Purification and characterization of ferredoxin-NADP+ reductase encoded by Bacillus subtilis yumC

• Published in: Archives of microbiology Vol. 182; no. 1; pp. 80 - 89
• Main Authors: SEO, Daisuke, KAMINO, Kei, INOUE, Kazuhito, SAKURAI, Hidehiro
• Format: Journal Article
• Language: English
• Published: Heidelberg Springer 01.09.2004; Berlin Springer Nature B.V
• Subjects: Amino acids; Bacillus subtilis - enzymology; Bacillus subtilis - genetics; Bacteria; Biological and medical sciences; Enzymes; Ferredoxin-NADP Reductase - chemistry; Ferredoxin-NADP Reductase - genetics; Ferredoxin-NADP Reductase - isolation & purification; Ferredoxins - metabolism; Fundamental and applied biological sciences. Psychology; Gram-positive bacteria; Microbiology; Phylogeny; Purification; Ferredoxin-NADP+ reductase; Microbiology; Enzyme; Bacillus subtilis; Oxidase; yumC; NADPH-thioredoxin reductase; Bacillaceae; Thioredoxin; reductase; Bacillales; NADPH oxidase; Bacteria; Ferredoxin-NADP; Oxidoreductases; Ferredoxin
• ISSN: 0302-8933; 1432-072X
• DOI: 10.1007/s00203-004-0701-5

From Bacillus subtilis cell extracts, ferredoxin-NADP+ reductase (FNR) was purified to homogeneity and found to be the yumC gene product by N-terminal amino acid sequencing. YumC is a approximately 94-kDa homodimeric protein with one molecule of non-covalently bound FAD per subunit. In a diaphorase assay with 2,6-dichlorophenol-indophenol as electron acceptor, the affinity for NADPH was much higher than that for NADH, with Km values of 0.57 microM vs >200 microM. Kcat values of YumC with NADPH were 22.7 s(-1) and 35.4 s(-1) in diaphorase and in a ferredoxin-dependent NADPH-cytochrome c reduction assay, respectively. The cell extracts contained another diaphorase-active enzyme, the yfkO gene product, but its affinity for ferredoxin was very low. The deduced YumC amino acid sequence has high identity to that of the recently identified Chlorobium tepidum FNR. A genomic database search indicated that there are more than 20 genes encoding proteins that share a high level of amino acid sequence identity with YumC and which have been annotated variously as NADH oxidase, thioredoxin reductase, thioredoxin reductase-like protein, etc. These genes are found notably in gram-positive bacteria, except Clostridia, and less frequently in archaea and proteobacteria. We propose that YumC and C. tepidum FNR constitute a new group of FNR that should be added to the already established plant-type, bacteria-type, and mitochondria-type FNR groups.