RESUMO
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 (â¼25 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.
Assuntos
Proteínas de Bactérias/química , Lactococcus lactis/enzimologia , Luz , Tiorredoxina Dissulfeto Redutase/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Estabilidade Enzimática/genética , Estabilidade Enzimática/efeitos da radiação , Escherichia coli/enzimologia , Escherichia coli/genética , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/genética , Flavina-Adenina Dinucleotídeo/metabolismo , Lactococcus lactis/genética , Espectrometria de Massas , Oxigênio/química , Oxigênio/metabolismo , Tiorredoxina Dissulfeto Redutase/genética , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/química , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMO
Understanding of how P-type ATPases work would greatly benefit from the elucidation of more high-resolution structures. The nucleotide-binding domain of Mg-ATPase was selected for structural studies because Mg-ATPase is closely related to eukaryotic Ca-ATPase and Na,K-ATPase while the nucleotide-binding domain itself has diverged substantially. Two fragments of Mg-ATPase were cloned in Escherichia coli and purified. The entire cytoplasmic loop (residues 367-673), consisting of the phosphorylation and nucleotide-binding domains, expressed well and was purified in large quantities. The smaller 19.5 kDa nucleotide-binding domain (residues 383-545) expressed less well but formed crystals that diffracted to a resolution of 1.53 A which will be used for molecular replacement.
