Arabidopsis NAD-malic enzyme functions as a homodimer and heterodimer and has a major impact during nocturnal metabolism

TRONCONI, M. A.; FAHNENSTICH, H.; GERRARD WHEELER, M. C.; ANDREO, C. S.; FLÜGGE, U. I.; DRINCOVICH, M. F.; MAURINO, V. G.

PLANT PHYSIOLOGY.

American Society of Plant Physiologists

Año: 2008 vol. 146 p. 1540 - 1552

0032-0889

<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; mso-bidi-font-size:10.0pt; font-family:Arial; mso-fareast-font-family:"Times New Roman"; mso-bidi-font-family:"Times New Roman"; mso-ansi-language:EN-GB; mso-fareast-language:EN-US;} @page Section1 {size:612.0pt 792.0pt; margin:72.0pt 90.0pt 72.0pt 90.0pt; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> Two genes encoding mitochondrial NAD-dependent-malic enzyme (NAD-ME) were identified in Arabidopsis (Arabidopsis thaliana). The encoded proteins are separated into the two clades found in plant NAD-ME phylogenetic trees. AtNAD-ME1 belongs to clade 1, which includes known a subunits with molecular masses of 65 kDa whilst AtNAD-ME2 clusters with the known b subunits with molecular masses of about 58 kDa. The separated recombinant proteins showed NAD-ME activity and presented comparable kinetic properties. Both AtNAD-ME1 and -2 are dimers in its active conformation in solution. Native-electrophoresis coupled to denaturing-electrophoresis revealed that in vivo AtNAD-ME is a dimer of non-identical subunits in Arabidopsis. Further support for this conclusion was obtained by reconstitution of the active dimer in vitro. Characterization of loss-of-function mutants for both AtNAD-ME indicated that in vivo each protein exhibits enzymatic activity. None of the single nor the double mutants showed a growth or a developmental phenotype suggesting that the NAD-ME activity is not essential for normal autotrophic development. Nevertheless, metabolic profiling of plants completely lacking NAD-ME activity indicated differential patterns of modifications in the light and dark periods and reveals a major participation of NAD-ME during nocturnal metabolism. This is the first time where the activity of the separated NAD-ME subunits has been measured in vitro and in vivo. This opens the possibility that the separated subunits could also produce active in vivo enzymes, at least in a particular tissue or under a particular situation.