Abstract
The NADPH-dependent homodimeric flavoenzyme thioredoxin reductase (TrxR) provides reducing equivalents to thioredoxin, a key regulator of various cellular redox processes. Crystal structures of photo-inactivated thioredoxin reductase (TrxR) from the Gram-positive bacterium Lactococcus lactis have been determined. These structures reveal novel molecular features that provide further insight into the mechanisms behind the sensitivity of this enzyme toward visible light. We propose that a pocket on the si-face of the isoalloxazine ring accommodates oxygen that reacts with photo-excited FAD generating superoxide and a flavin radical that oxidize the isoalloxazine ring C7α methyl group and a nearby tyrosine residue. This tyrosine and key residues surrounding the oxygen pocket are conserved in enzymes from related bacteria, including pathogens such as Staphylococcus aureus. Photo-sensitivity may thus be a widespread feature among bacterial TrxR with the described characteristics, which affords applications in clinical photo-therapy of drug-resistant bacteria.
Highlights
Reductase, whereas glutathione-negative bacteria such as Bacillus subtilis and Staphylococcus aureus lack this pathway[11,12]
thioredoxin reductase (TrxR) from L. lactis is inactivated by visible light in vitro, while the corresponding enzyme from E. coli is more resistant to photo-inactivation under these conditions[16]
In order to evaluate the sensitivity of native TrxR in an environment resembling the conditions in vivo, cell extracts of mid-late exponential phase cultures of L. lactis and E. coli K-12 were subjected to irradiation over a period of 12 h and TrxR activity was measured at different time points by use of a coupled assay with Trx, applying dithiobis(2-nitrobenzoic acid) (DTNB) as the final electron acceptor
Summary
Reductase, whereas glutathione-negative bacteria such as Bacillus subtilis and Staphylococcus aureus lack this pathway[11,12]. It was recently discovered that reducing equivalents for ribonucleotide reduction can be provided in a TrxR-independent manner through flavoredoxin[14] This may explain why knock-out mutants lacking either TrxR or Trx are viable even under oxidative stress conditions[15]. While investigating the biochemical properties of L. lactis TrxR (LlTrxR) we discovered that the enzyme is susceptible to photo-inactivation by visible light in an oxygen-dependent manner[16]. This inactivation coincided with a shift in the absorbance spectrum of the tightly bound FAD co-enzyme and oxidation of an isoalloxazine methyl group. We propose possible mechanisms that account for the observed oxidative damage of the enzyme
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