsky
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005415:Flavins / Q000378:metabolism; D005420:Flavoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010084:Oxidation-Reduction; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017279:Selenocysteine / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D050862:Oxidoreductases Acting on Sulfur Group Donors
chemical_list: D005415:Flavins; D005420:Flavoproteins; D011506:Proteins; D011994:Recombinant Proteins; D017279:Selenocysteine; C088825:GFER protein, rat; D050862:Oxidoreductases Acting on Sulfur Group Donors
keywords: Augmenter of liver regeneration; Charge-transfer; Disulfide; Flavin; Selenium; Selenocysteine insertion sequence