Characterization of the flavin reductase gene (fre) of Escherichia coli and construction of a plasmid for overproduction of the enzyme

Abstract

The enzyme NAD(P)H:flavin oxidoreductase (flavin reductase) catalyzes the reduction of soluble flavins by reduced pyridine nucleotides. In Escherichia coli it is part of a multienzyme system that reduces the Fe(III) center of ribonucleotide reductase to Fe(II) and thereby sets the stage for the generation by dioxygen of a free tyrosyl radical required for enzyme activity. Similar enzymes are known in other organisms and may more generally be involved in iron metabolism. We have now isolated the gene for the E. coli flavin reductase from a Agtll library. After DNA sequencing we found an open reading frame coding for a polypeptide of 233 amino acids, with a molecular weight of 26,212 and with an N-terminal segment identical to that determined by direct Edman degradation. The coding sequence is preceded by a weak ribosome binding site centered 8 nucleotides from the start codon and by a promoterlike sequence centered at a distance of 83 nucleotides. In a Kohara library the gene hybridized to position 3680 on the physical map ofE. coil. A bacterial strain that overproduced the enzyme approximately 100-fold was constructed. The translated amino acid sequence contained a potential pyridine nucleotide-binding site and showed 25% identity with the C-terminal part of one subunit (protein C) of methane monooxygenase from methanotropic bacteria that reduces the iron center of a second subunit (protein A) of the oxygenase by pyridine nucleotides.

Reduction of Cob(III)alamin to Cob(II)alamin in Salmonella enterica Serovar Typhimurium LT2

“…3). N-terminal sequence analysis of the first 15 amino acids of the purified protein yielded the sequence TTLSCKVTSVEAITD, a perfect match to the E. coli Fre (encoded by the fre gene) (11,29). This finding was surprising because this enzyme was not expected to catalyze the reduction of cob(II)alamin to cob(I)alamin given the extremely low redox potential of the cob(II)alamin/cob(I)alamin couple (E 0 Ј ϭ Ϫ0.61V [1,19]).…”

Structural prototypes for an extended family of flavoprotein reductases: Comparison of phthalate dioxygenase reductase with ferredoxin reductase and ferredoxin

“…[PI refers to a pterin-binding moiety. Other proteins identified as members of the family (Neidle et al, 1991;Andrews et al, 1992) are xylene monooxygenase reductase (Suzuki et al, 1991), toluate dioxygenase reductase (XylZ) (Neidle et al, 1991), vanillate demethylase reductase (Brunel & Davison, 1988), benzoate dioxygenase reductase (Neidle et al, 1991), naphthalene dioxygenase ferredoxin reductase (Simon et al, 1993), nitric oxide synthase (Bredt et al, 1991), bacterial hemoglobin-like protein (HMP) (Andrews et al, 1992), LuxC (Swartzman et al, 1990), ferrisiderophore reductase C (Fsrc) (Spyrou et al, 1991;Andrews et al, 1992), phenol hydroxylase component 5 (Nordlund et al, 1990), and cytochrome bLX5 (Segal et al, 1992 160 170 180 190 200 210 220 230 240 250 260 27 0 280 arating equivalent main-chain atoms (Kabsch, 1976). using only atomic separations of less than 3.0 A (see Methods).…”

The haemoglobin‐like protein (HMP) of Escherichia coli has ferrisiderophore reductase activity and its C‐terminal domain shares homology with ferredoxin NADP+ reductases

“…Alternatively, the haem moiety of HMP (FsrB) may be involved in oxygen-dependent regulation of FsrB activity. The ferric reductase activity of FsrB is probably mediated by flavinonucleotides, as is thought to be the case for FsrC-dependent ferric reduction [15]. HMP (FsrB) also possesses dihydropteridine reductase (DHPR) activity [S], although the HMP (FsrB) studied here is distinct from the previously purified DHPR of E. coli [36].…”