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.
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 K(m) 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.
Luo
Jing
J
Department of Biotechnology, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
Fukuda
Eriko
E
Takase
Hirofumi
H
Fushinobu
Shinya
S
Shoun
Hirofumi
H
Wakagi
Takayoshi
T
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000737:chemistry; D000345:Affinity Labels / Q000737:chemistry*; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003065:Coenzyme A / Q000378:metabolism*; D007658:Ketone Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D016944:Sulfolobus / Q000201:enzymology*
D000345:Affinity Labels; D019843:Archaeal Proteins; D021122:Protein Subunits; C032930:7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole; D007658:Ketone Oxidoreductases; C023096:2-oxoglutarate synthase; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; D009327:4-Chloro-7-nitrobenzofurazan; D008239:Lysine; D003065:Coenzyme A