Escherichia coli ferredoxin NADP+ reductase: activation of E. coli anaerobic ribonucleotide reduction, cloning of the gene (fpr), and overexpression of the protein

Abstract

A specific ribonucleoside triphosphate reductase is induced in anaerobic Escherichia cofi. This enzyme, as isolated, lacks activity in the test tube and can be activated anaerobically with S-adenosylmethionine, NADPH, and two previously uncharacterized E. coli fractions. The gene for one of these, previously named dAl, was cloned and sequenced. We found an open reading frame coding for a polypeptide of 248 amino acid residues, with a molecular weight of 27,645 and with an N-terminal segment identical to that determined by direct Edman degradation. In a Kohara library, the gene hybridized between positions 3590 and 3600 on the physical map ofE. coli. The deduced amino acid sequence shows a high extent of sequence identity with that of various ferredoxin (flavodoxin) NADP+ reductases. We therefore conclude that dAl is identical with E. coli ferredoxin (flavodoxin) NADP+ reductase. Biochemical evidence from a bacterial strain, now constructed and overproducing dAl activity up to 100-fold, strongly supports this conclusion. The sequence of the gene shows an apparent overlap with the reported sequence of mvrA, previously suggested to be involved in the protection against superoxide (M. Morimyo, J. Bacteriol. 170:2136-2142, 1988). We suggest that a frameshift introduced during isolation or sequencing of mvrA caused an error in the determination of its sequence.During anaerobic growth, Escherichia coli induces an enzyme that catalyzes the reduction of CTP to dCTP (7-9, 11). The gene for this enzyme was recently cloned (28) and found to be distinct from nrdA and nrdB, which code for the aerobic ribonucleoside diphosphate reductase (29). In the active state, both enzymes contain organic radicals as part of their protein structures and iron as a cofactor (19,29). In the aerobic enzyme, the radical is located on and the enzyme contains a diferric center with the iron ions linked by a ,u-oxo-bridge (29). A glycyl residue was suggested to harbor the organic radical of the anaerobic reductase, whose iron center consists of an iron-sulfur cluster (19,28).A difference between the two enzymes is that the aerobic reductase, as isolated, shows full activity and contains the radical in stable form, whereas the isolated anaerobic enzyme is inactive and lacks the radical. Instead, the enzyme activity and radical of the latter enzyme appear only after anaerobic incubation of the isolated protein with NADPH, S-adenosylmethionine, and two uncharacterized E. coli fractions, provisionally called dAl and RT (8

Probing the NADPH-binding site of Escherichia coli flavodoxin oxidoreductase

“…Alignment of the amino acid sequence of FLDR with other pyridine dinucleotide-dependent oxidoreductases shows that an Flavodoxin reductase NADPH recognition Figure 6 Amino acid alignment of FLDR (from E. coli, Swiss-Prot code P28861, [15]) with other members of the FNR family of enzymes Alignments were performed as described in the Experimental section, comparing other protein sequences with the region of FLDR from L139 to E190 (encompassing all three of the mutated arginines). Flavodoxin reductase arginine residues 144, 174 and 184 are highlighted in bold, as are positively charged amino acids (arginines and lysines) conserved in the aligned enzymes.…”

“…[14] was used for overexpression of the fldr gene. Plasmid pCL21 (a derivative of pET 16b, Novagen, Madison, WI, U.S.A.) was used for production of FLDR and was constructed by PCR amplification of the structural gene from plasmid pEE1010 (a gift from Dr Elizabeth Hagga/ rd-Ljungquist, Department of Chemistry, Karolinska Institute, Stockholm, Sweden), as previously reported [7,15]. The wild-type fldr gene was expressed under the control of an isopropyl β--thiogalactoside-inducible T7 promoter in pCL21.…”

Cysteines Involved in Radical Generation and Catalysis of Class III Anaerobic Ribonucleotide Reductase

“…22 was followed with some minor changes. The C1-a strain containing the plasmid pEE1010 was grown to stationary phase for expression of flavodoxin reductase, and the cells were then lysed with lysozyme treatment and freeze-thawing.…”

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

“…The first includes the plant-type FNRs that are monomeric enzymes also found in eukaryotic algae as well as in cyanobacteria, and have been intensively reviewed (Arakaki et al 1997). The second group is sometimes called the bacteria type and is composed of monomeric FNRs from some bacteria such as Escherichia coli (Bianchi et al 1993) and Azotobacter vinelandii (Isas et al 1995). There are significant similarities between these two groups of FNRs in 3-D structure (Prasad et al 1998) as well as in amino acid sequence (Arakaki et al 1997, Ceccarelli et al 2004).…”