Chlorophyll (Chl) biosynthesis, crucial to life on Earth, is tightly regulated because its precursors are phototoxic 1 . In flowering plants, the enzyme Light-dependent Protochlorophyllide OxidoReductase (LPOR) captures photons to catalyze the penultimate reaction: the reduction of a double-bond within protochlorophyllide (Pchlide) to generate chlorophyllide (Chlide) 2,3 . In darkness, LPOR oligomerizes to facilitate photon energy transfer and catalysis 4,5 . However, the complete 3D structure of LPOR, the higher-order architecture of LPOR oligomers, and the implications of these self-assembled states for catalysis, including how LPOR positions Pchlide and the cofactor NADPH, remain unknown. Here we report the atomic structure of LPOR assemblies by electron cryo-microscopy (cryoEM). LPOR polymerizes with its substrates into helical filaments around constricted lipid bilayer tubes. Portions of LPOR and Pchlide insert into the outer membrane leaflet, targeting the product, Chlide, to the membrane for the final reaction site of chlorophyll biosynthesis. In addition to its crucial photocatalytic role, we show that in darkness LPOR filaments directly shape membranes into high-curvature tubules with the spectral properties of the prolammelar body, whose light-triggered disassembly provides lipids for thylakoid assembly. Our structure of the catalytic site, moreover, challenges previously proposed reaction mechanisms 6 . Together, our results reveal a new and unexpected synergy between photosynthetic membrane biogenesis and chlorophyll synthesis in plants orchestrated by LPOR.

This Current Topic focuses on light-dependent protochlorophyllide oxidoreductase (POR, EC 1.3.1.33). POR catalyzes the penultimate reaction of chlorophyll biosynthesis, i.e., the light-triggered reduction of protochlorophyllide to chlorophyllide. In this reaction, the chlorin ring of the chlorophyll molecule is formed, which is crucial for photosynthesis. POR is one of very few enzymes that are driven by light; however, it is unique in the need for its substrate to absorb photons to induce the conformational changes in the enzyme, which are required for its catalytic activation. Moreover, the enzyme is also involved in the negative feedback of the chlorophyll biosynthesis pathway and controls chlorophyll content via its light-dependent activity. Even though it has been almost 70 years since the first isolation of active POR complexes, our knowledge of them has markedly advanced in recent years. In this review, we summarize the current state of knowledge of POR, including the phylogenetic roots of POR, the mechanisms of the regulation of POR genes expression, the regulation of POR activity, the import of POR into plastids, the role of POR in PLB formation, and the molecular mechanism of protochlorophyllide reduction by POR. To the best of our knowledge, no previous review has compiled such a broad set of recent findings about POR.

Light-dependent protochlorophyllide oxidoreductase (POR) is a plant enzyme involved in the chlorophyll biosynthesis pathway. POR reduces one of the double bonds of the protochlorophyllide (Pchlide) using NADPH and light. In the present study, we found out that phosphatidylglycerol and sulfoquinovosyl diacylglycerol are allosteric regulators of the nucleotide binding, which increase the affinity towards NADPH a 100-fold. Moreover, we showed for the first time that NADH can, like NADPH, form active complexes with Pchlide and POR, however, at much higher concentrations. Additionally, monogalactosyldiacylglycerol (MGDG) was shown to be the main factor responsible for the red shift of the fluorescence emission maximum of Pchlide:POR:NADPH complexes. Importantly, the emission maximum at 654 nm was obtained only for the reaction mixtures supplemented with MGDG and at least one of the negatively charged plant lipids. Moreover, the site-directed mutagenesis allowed us to identify amino acid residues that may be responsible for lipid binding and Pchlide coordination. Our experiments allowed us to identify six different Pchlide:POR complexes that differ in the fluorescence emission maxima of the pigment. The results presented here reveal the contribution of thylakoid lipids in the regulation of the chlorophyll biosynthesis pathway; however, the molecular mechanisms of this process are to be clarified.

Photoactive Pchlide-POR-NADPH complexes were reconstituted using protochlorophyllide (Pchlide) and recombinant light-dependent protochlorophyllide oxidoreductase (POR) proteins, His₆-PORA, His₆-PORB and His₆-PORC, from Arabidopsis thaliana. We did not observe any differences in the kinetics of the protochlorophyllide photoreduction at room temperature among the PORA, PORB and PORC proteins. In contrast, the PORC protein showed lower yield of Chlide formation than PORA and PORB when preincubated in the dark for 30 min and then illuminated for a short time. The most significant observation was that reconstituted Pchlide-POR-NADPH complexes showed fluorescence maxima at 77 K similar to those observed for highly aggregated Pchlide-POR-NADPH complexes in prolamellar bodies (PLBs) in vivo. Homology models of PORA, PORB and PORC of Arabidopsis thaliana were developed to compare predicted structures of POR isoforms. There were only slight structural differences, mainly in the organisation of helices and loops, but not in the shape of whole molecules. This is the first comparative analysis of all POR isoforms functioning at different stages of A. thaliana development.

Light-dependent protochlorophyllide oxidoreductase (LPOR) catalyzes the reduction of protochlorophyllide to chlorophyllide, which is a key reaction for angiosperm development. Dark operative light-independent protochlorophyllide oxidoreductase (DPOR) is the other enzyme able to catalyze this reaction, however, it is not present in angiosperms. LPOR, which evolved later than DPOR, requires light to trigger the reaction. The ancestors of angiosperms lost DPOR genes and duplicated the LPORs, however, the LPOR evolution in angiosperms has not been yet investigated. In the present study, we built a phylogenetic tree using 557 nucleotide sequences of LPORs from both bacteria and plants to uncover the evolution of LPOR. The tree revealed that all modern sequences of LPOR diverged from a single sequence ∼1.36 billion years ago. The LPOR gene was then duplicated at least 10 times in angiosperms, leading to the formation of two or even more LPOR isoforms in multiple species. In the case of Arabidopsis thaliana, AtPORA and AtPORB originated in one duplication event, in contrary to the isoform AtPORC, which diverged first. We performed biochemical characterization of these isoforms in vitro, revealing differences in the lipid-driven properties. The results prone us to hypothesize that duplication events of LPOR gave rise to the isoforms having different lipid-driven activity, which may predispose them for functioning in different locations in plastids. Moreover, we showed that LPOR from Synechocystis operated in the lipid-independent manner, revealing differences between bacterial and plant LPORs. Based on the presented results, we propose a novel classification of LPOR enzymes based on their biochemical properties and phylogenetic relationships.