Mutagenesis of the proposed iron-sulfur cluster binding ligands in Escherichia coli biotin synthase

• Published in: FEBS letters Vol. 466; no. 2; pp. 372 - 376
• Main Authors: Hewitson, Kirsty S, Baldwin, Jack E, Shaw, Nicholas M, Roach, Peter L
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 28.01.2000
• Subjects: aRR AE, anaerobic ribonucleotide reductase activating enzyme; Base Sequence; Biotin synthase; DNA Primers; DTT, dithiothreitol; Escherichia coli - enzymology; Fe-S, iron-sulfur cluster; Iron - metabolism; Iron-sulfur cluster; Iron-Sulfur Proteins - metabolism; Ligands; Mutagenesis of iron-sulfur cluster ligand; Mutagenesis, Site-Directed; pfl AE, pyruvate formate lyase activating enzyme; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; SAM, S-adenosylmethionine; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; Spectrophotometry, Ultraviolet; Sulfurtransferases - genetics; Sulfurtransferases - isolation & purification; Sulfurtransferases - metabolism; SAM, S-adenosylmethionine; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; Biotin synthase; Fe-S, iron-sulfur cluster; Iron-sulfur cluster; Mutagenesis of iron-sulfur cluster ligand; pfl AE, pyruvate formate lyase activating enzyme; aRR AE, anaerobic ribonucleotide reductase activating enzyme; DTT, dithiothreitol
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(00)01101-7

Biotin synthase (BioB) is a member of a family of enzymes that includes anaerobic ribonucleotide reductase and pyruvate formate lyase activating enzyme. These enzymes all use S-adenosylmethionine during turnover and contain three highly conserved cysteine residues that may act as ligands to an iron-sulfur cluster required for activity. Three mutant enzymes of BioB have been made, each with one cysteine residue (C53, 57, 60) mutated to alanine. All three mutant enzymes were inactive, but they still exhibited the characteristic UV-visible spectrum of a [2Fe-2S] 2+ cluster similar to that of the wild-type enzyme.