Demethylmenaquinol is a substrate of Escherichia coli nitrate reductase A (NarGHI) and forms a stable semiquinone intermediate at the NarGHI quinol oxidation site.

Quinones are essential building blocks of respiration, a universal process dedicated to efficient harvesting of environmental energy and its conversion into a transmembrane chemiosmotic potential. Quinones differentiate mostly by their midpoint redox potential. As such, γ-proteobacteria such as Escherichia coli are characterized by the presence of demethylmenaquinone (DMK) with an intermediate redox potential between low-potential (menaquinone) and high-potential (ubiquinone) quinones. In this study, we show that demethylmenaquinol (DMKH2) is a good substrate for nitrate reductase A (NarGHI) in nitrate respiration in E. coli. Kinetic studies performed with quinol analogs on NarGHI show that removal of the methyl group on the naphthoquinol ring impacts modestly the catalytic constant but not the KM. EPR-monitored redox titrations of NarGHI-enriched membrane vesicles reveal that endogeneous demethylmenasemiquinone (DMSK) intermediates are stabilized in the enzyme. The measured midpoint potential of the DMK/DMKH2 redox couple in NarGHI (E'm,7.5 (DMK/DMKH2) ~-70mV) is significantly lower than that previously measured for unbound species. High resolution pulsed EPR experiments demonstrate that DMSK are formed within the NarGHI quinol oxidation site. Overall, our results provide the first characterization of a protein-bound DMSK and allows for comparison for distinct use of three quinones at a single Q-site in NarGHI.

Cloning and molecular characterization of three lysophosphatidic acid acyltransferases expressed in flax seeds.

In the context of the growing demand for α-linolenic acid due to its high nutritional value as a polyunsaturated fatty acid, we have investigated the contribution of 2-lysophosphatidic acid acyltransferase (LPAAT) enzymes from flax (Linum usitatissimum) in the accumulation of α-linolenic acid into the oil fraction of flax seed. We have isolated the cDNAs encoding three class A microsomal LPAAT2 isoforms from developing flax seeds. The three isoforms, denominated LPAAT2A, LPAAT2A2 and LPAAT2B, are able to complement the LPAAT deficient JC201 E. coli mutant, confirming their functionality. We have performed enzymatic assays showing that the specific activity of the LPAAT2A isoform is significantly higher than that of the LPAAT2A2 and LPAAT2B toward the unsaturated oleic, linoleic and linolenic acids. Moreover, LPAAT2A presents in vitro a high specificity and selectivity for linoleic and linolenic acids as compared to saturated fatty acids. The three isoforms are expressed during all the stages of seed development and in stem and leaf tissues, as shown by an analysis of the transcription level of the corresponding genes. The heterologous expression of LPAAT2A in Arabidopsis seeds leads to an increase in the accumulation of linoleic and linolenic acids in the oil fraction of the seeds from two transgenic lines.