Role of a highly conserved YPITP motif in 2-oxoacid:ferredoxin oxidoreductase: heterologous expression of the gene from Sulfolobus sp.strain 7, and characterization of the recombinant and variant enzymes.
2-Oxoacid:ferredoxin oxidoreductase from Sulfolobus sp. strain 7, an aerobic and thermoacidophilic crenoarchaeon, catalyses the coenzyme A-dependent oxidative decarboxylation of pyruvate and 2-oxoglutarate, a cognate Zn-7Fe-ferredoxin serving as an electron acceptor. It comprises two subunits, a (632 amino acids) and b (305 amino acids). To further elucidate its structure and function, we constructed a gene expression system. The wild-type recombinant enzyme was indistinguishable from the natural one in every criterion investigated. A series of variants was constructed to elucidate the role of the YPITP-motif (residues 253-257) in subunit a, which is conserved universally in the 2-oxoacid:ferredoxin oxidoreductase (OFOR) family. Single amino-acid replacements at Y253 and P257 by other amino acids caused a drastic loss of enzyme activity. T256, the hydroxyl group of which has been proposed to be essential for binding of the 2-oxo group of the substrate in the Desulfovibrio africanus enzyme, was unexpectedly replaceable with Ala, the kcat and Km for 2-oxoglutarate being approximately 33% and approximately 51%, respectively, as compared with that of the wild-type enzyme. Replacement at other positions resulted in a significant decrease in the kcat of the reaction while the Km for 2-oxoacid was only slightly affected. Thus, the YPITP-motif is essential for the turnover of the reaction rather than the affinity toward 2-oxoacid.
Fukuda
E
E
Department of Biotechnology, The University of Tokyo, Japan.
Kino
H
H
Matsuzawa
H
H
Wakagi
T
T
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D003065:Coenzyme A / Q000378:metabolism; D017124:Conserved Sequence; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D007658:Ketone Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016944:Sulfolobus / Q000201:enzymology / Q000235:genetics*
D011994:Recombinant Proteins; D007658:Ketone Oxidoreductases; C023096:2-oxoglutarate synthase; D003065:Coenzyme A