Ogrodzinska, W. | Search bioRxiv

bioRxiv2024

Mobility of Four Structural Regions Drives Isoform-Specific Properties of Plant LPOR

Light-dependent protochlorophyllide oxidoreductase (LPOR) is a photocatalytic enzyme in the chlorophyll (Chl) biosynthetic pathway that underwent duplications in angiosperms, resulting in the emergence of multiple isoforms across various plant species. The physiological roles of these LPOR homologs remained unclear, so we selected six plant species with different number of isoforms of the enzyme and characterized their properties in vitro. Our findings revealed that these isoforms vary in their affinity for the reaction product, chlorophyllide (Chlide), as well as for NADPH under lipid-free conditions and in reaction mixtures supplemented with plant lipids. Additionally, we observed differences in their oligomerization behavior. Our experimental approach generated a dataset comprising several hundred pairs of spectra, recorded before and after reaction-triggering illumination. This data was used to analyze the correlation between fluorescence emission maxima before and after photoconversion. The analysis showed that some isoforms rapidly release Chlide after the reaction, while others retain the pigment in the binding pocket, especially at high NADPH concentrations. These results suggest that LPOR isoforms differ in their rates of Chlide release and complex disassembly, potentially influencing the overall rate of the Chl biosynthetic pathway, even in mature leaves. We further analyzed the flexibility of these isoforms using AlphaFold2 predictions, identifying four regions of the enzyme that are particularly mobile. Two of these regions are involved in pigment binding, while the other two play a role in oligomerization. Based on these findings, we propose a model of conformational changes that drive the formation of LPOR oligomers.

biochemistry↗

bioRxiv2024

LPOR and the membranes - evolutionary pathway towards prolamellar body formation

Light-dependent protochlorophyllide oxidoreductase (LPOR) has captivated the interest of the research community for decades. One reason is the photocatalytic nature of the reaction catalyzed by the enzyme, and the other is the involvement of LPOR in the formation of a paracrystalline lattice called a prolamellar body (PLB) that disintegrates upon illumination, initiating a process of photosynthetic membrane formation. In this paper, we have integrated three traditional methods previously employed to study the properties of the enzyme to investigate how LPOR evolved and how PLB forms. We found that in cyanobacteria, LPOR activity appears to be independent of lipids, with membrane interaction primarily affecting the enzyme post-reaction, with MGDG and PG having opposite effects on SynPOR. In contrast, plant isoforms exhibit sequence alterations, rendering the enzyme effective in substrate binding mainly in the presence of anionic lipids, depending on residues at positions 122, 312, and 318. Moreover, we demonstrated that the interaction with MGDG could initially serve as enhancement of the substrate specificity towards monovinyl-protochlorophyllide (Pchlide). We have shown that the second LPOR isoforms of eudicots and monocots accumulated mutations that made these variants less and more dependent on anionic lipids, respectively. Finally, we have shown that in the presence of Pchlide, NADP+, and the lipids, plant but not cyanobacterial LPOR homolog remodel membranes into the cubic phase. The cubic phase is preserved if samples supplemented with NADP+ are enriched with NADPH. The results are discussed in the evolutionary context, and the model of PLB formation is presented. SignificanceLPOR is a unique enzyme with photocatalytic properties, developed by cyanobacteria and inherited by algae and plants. In this study, we investigated the properties of the cyanobacterial homolog, revealing that two lipids, PG and MGDG, have opposite effects on enzyme activity. Additionally, we identified mutations in plant isoforms that render the enzyme dependent on anionic lipids. Moreover, we demonstrated that in the presence of NADP+, the plant homolog remodels lipids into a cubic phase, which appears to be the initial step of prolamellar body (PLB) formation. PLB is a unique paracrystalline arrangement of lipids and proteins found in immature chloroplasts, which disintegrates upon illumination, initiating photosynthetic membrane formation.

biochemistry↗