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

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

Isolation of Adaptive Genes from Shorea curtisii Dyer ex King via PCR

Two pairs of primers for heat shock protein 90 (HSP90) and abscisic acid induced guard cell protein (ABGI) which are adaptive genes responsible for heat stress and drought stress, respectively were designed and synthesized based on the sequence of HSP90 in Catharanthus roseus and ABGI in Vicia faba. These primers were used in amplification of HSP90 and ABGI from the genomic DNA of Shorea curtisii via polymerase chain reaction (PCR). Optimization of PCR produced single distinct DNA fragment with approximately 750bp HSP90 and 830bp ABGI. Verification of PCR inserts from recombinant plasmids was conducted via restriction digestion method using EcoR1 and PCR method using the same primer sets, and also T7/SP6 Promoter primers for further confirmation purpose. Restriction digestion successfully detected HSP90 PCR insert but failed to detect ABGI PCR insert with desired size. PCR fragments with size about 750bp and 830bp, which matched the size of HSP90 PCR insert and ABGI PCR insert respectively, were re-amplified from respective recombinant plasmids using the same primer sets. By using T7/SP6 Promoter Primers in HSP90 PCR insert and ABGI PCR insert verification, the amplified DNA fragment was about 920bp and 1000bp respectively. The recombinant plasmids were sent for DNA sequencing of PCR products in HSP90 and ABGI amplification. The DNA sequencing was in progress and sequence analysis was not able to be performed

Mitochondrial sulphydryl oxidase Erv1: both shuttle cysteine residues are required for its function with distinct roles

Erv1 is an essential component of the mitochondrial import and assembly (MIA) pathway, playing an important role in the oxidative folding of mitochondrial intermembrane space proteins. In the MIA pathway, Mia40, a sulphydryl oxidoreductase with a CPC motif at its active site, oxidises newly-imported substrate proteins. Erv1, a FAD-dependent sulphydryl oxidase, in turn reoxidises Mia40 via its N-terminal Cys30-Cys33 shuttle disulphide. However, it is unclear how the two shuttle cysteines of Erv1 relay electrons from Mia40 CPC to the Erv1 active-site Cys130-Cys133 disulphide. In this study, using yeast genetic approaches we showed that both shuttle cysteine residues of Erv1 are required for cell growth. In organelle and in vitro studies confirmed that both shuttle cysteines were indeed required for import of MIA-pathway substrates and Erv1 enzyme function to oxidise Mia40. Furthermore, our results revealed that the two shuttle cysteines of Erv1 are functionally distinct. While Cys33 is essential for forming the intermediate disulphide Cys33-Cys130’ and transferring electrons to the redox active-site directly, Cys30 plays two important roles: (i) dominantly interacts and receives electrons from Mia40 CPC, and (ii) resolves the Erv1 Cys33-Cys130 intermediate disulphide. Taken together, we conclude that both shuttle cysteines are required for Erv1 function, and play complementary but distinct roles to ensure rapid turnover of active Erv1

Mitochondrial thiol oxidase Erv1:Both shuttle cysteine residues are required for its function with distinct roles

10.1042/BJ20131540Biochemical Journal4602199-21

The disease-associated mutation of the mitochondrial thiol oxidase Erv1 impairs cofactor binding during its catalytic reaction

10.1042/BJ20140679Biochemical Journal4643449-45