A bimodular oxidoreductase mediates the specific reduction of phylloquinone (vitamin K1) in chloroplasts

Abstract

SUMMARYPlants and certain species of cyanobacteria are the only organisms capable of synthesizing phylloquinone (vitamin K 1 for vertebrates), which they use as an electron carrier during photosynthesis. Recent studies, however, have identified a plastidial pool of non-photoactive phylloquinone that could be involved in additional cellular functions. Here, we characterized an Arabidopsis bimodular enzyme -the At4g35760 gene product -comprising an integral domain homologous to the catalytic subunit of mammalian vitamin K epoxide reductase (VKORC1, EC 1.1.4.1) that is fused to a soluble thioredoxin-like moiety. GFP-fusion experiments in tobacco mesophyll cells established that the plant protein is targeted to plastids, and analyses of transcript and protein levels showed that expression is maximal in leaf tissues. The fused and individual VKORC1 domains were separately expressed in yeast, removing their chloroplast targeting pre-sequence and adding a C-terminal consensus signal for retention in the endoplasmic reticulum. The corresponding microsomal preparations were equally effective at mediating the dithiotreitol-dependent reduction of phylloquinone and menaquinone into their respective quinol forms. Strikingly, unlike mammalian VKORC1, the Arabidopsis enzyme did not reduce phylloquinone epoxide, and was resistant to inhibition by warfarin. The isoprenoid benzoquinone conjugates plastoquinone and ubiquinone were not substrates, establishing that the plant enzyme evolved strict specificity for the quinone form of naphthalenoid conjugates. In vitro reconstitution experiments established that the soluble thioredoxin-like domain can function as an electron donor for its integral VKORC1 partner.

Lumen Thiol Oxidoreductase1, a Disulfide Bond-Forming Catalyst, Is Required for the Assembly of Photosystem II inArabidopsis

“…Previous studies with mycobacterial and cyanobacterial VKOR-like proteins have demonstrated that the thioredoxin-like domain carries DsbAlike activity, while the VKOR-like central domain is functionally equivalent to DsbB (Singh et al, 2008;Dutton et al, 2010;Wang et al, 2011). In vitro assays show that the Arabidopsis LTO1 thioredoxin-like domain can transfer electrons to its VKOR-like central domain (Furt et al, 2010). This supports the view that the plastid protein operates in a manner similar to bacterial VKORlike proteins in regard to thiol-disulfide chemistry.…”

“…In vitro enzymatic assays indicate that the Arabidopsis VKOR-like protein is active in reducing phylloquinone but not PQ, two quinones found in plastids (Furt et al, 2010). The role of phylloquinone as a structural cofactor tightly bound to PSI is well documented (Brettel, 1997).…”

“…However, its function in plastid biogenesis has so far remained elusive (Furt et al, 2010). In this article, we investigated the function of plastid Lumen Thiol Oxidoreductase1 (LTO1), the Arabidopsis VKOR-like protein.…”

“…In the plant Arabidopsis thaliana, a VKOR-like protein localizes to the plastid (Furt et al, 2010) and is detected at the thylakoid membrane by proteomic analysis (Zybailov et al, 2008). However, its function in plastid biogenesis has so far remained elusive (Furt et al, 2010).…”

“…The VKOR-like proteins were defined based on the presence of a redox domain containing two Cys pairs. This domain is related to the one present in VKOR, an integral membrane protein of the endoplasmic reticulum (ER) (Goodstadt and Ponting, 2004;Dutton et al, 2008;Furt et al, 2010). VKOR is well studied for its involvement in the reduction of vitamin K, a phylloquinone required as a cofactor for the g-carboxylation of clotting factors in blood (Tie and Stafford, 2008).…”