Abstract
Erv1 is an FAD-dependent thiol oxidase of the ERV (essential for respiration and viability)/ALR (augmenter of liver regeneration) sub-family and an essential component of the mitochondrial import and assembly pathway. Erv1 contains six tryptophan residues, which are all located in the highly conserved C-terminal FAD-binding domain. Though important structural roles were predicted for the invariable Trp(95), no experimental study has been reported. In the present study, we investigated the structural and functional roles of individual tryptophan residues of Erv1. Six single tryptophan-to-phenylalanine yeast mutant strains were generated and their effects on cell viability were tested at various temperatures. Then, the mutants were purified from Escherichia coli. Their effects on folding, FAD-binding and Erv1 activity were characterized. Our results showed that Erv1(W95F) has the strongest effect on the stability and function of Erv1 and followed by Erv1(W183F). Erv1(W95F) results in a decrease in the Tm of Erv1 by 23°C, a significant loss of the oxidase activity and thus causing cell growth defects at both 30°C and 37°C. Erv1(W183F) induces changes in the oligomerization state of Erv1, along with a pronounced effect on the stability of Erv1 and its function at 37°C, whereas the other mutants had no clear effect on the function of Erv1 including the highly conserved Trp(157) mutant. Finally, computational analysis indicates that Trp(95) plays a key role in stabilizing the isoalloxazine ring to interact with Cys(133). Taken together, the present study provided important insights into the molecular mechanism of how thiol oxidases use FAD in catalysing disulfide bond formation.
Highlights
Erv1 is an essential component of the mitochondrial import and assembly (MIA) pathway, playing a critical role during import and oxidative folding of the mitochondrial intermembrane space (IMS) proteins [1,2,3]
Effects of Erv1 tryptophan mutations on yeast cell viability To investigate the requirement of individual tryptophan residues of Erv1 in its function in vivo, we generated six tryptophan mutant yeast strains by expressing plasmid-encoded erv1 single tryptophan-to-phenylalanine mutants, in an ERV1 knockout strain erv1, isogenic to the WT Trp303, as described previously [24]
We investigated the structural and functional roles of all six tryptophan residues of Erv1, using yeast genetic, biochemical and computational methods, with focus on the highly conserved Trp95
Summary
Erv1 (essential for respiration and viability 1) is an essential component of the mitochondrial import and assembly (MIA) pathway, playing a critical role during import and oxidative folding of the mitochondrial intermembrane space (IMS) proteins [1,2,3]. Structural and functional study of the corresponding Erv1R182H mutant showed that Arg182 plays an important role in the folding and FAD binding of Erv1 [25].
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