The catalytic mechanism of vitamin K epoxide reduction in a cellular environment

Shen, Guomin; Cui, Weidong; Cao, Qing; Gao, Meng; Liu, Hongli; Su, Gaigai; Gross, Michael L.; Li, Weikai

doi:10.1074/jbc.ra120.015401

Cited by 10 publications

(18 citation statements)

References 30 publications

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“…Compared to engineered SsVKOR, HsVKOR also contains additional structural features, such as the helical extension of TM1 [23], that we have not explored. Finally, the redox potential of catalytic cysteines in the engineered SsVKOR and HsVKOR can be different, which will result in different levels of active site cysteines in reduced state that contributes to a large portion of overall HsVKOR activity [31].…”

Section: Discussionmentioning

confidence: 99%

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

Shen

Cao

et al. 2022

The FEBS Journal

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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.

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Section: Discussionmentioning

confidence: 99%

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

Shen

Cao

et al. 2022

The FEBS Journal

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“…Correlation between MS and biochemical data suggested that the PO and R states contribute about equally to the cellular HsVKOR activity. 87 Although the R state is of low abundance, its specific activity is approximately 8-fold higher than the PO state. 87 The complicated interplay between warfarin inhibition and catalysis underlies warfarin response (Figure 3C).…”

Section: Vkor Catalysis and Warfarin Inhibition Act On Different Cell...mentioning

confidence: 99%

“…For the KO binding, protection of Cys16 and Cys85 was observed only with Cys43Ala mutation that stabilizes the PO state (Figure 3B). 69,87 The wild‐type HsVKOR does not show this protection effect, probably because the catalysis continuously resolves the PO state. Unlike warfarin inhibition, HsVKOR catalysis is inactive at the O state because this reductase is fully oxidized.…”

Section: Cellular and Structural Mechanisms Of Vitamin K Antagonizationmentioning

confidence: 99%

“…Unlike warfarin inhibition, HsVKOR catalysis is inactive at the O state because this reductase is fully oxidized. Correlation between MS and biochemical data suggested that the PO and R states contribute about equally to the cellular HsVKOR activity 87 . Although the R state is of low abundance, its specific activity is approximately 8‐fold higher than the PO state 87 …”

Section: Cellular and Structural Mechanisms Of Vitamin K Antagonizationmentioning

confidence: 99%

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Structural and cellular basis of vitamin K antagonism

Liu

Shen

2022

Journal of Thrombosis and Haemostasis

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Vitamin K antagonists (VKAs), such as warfarin, are oral anticoagulants widely used to treat and prevent thromboembolic diseases. Therapeutic use of these drugs requires frequent monitoring and dose adjustments, whereas overdose often causes severe bleeding. Addressing these drawbacks requires mechanistic understandings at cellular and structural levels. As the target of VKAs, vitamin K epoxide reductase (VKOR) generates the active, hydroquinone form of vitamin K, which in turn drives the γ-carboxylation of several coagulation factors required for their activity. Crystal structures revealed that VKAs inhibit VKOR via mimicking its catalytic process. At the active site, two strong hydrogen bonds that facilitate the catalysis also afford the binding specificity for VKAs. Binding of VKAs induces a global change from open to closed conformation. Similar conformational change is induced by substrate binding to promote an electron transfer process that reduces the VKOR active site. In the cellular environment, reducing partner proteins or small reducing molecules may afford electrons to maintain the VKOR activity. The catalysis and VKA inhibition require VKOR in different cellular redox states, explaining the complex kinetics behavior of VKAs. Recent studies also revealed the mechanisms underlying warfarin resistance, warfarin dose variation, and antidoting by vitamin K. These mechanistic understandings may lead to improved anticoagulation strategies targeting the vitamin K cycle.

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“…This result facilitates the construction of warfarin‐hVKOR model from MD simulations. Further NEM footprinting analysis of hVKOR with its substrate vitamin K epoxide elucidates the reactive states of hVKOR in the cellular environment [97].…”

Section: Ms‐based Techniques For Footprinting Mpsmentioning

confidence: 99%

Advances in mass spectrometry‐based footprinting of membrane proteins

Gross

2022

Proteomics

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Structural biology is entering an exciting time where many new high‐resolution structures of large complexes and membrane proteins (MPs) are determined regularly. These advances have been driven by over 15 years of technological improvements, first in macromolecular crystallography, and recently in cryo‐electron microscopy. Obtaining information about MP higher order structure and interactions is also a frontier, important but challenging owing to their unique properties and the need to choose suitable detergents/lipids for their study. The development of mass spectrometry (MS), both instruments and methodology in the past 10 years, has also advanced it as a complementary method to study MP structure and interactions. In this review, we discuss advances in MS‐based footprinting for MPs and highlight recent methodologies that offer new promise for MP study by chemical footprinting and mass spectrometry.

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The catalytic mechanism of vitamin K epoxide reduction in a cellular environment

Cited by 10 publications

References 30 publications

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

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

Structural and cellular basis of vitamin K antagonism

Advances in mass spectrometry‐based footprinting of membrane proteins

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