1 Department of Systems Biology, Technical University of Denmark2 Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark3 Department of Mechanical Engineering, Technical University of Denmark4 Materials and Surface Engineering, Department of Mechanical Engineering, Technical University of Denmark5 Enzyme and Protein Chemistry, Department of Biotechnology and Biomedicine, Technical University of Denmark
Thioredoxins (Trx) are small ubiquitous disulfide oxidoreductases involved in thiol redox control in all kingdoms of life and provides reducing equivalents to various enzymes (e.g. ribonucleotide reductase, methionine sulfoxide reductase and peroxiredoxins). Oxidized Trx is recycled by NADPH-dependent thioredoxin reductase (NTR) in order to complete its catalytic cycle. Glutathione-dependent glutaredoxin complements Trx in many organisms. This thesis focuses on disulfide reduction pathways in Lactococcus lactis, an important industrial microorganism used traditionally for cheese and buttermilk production. L. lactis lacks glutathione and glutaredoxin, but it a contains Trx system consisting of an NTR (LlTrxB), a classical Trx (LlTrxA) containing the conserved WCGPC active site motif, a Trx-like protein (LlTrxD) containing a WCGDC active site motif and a redoxin (LlNrdH) providing electrons to class Ib ribonucleotide reductase (NrdEF). Physiological functions of LlTrxA and LlTrxD were studied using ΔtrxA, ΔtrxD and ΔtrxAΔtrxD mutant strains of L. lactis ssp. cremoris MG1363 exposed to various stress conditions and comparing them to the wild type (wt) strain. These experiments revealed that the ΔtrxA genotype caused about 30% growth inhibition at non-stressed conditions and significantly increased sensitivity to oxidants (e.g. H2O2, diamide), while deletion of trxD displayed an effect predominantly in the ΔtrxAΔtrxD mutant. The ΔtrxD mutant exhibited a significantly higher sensitivity only in case of exposure to sodium arsenate and potassium tellurite. Arsenate detoxicification involves arsenate reductase (ArsC), an established Trx target in Bacillus subtilis. The sensitivity of the ΔtrxD mutant may indicate that ArsC is reduced by TrxD in L. lactis. Comparison of protein profiles of the wt, ΔtrxA and ΔtrxD mutants by difference gel electrophoresis (DIGE) revealed significant changes between ΔtrxA and wt. Higher levels of several oxidative stress-related proteins (e.g. glutathione peroxidase) were observed in the ΔtrxA mutant. Proteomic analysis (pulse labeling by [35S]-L-methionine) of the ΔtrxD mutant vs. wt upon exposure to sodium arsenate showed down-regulation of several ATPases (DnaK and GroEL) and GTPases (Ef-G, Ef-Ts) concomitantly with up-regulation of enzymes involved in aerobiosis and nucleotide metabolism in the ΔtrxD mutant. The ΔtrxAΔtrxD deletion mutant is viable, in agreement with a previous study showing that NTR in L. lactis is not essential. Therefore, the presence of an additional thiol redox system is hypothesized. Biochemical studies demonstrated that recombinant LlTrxA, LlTrxD and LlNrdH are substrates for LlNTR, while only LlTrxA and LlNrdH are efficiently reduced by E. coli NTR. LlTrxA appears to have a higher redox potential (-259 mV) compared to E. coli EcTrx1 (-270 mV) but similar reactivity as EcTrx1 towards insulin disulfides and the alkylation reagent iodoacetamide (IAM). LlTrxD exhibited a high redox potential (-243 mV) and about 100-fold higher reactivity towards IAM than LlTrxA and EcTrx1, but no activity towards insulin was observed. LlNrdH showed a higher redox potential (-238 mV) compared to E. coli NrdH (-248 mV) and a lower reactivity towards insulin compared to LlTrxA.
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Hägglund, Per, Svensson, Birte
Department of Systems Biology, Technical University of Denmark, 2013