The NADH-dependent reductase of a putative multicomponent tetrahydrofuran mono-oxygenase contains a covalently bound FAD.
NADH-cytochrome c oxidoreductase activity specifically expressed during growth on tetrahydrofuran was detected in cell extracts of Pseudonocardia sp. strain K1. The enzyme catalyzing this reaction was purified to apparent homogeneity by a three-step purification procedure. It was characterized as a monomer of apparent molecular mass 40 kDa. Spectroscopic studies indicated that it contains an iron-sulfur cluster and a flavin cofactor. An amount of 1 mol of flavin and 1 mol of iron was determined per mol of homogeneous protein. The N-terminal amino-acid sequence exhibited great similarity to the reductase component of various oxygenases. Cloning and sequencing of the corresponding gene designated as thmD revealed an ORF encoding a protein of 360 amino acids. An overall similarity of up to 38% was obtained to the NAD(P)H-acceptor reductase of several binuclear iron-containing mono-oxygenases. Conserved sequence motifs were identified that were similar to the chloroplast-type ferredoxin 2Fe-2S centre and to nucleotide-binding domains. Studies on the flavin cofactor showed that it could not be removed from the protein by denaturation, indicating a covalent attachment. Spectroscopic studies revealed that the flavin is at the FAD level and covalently bound to the protein via the flavin 8alpha-methyl group. Thus, the isolated reductase component is the first enzyme of this type for which a covalent attachment of the flavin has been observed.
Thiemer
B
B
Institut für Mikrobiologie, Martin-Luther-Universität Halle, Germany.
Andreesen
J R
JR
Schräder
T
T
D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001665:Binding Sites; D002474:Cell-Free System; D003001:Cloning, Molecular; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D020618:Methylococcus / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009243:NAD / Q000378:metabolism*; D009245:NADH Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009615:Nocardia / Q000201:enzymology; D016366:Open Reading Frames; D011549:Pseudomonas / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D006899:Mixed Function Oxygenases; C433514:tetrahydrofuran monooxygenase; D009245:NADH Dehydrogenase