Substrate recognition by 2-oxoacid:ferredoxin oxidoreductase from Sulfolobus sp. strain 7.
2-Oxoacid:ferredoxin oxidoreductase (OFOR) catalyzes the coenzyme A-dependent oxidative decarboxylation of 2-oxoacids, at an analogous metabolic position to 2-oxoacid dehydrogenase multienzyme complex. The enzyme from Sulfolobus sp. strain 7, a thermoacidophilic crenarchaeon, is a heterodimer comprising two subunits, a (632 amino acids) and b (305 amino acids). In contrast to other OFORs, the Sulfolobus enzyme shows a broad specificity for 2-oxoacids such as pyruvate and 2-oxoglutarate. Based on careful multiple alignment of this enzyme family and on the reported three-dimensional structure of the homodimeric pyruvate:ferredoxin oxidoreductase (POR) from Desulfovibrio africanus, we selected five amino acids, T256, R344 and T353 of subunit-a, and K49 and L123 of subunit-b, as candidate 2-oxoacid recognizing residues. To identify the residues determining the 2-oxoacid specificity of the enzyme family, we performed point mutations of these five amino acids, and characterized the resulting mutants. Analyses of the mutants revealed that R344 of subunit-a of the enzyme was essential for the activity, and that K49R and L123N of subunit-b drastically affected the enzyme specificity for pyruvate and 2-oxoglutarate, respectively. Replacement of the five residues resulted in significant changes in both K(m) and V(max), indicating that these amino acids are clearly involved in substrate recognition and catalysis.
Fukuda
Eriko
E
Department of Biotechnology, The University of Tokyo, 1-1-1, Yayoi, Bunkyo, Tokyo 113-8657, Japan.
Wakagi
Takayoshi
T
D000595:Amino Acid Sequence; D001665:Binding Sites; D002087:Butyrates / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D007658:Ketone Oxidoreductases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids; D019289:Pyruvic Acid / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D016944:Sulfolobus / Q000201:enzymology* / Q000235:genetics
D002087:Butyrates; D007656:Ketoglutaric Acids; C005087:alpha-ketobutyric acid; D019289:Pyruvic Acid; D007658:Ketone Oxidoreductases; C023096:2-oxoglutarate synthase