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Role of tryptophan residues of Erv1: Trp95 and Trp183 are important for its folding and oxidase function

Role of tryptophan residues of Erv1: Trp95 and Trp183 are important for its folding and oxidase function

Wang, Qi, Ang, Swee Kim, Ceh-Pavia, Efrain, Pang, Jiayun and Lu, Hui (2015) Role of tryptophan residues of Erv1: Trp95 and Trp183 are important for its folding and oxidase function. Bioscience Reports, 35 (4):e00244. ISSN 0144-8463 (Print), 1573-4935 (Online) (doi:10.1042/BSR20150144)

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Abstract

Erv1 is an FAD-dependent sulphydryl oxidase of the ERV/ALR 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 Trp95, no experimental study has been reported. In this study, we investigated the structural and functional roles of individual Trp residues of Erv1. Six single Trp-to-Phe yeast mutant strains were generated and their effects on cell viability were tested at various temperatures. Then, the mutants were purified from E. coli. Their effects on folding, FAD-binding, and Erv1 activity were characterised. Our results showed that Erv1W95F has the strongest effect on the stability and function of Erv1, and followed by Erv1W183F. Erv1W95F results in a decrease of 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. Erv1W183F induces changes in the oligomerisation state of Erv1, along with a pronounced effect on the stability of Erv1 and its function at 37°C, whilst the other mutants had no clear effect on the function of Erv1 including the highly conserved Trp157 mutant. Finally, computational analysis indicates that Trp95 plays a key role in stabilising the isoalloxazine ring to interact with Cys133. Taken together, this study provided important insights into the molecular mechanism of how sulfhydryl oxidases use FAD in catalyzing disulfide bond formation.

Item Type: Article
Additional Information: © 2015 Authors. This is an open access article published by Portland Press Limited and distributed under the Creative Commons Attribution Licence 3.0.
Uncontrolled Keywords: sulphydryl oxidase, FAD binding, tryptophan residue, protein folding, mitochondria
Subjects: Q Science > QR Microbiology
Faculty / Department / Research Group: Faculty of Engineering & Science
Faculty of Engineering & Science > Department of Pharmaceutical, Chemical & Environmental Sciences
Last Modified: 02 May 2017 15:23
Selected for GREAT 2016: None
Selected for GREAT 2017: GREAT a
Selected for GREAT 2018: None
URI: http://gala.gre.ac.uk/id/eprint/13822

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