Intramembrane Thiol Oxidoreductases: Evolutionary Convergence and Structural Controversy

Abstract

During oxidative protein folding, the disulfide-bond formation is catalyzed by thioloxidoreductases. Through dedicated relay pathways, the disulfide is generated in donor enzymes, passed to carrier enzymes, and subsequently delivered to target proteins. The eukaryotic disulfide donors are flavoenzymes, Ero1 in endoplasmic reticulum and Erv1 in mitochondria. In prokaryotes, disulfide generation is coupled to quinone reduction, catalyzed by intramembrane donor enzymes, DsbB and VKOR. To catalyze de novo disulfide formation, these different disulfide donors show striking structural convergences in several levels. They share a four-helix-bundle core structure at their active site, which contains a CXXC motif at a helical end. They have also evolved a flexible loop with shuttle cysteines to transfer electrons to the active site and relay the disulfide bond to the carrier enzymes. Studies of the prokaryotic VKOR, however, have stirred debate of whether the human homolog adopts the same topology with four transmembrane helices and uses the same electron-transfer mechanism. The controversies have recently been resolved by investigating the human VKOR structure and catalytic process in living cells with a mass spectrometry based approach. Structural convergence is found between human VKOR and the disulfide donors to underlie the cofactor reduction, disulfide generation, and electron transfer.

VKORC1L1, An Enzyme Mediating the Effect of Vitamin K in Liver and Extrahepatic Tissues

“…We note, most importantly, that the study of intact human VKORC1, using live cell cysteine labeling in combination with mass spectrometry, convincingly showed that a major fraction of Cys43, Cys51, Cys132 and Cys135 is oxidized in living cells, strongly suggesting that they are all located in the oxidative ER lumen [ 17 ]. Together with the structural data, these results support the four-TM model for human VKORC1 [ 35 ]. Importantly, biochemical and structural modeling predict that human VKORC1L1 is also organized as a four-TM protein ( Figure 2 A) and that the two loop cysteine residues of VKORC1L1 are essential for its activity [ 30 , 33 ].…”

“…Indeed, another model has been proposed for human VKORC1, in which the protein contains only three transmembrane helixes and where Cys43 and Cys51 are localized in the cytosol. A critical evaluation of the technical details, which could explain the discrepancy between some biochemical data and the structural biology predictions, has been published recently [ 35 ]. We note, most importantly, that the study of intact human VKORC1, using live cell cysteine labeling in combination with mass spectrometry, convincingly showed that a major fraction of Cys43, Cys51, Cys132 and Cys135 is oxidized in living cells, strongly suggesting that they are all located in the oxidative ER lumen [ 17 ].…”

Structure and Electron-Transfer Pathway of the Human Methionine Sulfoxide Reductase MsrB3

“…The resulting reduced cysteines in the loop then reduce the Ero1 FAD-proximal disulfide, effectively shuttling electrons from PDI to the Ero1 active site. Another example of a shuttle disulfide in the ER occurs in the transmembrane protein VKOR (29). The VKOR CX 7 C shuttle disulfide transfers electrons from membrane-bound PDI family proteins to the VKOR active-site disulfide next to the quinone cofactor, from where they can be used to reduce vitamin K epoxide (43).…”

A unified evolutionary origin for the ubiquitous protein transporters SecY and YidC

“…Perhaps the most extensive known case of structural convergence in functionally similar helical IMPs occurred among thiol oxidoreductases. Four analogous families all use four-helix bundles to bind their redox cofactors, despite two being IMPs and two being cytoplasmic (Li et al, 2018). But they are nonetheless easily distinguishable because they connect those four helices in different orders.…”