# Characterization of enzymes in the vitamin K cycle

> **NIH NIH R01** · UNIV OF NORTH CAROLINA CHAPEL HILL · 2021 · $530,546

## Abstract

Project Summary/Abstract
The vitamin K cycle enzymes: gamma-glutamyl carboxylase (GGCX), vitamin K epoxide reductase (VKOR) and
vitamin K reductase (VKR) are responsible for the post-translational carboxylation of vitamin K-dependent (VKD)
proteins into their biologically active forms. Carboxylation is mainly associated with blood coagulation, as four
coagulation factors (factors II, VII, IX, and X) and three anticoagulant proteins (proteins C, S, and Z) require
carboxylation for their function. With the discovery of new VKD proteins and their new biological functions, the
importance of carboxylation has been expanded to vascular calcification, bone development, glucose
metabolism, and cell proliferation. GGCX is the enzyme that directly modifies VKD proteins to their functional
forms. Genetic variations in GGCX have been identified in patients with vitamin K-related disorders. However, it
is not clear why some GGCX mutations cause bleeding disorders while other mutations result in non-bleeding
symptoms. Additionally, one GGCX mutation can sometimes cause two distinct clinical phenotypes. However, it
remains unclear as to why the administration of vitamin K can ameliorate one symptom but not the other. VKOR
is a regulatory enzyme of the vitamin K cycle and the target of the oral anticoagulant, warfarin. The mechanism
of VKOR's active site regeneration remains elusive. Recent studies have shown that superwarfarin poisonings
(an anticoagulant more powerful than warfarin) are a growing public health concern and that vitamin K therapy
for that is costly and inefficient. The enzyme that reduces vitamin K to its hydroquinone form to support VKD
carboxylation is VKR. Despite decades of effort, the identity of VKR is still unknown. Our long-term research goal
is to understand in detail the structure and function relationship of all vitamin K cycle enzymes within their native
milieu for better control coagulation and other related physiological processes. The current proposal aims to
solve the main questions remaining in the field (as mentioned above). To accomplish these goals, we propose
the following specific aims: Specific Aim 1 - we will use our recently established CRISPR (Clustered Regularly
Interspaced Short Palindromic Repeats)-Cas9 knockout reporter cell lines to study the effect of all currently
identified naturally occurring GGCX mutations on the carboxylation of different VKD proteins associated with
distinct clinical phenotypes; Specific Aim 2 - we will apply genetic code expansion technology to clarify VKOR's
active site regeneration and design and synthesize novel vitamin K derivatives to rescue superwarfarin
poisonings; and Specific Aim 3 - we will use the genome-wide CRISPR-Cas9 knockout library to screen for the
unknown enzyme, VKR. We expect that information derived from these studies will help us understand how the
three vitamin K cycle enzymes contribute to the complex mechanisms of carboxylation, thereby gaining new
therapeutic insi...

## Key facts

- **NIH application ID:** 10085669
- **Project number:** 5R01HL131690-06
- **Recipient organization:** UNIV OF NORTH CAROLINA CHAPEL HILL
- **Principal Investigator:** DARREL W STAFFORD
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $530,546
- **Award type:** 5
- **Project period:** 2016-06-01 → 2024-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10085669

## Citation

> US National Institutes of Health, RePORTER application 10085669, Characterization of enzymes in the vitamin K cycle (5R01HL131690-06). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10085669. Licensed CC0.

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