title: Role of tryptophan residues of Erv1: Trp95 and Trp183 are important for its folding and oxidase function.
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.
authors.affiliation: Manchester Institute of Biotechnology, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, U.K.
authors.forename: Qi
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Manchester Institute of Biotechnology, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, U.K. Current address: Department of Microbiology, Yong Loo Lin School of Medicine, National University Health System, 5 Science Drive 2, National University of Singapore, Singapore 117597, Singapore.
authors.forename: Swee Kim
authors.initials: SK
authors.lastname: Ang
authors.affiliation: Manchester Institute of Biotechnology, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, U.K.
authors.forename: Efrain
authors.initials: E
authors.lastname: Ceh-Pavia
authors.affiliation: Department of Pharmaceutical, Chemical and Environmental Sciences, Faculty of Engineering and Science, University of Greenwich, Medway Campus, Central Avenue, Chatham Maritime, Kent ME4 4TB, U.K.
authors.forename: Jiayun
authors.initials: J
authors.lastname: Pang
authors.affiliation: Manchester Institute of Biotechnology, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, U.K. hui.lu@manchester.ac.uk.
authors.forename: Hui
authors.initials: H
authors.lastname: Lu
mesh_terms: D019943:Amino Acid Substitution; D002384:Catalysis; D003198:Computer Simulation; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D020125:Mutation, Missense; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D024101:Mitochondrial Proteins; D029701:Saccharomyces cerevisiae Proteins; D014364:Tryptophan; D050862:Oxidoreductases Acting on Sulfur Group Donors; C073790:ERV1 protein, S cerevisiae
keywords: flavin-adenine dinucleotide (FAD) binding; mitochondria; protein folding; thiol oxidase; tryptophan residue