Structural features determining the vitamin K epoxide reduction activity in the VKOR family of membrane oxidoreductases

Abstract

Vitamin K epoxide reductases (VKORs) are a large family of integral membrane enzymes found from bacteria to humans. Human VKOR, specific target of warfarin, has both the epoxide and quinone reductase activity to maintain the vitamin K cycle. Bacterial VKOR homologs, however, are insensitive to warfarin inhibition and are quinone reductases incapable of epoxide reduction. What affords the epoxide reductase activity in human VKOR remains unknown. Here, we show that a representative bacterial VKOR homolog can be converted to an epoxide reductase that is also inhibitable by warfarin. To generate this new activity, we first substituted several regions surrounding the active site of bacterial VKOR by those from human VKOR based on comparison of their crystal structures. Subsequent systematic substitutions narrowed down to merely eight residues, with the addition of a membrane anchor domain, that are responsible for the epoxide reductase activity. Substitutions corresponding to N80 and Y139 in human VKOR provide strong hydrogen bonding interactions to facilitate the epoxide reduction. The rest of six substitutions increase the size and change the shape of the substrate‐binding pocket, and the membrane anchor domain stabilizes this pocket while allowing certain flexibility for optimal binding of the epoxide substrate. Overall, our study reveals the structural features of the epoxide reductase activity carried out by a subset of VKOR family in the membrane environment.

Inhibition of FSP1: A new strategy for the treatment of tumors (Review)

“…VK exists in two forms in nature: The first, called phylloquinone (also known as VK1), is found in photosynthetic organisms, such as green plants, cyanobacteria and algae; the other, namely menadione (also known as VK2), is found in animals and bacteria. VK is a redox-active naphthoquinone ( 71 , 72 ), which can be converted into its corresponding hydroquinone VKH2 and is found mainly in the mature VK cycle ( 61 , 73 ). Recently, Mishima et al ( 74 ) found that FSP1 reduces VK in the manner of ubiquitin ketone.…”

Structural and cellular basis of vitamin K antagonism

“…57,63,99,100 To clarify this issue, the SsVKOR homolog was converted to a warfarin-sensitive epoxide reductase (same as HsVKOR) with merely eight mutations and with addition of a membrane anchor domain. 101 This combination introduces the two key hydrogen bonds at the active site and changes the shape and flexibility of this pocket. These structural features largely determine…”

“…These key differences were taken also as evidence that HsVKOR and SsVKOR adopt completely different structural folds 57,63,99,100 . To clarify this issue, the SsVKOR homolog was converted to a warfarin‐sensitive epoxide reductase (same as HsVKOR) with merely eight mutations and with addition of a membrane anchor domain 101 . This combination introduces the two key hydrogen bonds at the active site and changes the shape and flexibility of this pocket.…”

Fractal Analysis of Tight Sandstone Petrophysical Properties in Unconventional Oil Reservoirs with NMR and Rate-Controlled Porosimetry

“…The 3D capillary model uses the relation between the pore size and pore number to determine the fractal dimension. The pore number N (> r ) with a pore radius larger than r can be obtained from the total mercury saturation V Hg ( r ) ,, where l is the core length. Figure a shows the process of fractal dimension calculation using the 3D capillary model, and the fractal dimension can be obtained from the slope of the log N (> r ) – log r curve in a log–log plot.…”