Identification of the lysine residue responsible for coenzyme A binding in the heterodimeric 2-oxoacid:ferredoxin oxidoreductase from Sulfolobus tokodaii, a thermoacidophilic archaeon, using 4-fluoro-7-nitrobenzofurazan as an affinity label

• Published in: Biochimica et biophysica acta 2009-02, Vol.1794 (2), p.335-340
• Main Authors: Luo, Jing, Fukuda, Eriko, Takase, Hirofumi, Fushinobu, Shinya, Shoun, Hirofumi, Wakagi, Takayoshi
• Format: Article
• Language: English
• Subjects: 2-Oxoacid:ferredoxin oxidoreductase; 4-Chloro-7-nitrobenzofurazan - analogs & derivatives; 4-Chloro-7-nitrobenzofurazan - chemistry; Affinity label; Affinity Labels - chemistry; Amino Acid Sequence; Archaea; Archaeal Proteins - chemistry; Archaeal Proteins - metabolism; Binding Sites; Chromatography, High Pressure Liquid; CoA; Coenzyme A - metabolism; Ketone Oxidoreductases - chemistry; Ketone Oxidoreductases - metabolism; Lysine - metabolism; Metalloendopeptidases - metabolism; Models, Molecular; Molecular Sequence Data; NBD-F; Protein Binding; Protein Multimerization; Protein Subunits - chemistry; Protein Subunits - metabolism; Sulfolobus; Sulfolobus - enzymology; Sulfolobus tokodaii
• ISSN: 1570-9639; 0006-3002; 1878-1454
• DOI: 10.1016/j.bbapap.2008.10.006

The heterodimeric 2-oxoacid:ferredoxin oxidoreductase (StOFOR) from Sulfolobus tokodaii, a thermoacidophilic archaeon, was inactivated by low concentrations of 4-fluoro-7-nitrobenzofurazan (NBD-F), with concomitant increase in fluorescence in subunit-b. The inactivation was prevented by CoA, suggesting that NBD-F covalently bound to the Lys which is responsible for CoA binding. The NBD-labeled subunit-b was isolated and digested with endoproteinase Lys-C. The resulting polypeptide mixture was separated by reverse phase HPLC and the fluorescent fraction was isolated. Amino acid sequencing of the fraction revealed that it comprised a mixture of two polypeptides containing Lys125 and Lys173, respectively. Two StOFOR mutants, K125A and K173A, were constructed, expressed and purified. K125A showed a large increase in the Km value for CoA and showed poor inactivation by NBD-F, compared with K173A and wild type StOFOR, indicating Lys125 in subunit-b is the critical residue that interacts with CoA.