title: Lactococcus lactis thioredoxin reductase is sensitive to light inactivation.
abstract: Thioredoxin, involved in numerous redox pathways, is maintained in the dithiol state by the nicotinamide adenine dinucleotide phosphate-dependent flavoprotein thioredoxin reductase (TrxR). Here, TrxR from Lactococcus lactis is compared with the well-characterized TrxR from Escherichia coli. The two enzymes belong to the same class of low-molecular weight thioredoxin reductases and display similar kcat values (∼25 s(-1)) with their cognate thioredoxin. Remarkably, however, the L. lactis enzyme is inactivated by visible light and furthermore reduces molecular oxygen 10 times faster than E. coli TrxR. The rate of light inactivation under standardized conditions (λmax=460 nm and 4 °C) was reduced at lowered oxygen concentrations and in the presence of iodide. Inactivation was accompanied by a distinct spectral shift of the flavin adenine dinucleotide (FAD) that remained firmly bound. High-resolution mass spectrometric analysis of heat-extracted FAD from light-damaged TrxR revealed a mass increment of 13.979 Da, relative to that of unmodified FAD, corresponding to the addition of one oxygen atom and the loss of two hydrogen atoms. Tandem mass spectrometry confined the increase in mass of the isoalloxazine ring, and the extracted modified cofactor reacted with dinitrophenyl hydrazine, indicating the presence of an aldehyde. We hypothesize that a methyl group of FAD is oxidized to a formyl group. The significance of this not previously reported oxidation and the exceptionally high rate of oxygen reduction are discussed in relation to other flavin modifications and the possible occurrence of enzymes with similar properties.
authors: Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark , Building 224, Søltofts Plads, DK-2800 Kongens Lyngby, Denmark.
authors: Olof
authors: O
authors: Björnberg
authors: Thibault
authors: T
authors: Viennet
authors: Nicklas
authors: N
authors: Skjoldager
authors: Aida
authors: A
authors: Ćurović
authors: Kristian Fog
authors: KF
authors: Nielsen
authors: Birte
authors: B
authors: Svensson
authors: Per
authors: P
authors: Hägglund
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability / Q000235:genetics / Q000528:radiation effects; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013294:Lactococcus lactis / Q000201:enzymology* / Q000235:genetics; D008027:Light; D013058:Mass Spectrometry; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005182:Flavin-Adenine Dinucleotide; D013879:Thioredoxins; D013880:Thioredoxin-Disulfide Reductase; D010100:Oxygen