A three-way dance: NAD-ME switches subunits to perform different tasks

Hüdig and Tronconi et al. reveal that the housekeeping and C₄-decarboxylase functions of a C₄ enzyme are performed by isoforms that combine the same α subunit with different β subunits. Plant Cell https://doi.org/10.1093/plcell/koab265

 Meike Hüdig, Molecular Plant Physiology, University of Bonn; Marcos A. Tronconi, Centro de Estudios Fotosintéticos y Bioquímicos, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina. Veronica Maurino, Molecular Plant Physiology, University of Bonn

Background: C₄ photosynthesis is an efficient improvement of the standard CO₂ fixation pathway that evolved over 60 times. This auxiliary metabolic route prevents Rubisco oxygenase activity by enriching CO₂ in its surroundings. C₄-NAD-malic enzyme (NAD-ME) releases the prefixed CO₂ to feed Rubisco in about one-third of these C₄ lineages. Furthermore, in every cell, NAD-ME functions in mitochondrial malate respiration, a housekeeping role. Most C₄ enzymes originated by gene duplication and subsequent neofunctionalization of one housekeeping gene copy. However, the evolution of C₄-NAD-ME showed sub-functionalization and differences in the frequency of gene duplication of the two paralogous α- and β-NAD-ME genes that encode the subunits of NAD-ME. We used two independent C₄-NAD-ME lineages from the Cleomaceae (Gynandropsis gynandra, see image, and Cleome angustifolia) and a C₃ sister species (Tarenaya hassleriana) to unravel the mystery around the elusive C₄-NAD-ME.

Question: Given that the evolution of C₄-NAD-ME turned out to be an exceptional case, we wanted to know if there is a specific NAD-ME version to perform this additional task and, if so, how it differs from its housekeeping NAD-ME.

Findings: NAD-ME is comprised of two different α and β protein subunits. In both G. gynandra and C. angustifolia, the β subunit was duplicated (β1 and β2), resulting in two enzymatic entities, NAD-MEα/β1 and NAD-MEα/β2. The C₄-decarboxylase role is conducted by the NAD-MEα/β1 heteromer, a highly efficient enzymatic entity exclusively present in photosynthetic tissues. Unlike the β2 subunit, β1 is a non-catalytic protein forming more rigid contacts with the associated α-subunit; it is also responsible for activating C₄-NAD-ME by aspartate, an important C₄ metabolite. The C₄ decarboxylase coexists with the housekeeping NAD-MEα/β2 in mitochondria. In silico simulations suggest that positively selected amino acid substitutions in the β1 subunit of both C₄ species enables the differential assembly of the subunits to form independent enzymatic entities acting in the same cell compartment.

Next steps: Our work demonstrates a possible evolutionary route of heteromeric proteins. We want to explore the identified changes in the β subunits to pinpoint the properties to single amino acids. This could help to engineer existing NAD-ME with new C₄ properties.

Meike Hüdig, Marcos A. Tronconi, Juan P. Zubimendi, Tammy L. Sage, Gereon Poschmann, David Bickel, Holger Gohlke, Veronica G. Maurino. (2022). Respiratory and C₄-photosynthetic NAD-malic enzyme coexist in bundle sheath cell mitochondria and evolved via association of differentially adapted subunits. https://doi.org/10.1093/plcell/koab265