The
vitamin K epoxide reductase (VKORC1) enzyme is of primary importance
in many physiological processes,
i.e., blood coagulation, energy metabolism, and arterial
calcification prevention, due to its role in the vitamin K cycle.
Indeed, VKORC1 catalyzes
reduction of vitamin K epoxide to quinone and then to hydroquinone.
However, the three-dimensional VKORC1 structure remains experimentally
undetermined, because of the endoplasmic reticulum membrane location
of this enzyme. Here we present a molecular modeling investigation
of the VKORC1 enzymatic site structure and function, supported by in vitro enzymatic assays. Four VKORC1 mutants were designed in silico (F55G, F55Y, N80G, and F83G) based on a previous
study that identified residues
F55, N80, and F83 as being crucial for vitamin K epoxide binding.
F55G, N80G, and F83G nonconservative mutants were all predicted to
be inactive by molecular modeling
analyses. However, the F55Y conservative mutant was expected to be
active compared to wild-type VKORC1. In vitro enzymatic
assays performed on recombinant proteins assessed our molecular modeling
hypotheses and led us to describe the role of accurate VKORC1 active
site residues with respect to VKORC1. Residues F55, N80, and F83 appeared
to act in a concerted
manner to keep vitamin K epoxide close to the C135 catalytic residue.
Residues F55 and N80 prevent naphthoquinone head rotation away from
the active site, assisted by residue F83 that prevents vitamin K from
sliding outside the enzymatic pocket, through hydrophobic tail stabilization.
Our results thus highlighted the specific functions of VKORC1 catalytic
pocket residues and evidenced the ability of our structural model
to predict biological effects of VKORC1 mutations.
Leptospirosis is a re-emergent worldwide zoonosis. It is endemic in Martinique where transmission conditions are favourable. Humans are usually infected through contact with water contaminated with urine of rodents. Recent human leptospirosis outbreaks in Martinique require today effective rodent management to prevent leptospirosis transmission. Nowadays, use of anticoagulant rodenticides (AR) is the main method implemented to control rodent populations. Nevertheless, intensive use of these AR has selected worldwide many VKORC1-based resistant rodent strains to AR. Our aim was to characterize the sensitivity of Martinique commensal rodents to AR to better prevent leptospirosis transmission. Resistance of house mice to first-generation and in rare cases even to second-generation ARs were clearly demonstrated in Martinique with the detection of the Y139C mutation with a very high allelic frequency of 40% and the A26T/Y139C double-mutation with an allelic frequency of 0.9%. In black rat, the most prevalent rodent in Martinique, 3 new Vkorc1 coding mutations were detected, the H68N, A115T and S149N mutations associated with moderate resistance to first generation AR. Therefore, rodent management in Martinique must be carried carefully to avoid resistance diffusion and maintain long-term effective rodent management, to be able to efficiently prevent leptospirosis transmission.
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