Arabidopsis Thaliana Nqr (Nad(P)H:Quinone Oxidoreductase) Reduces Hexavalent Chromium

Arabidopsis thaliana NQR (NAD(P)H:quinone oxidoreductase) reduces hexavalent chromium

Sparla F, Peciccia M, Pupillo P, Trost P.

Department of Biology – Laboratory of Molecular Plant Physiology – University of Bologna

Plant NAD(P)H:quinone oxidoreductase (NQR) has long been considered a functional homologous to animal DT-diaphorase. Like the animal enzyme, plant NQR reduces quinone substrates directly to hydroquinones by a two-electron reaction mechanism, thereby avoiding the formation of reactive oxygen species. In spite of their biochemical similarity, the plant enzyme differs from DT-diaphorase in molecular structure, flavin cofactor, stereospecificity and sensitivity to inhibitors. Moreover, while DT-diaphorases are involved in antioxidative responses and their gene expression is influenced by several factors, plant NQR seems to be a housekeeping enzyme, unlikely involved as such in antioxidative defence. In fact, both Northern Blot and RT-PCR analysis have shown that NQR gene expression is not induced by pathogens (Pseudomonas syringae, Peronospora), by signal molecules involved in plant-pathogen interactions (salicylic acid, H2O2, methyl jasmonate), by hormones (2,4-D, ethylene), by high light treatments, by salt stress (NaCl, KCl), by heavy metals (CsCl, LiCl, K2CrO4), by wounding, by cinnamic acids (sinapinic, coumaric, caffeic acid) or by menadione.

Surprisingly, NQR amino acid sequence resulted 33-38% identical to five prokaryotic chromate reductases recently deposited in the Genebank, prompting us to focus our attention on chromate as a potential alternative electron acceptor for NQR. Chromate compounds have a widespread industrial application and chromate salts may pollute the environment through urban, industrial and nuclear waste. Unfortunately soluble Cr(VI) compounds are extremely toxic and exhibits mutagenic and carcinogenic effects on biological systems. Due to its high solubility, Cr(VI) does not remain confined in the site of initial contamination. On the contrary the reduced form of chromate, Cr(III), is less toxic and less soluble. Different bacterial chromate reductases have been characterized to date. Due to their capacity to convert Cr(VI) to less toxic Cr(III), their use has been proposed to remove chromate from contaminated areas.

In order to understand whether NQR could function as a chromate reductase, A. thaliana plants (grown on soil for 10 weeks at 22 C, with 15-h light/9-h dark cycle) were homogenized, filtered and centrifuged for 1 hour at 100.000g. About 20 % of both NADPH:duroquinone and NADPH:chromate reductase activities were found associated to the pellet, probably trapped within sealed vesicles. The supernatant was loaded on an anion-exchange column and eluted by a linear KCl gradient. Interestingly, both NADP:duroquinone and NADPH-chromate reductase activities co-eluted in the same two peaks, and both activities were partially inhibited by anti-NQR antibodies, strongly suggesting that NQR is responsible for the bulk of chromate reductase activity detectable in the soluble fraction of Arabidopsis plants. Like prokaryotic chromate reductases, NQR is a soluble protein and is able to use both NADH and NADPH as electron donors.

Arabidopsis NQR has been cloned and heterologously expressed in E. coli. We have found that also recombinant NQR is a kinetically efficient chromate reductase. Kinetic parameters, as well as the capacity of NQR to confer to higher plants a certain degree of resistance to chromate salts, and the capacity to detoxify chromate are currently under investigation.