Probing Light-Dependent Regulation of the Calvin Cycle Using a Multi-Omics Approach

Photoautotrophic microorganisms are increasingly explored for the conversion of atmospheric carbon dioxide into biomass and valuable products. The Calvin-Benson-Bassham (CBB) cycle is the primary metabolic pathway for net CO2 fixation within oxygenic photosynthetic organisms. The cyanobacteria, Synechocystis sp. PCC 6803, is a model organism for the study of photosynthesis and a platform for many metabolic engineering efforts. The CBB cycle is regulated by complex mechanisms including enzymatic abundance, intracellular metabolite concentrations, energetic cofactors and post-translational enzymatic modifications that depend on the external conditions such as the intensity and quality of light. However, the extent to which each of these mechanisms play a role under different light intensities remains unclear. In this work, we conducted non-targeted proteomics in tandem with isotopically non-stationary metabolic flux analysis (INST-MFA) at four different light intensities to determine the extent to which fluxes within the CBB cycle are controlled by enzymatic abundance. The correlation between specific enzyme abundances and their corresponding reaction fluxes is examined, revealing several enzymes with uncorrelated enzyme abundance and their corresponding flux, suggesting flux regulation by mechanisms other than enzyme abundance. Additionally, the kinetics of 13C labeling of CBB cycle intermediates and estimated inactive pool sizes varied significantly as a function of light intensity suggesting the presence of metabolite channeling, an additional method of flux regulation. These results highlight the importance of the diverse methods of regulation of CBB enzyme activity as a function of light intensity, and highlights the importance of considering these effects in future kinetic models.

Transport Proteins Enabling Plant Photorespiratory Metabolism

Photorespiration (PR) is a metabolic repair pathway that acts in oxygenic photosynthetic organisms to degrade a toxic product of oxygen fixation generated by the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase. Within the metabolic pathway, energy is consumed and carbon dioxide released. Consequently, PR is seen as a wasteful process making it a promising target for engineering to enhance plant productivity. Transport and channel proteins connect the organelles accomplishing the PR pathway—chloroplast, peroxisome, and mitochondrion—and thus enable efficient flux of PR metabolites. Although the pathway and the enzymes catalyzing the biochemical reactions have been the focus of research for the last several decades, the knowledge about transport proteins involved in PR is still limited. This review presents a timely state of knowledge with regard to metabolite channeling in PR and the participating proteins. The significance of transporters for implementation of synthetic bypasses to PR is highlighted. As an excursion, the physiological contribution of transport proteins that are involved in C4 metabolism is discussed.

Molecular basis for metabolite channeling in a ring opening enzyme of the phenylacetate degradation pathway

Substrate channeling is a mechanism for the internal transfer of hydrophobic, unstable or toxic intermediates from the active site of one enzyme to another. Such transfer has previously been described to be mediated by a hydrophobic tunnel, the use of electrostatic highways or pivoting and by conformational changes. The enzyme PaaZ is used by many bacteria to degrade environmental pollutants. PaaZ is a bifunctional enzyme that catalyzes the ring opening of oxepin-CoA and converts it to 3-oxo-5,6-dehydrosuberyl-CoA. Here we report the structures of PaaZ determined by electron cryomicroscopy with and without bound ligands. The structures reveal that three domain-swapped dimers of the enzyme form a trilobed structure. A combination of small-angle X-ray scattering (SAXS), computational studies, mutagenesis and microbial growth experiments suggests that the key intermediate is transferred from one active site to the other by a mechanism of electrostatic pivoting of the CoA moiety, mediated by a set of conserved positively charged residues.

Reengineering of the human pyruvate dehydrogenase complex: from disintegration to highly active agglomerates

The pyruvate dehydrogenase complex (PDC) plays a central role in cellular metabolism and regulation. As a metabolite-channeling multi-enzyme complex it acts as a complete nanomachine due to its unique geometry and by coupling a cascade of catalytic reactions using ‘swinging arms'. Mammalian and specifically human PDC (hPDC) is assembled from multiple copies of E1 and E3 bound to a large E2/E3BP 60-meric core. A less restrictive and smaller catalytic core, which is still active, is highly desired for both fundamental research on channeling mechanisms and also to create a basis for further modification and engineering of new enzyme cascades. Here, we present the first experimental results of the successful disintegration of the E2/E3BP core while retaining its activity. This was achieved by C-terminal α-helixes double truncations (eight residues from E2 and seven residues from E3BP). Disintegration of the hPDC core via double truncations led to the formation of highly active (approximately 70% of wildtype) apparently unordered clusters or agglomerates and inactive non-agglomerated species (hexamer/trimer). After additional deletion of N-terminal ‘swinging arms’, the aforementioned C-terminal truncations also caused the formation of agglomerates of minimized E2/E3BP complexes. It is likely that these ‘swinging arm’ regions are not solely responsible for the formation of the large agglomerates.