Photocatalytic LPOR forms helical lattices that shape membranes for chlorophyll synthesis

Abstract

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.

The Role of Membranes and Lipid-Protein Interactions in the Mg-Branch of Tetrapyrrole Biosynthesis

“…It has to be noted that the enzymatically active LPOR complexes have unique spectral and biochemical properties that are hardly reconstituted in vitro , especially in the absence of lipids (Gabruk et al, 2017 ). However, recent in vitro reconstitution studies successfully yielded crystal structures of oligomers: in case of cyanobacterial LPOR octamers were reported (Zhang et al, 2021 ) while in case of Arabidopsis LPOR helical structures associated with lipids were observed (Nguyen et al, 2021 ).…”

“…Data indicate the role of carotenoids [zeaxanthin and violaxanthin (Ouazzani Chahdi et al, 1998 ); neoxanthin and violaxanthin (Bykowski et al, 2020 ); the accumulation of poly-cis xanthophylls (Park et al, 2002 ; Cuttriss et al, 2007 )] and lipids [MGDG (Aronsson et al, 2008 ; Fujii et al, 2017 ); and MGDG, PG, and SQDG (Gabruk et al, 2017 ; Nguyen et al, 2021 )] in the formation of the photoactive enzyme complexes and the PLBs. Similarly, carotenoids (Denev et al, 2005 ) were suggested to be involved in the membrane association of LPOR.…”

“…Lipid biosynthesis mutants had hindered LPOR activity, no LPOR oligomerization and abnormal PLB formation (Fujii et al, 2017 , 2018 , 2019b ) which again outlines the strong relation between the LPOR oligomers, plastid lipids and inner membrane structures. Cryo electron microscopic investigations on in vitro reconstituted LPOR revealed that LPOR oligomers form helical filaments around lipid bilayer tubes and are inserted in the outer leaflet of the membranes (Nguyen et al, 2021 ).…”

SPIRE, Surface Projection Image Recognition Environment for bicontinuous phases: application for plastid cubic membranes

“…The number of PLB building blocks play a crucial role in maintaining its structure, including protochlorophyllide:light-dependent protochlorophyllide oxidoreductase:NADPH complex as well as particular galactolipids and carotenoids (Floris and Kühlbrandt, 2021;Bykowski et al, 2020;Cazzonelli et al, 2020;Franck et al, 2000;Fujii et al, 2019;Nguyen et al, 2021;Sperling et al, 1998). Although the role of light-dependent protochlorophyllide oxidoreductase in membrane tubulation was proven recently using electron cryo-tomography techniques (Nguyen et al, 2021;Floris and Kühlbrandt, 2021), factors governing the transition of tubular arrangements into cubic configuration remain elusive (Wietrzynski and Engel, 2021). The role of already recognized structure-remodeling proteins of plastid membranes (CURT1, VIPP1) in this process cannot be excluded and require further investigation (Armbruster et al, 2013;Gupta et al, 2020).…”

“…The cubic arrangement of the PLB is characterized by an imbalanced proportion of water channels and a relatively small length-scale compared to other naturally-occurring cubic membranes; a single membrane separates both sides of the structure. Note that recent results indicated that PLB membrane is densely decorated with light-dependent protochlorophyllide oxidoreductase protein, which role in membrane tubulation has been proven in in vitro studies (Nguyen et al, 2021;Floris and Kühlbrandt, 2021). Apart from marked elements, the presented photographs, and electron micrographs are not shown to scale; mesophyll cell and etioplast are free-form selected manually from Transmission Electron Microscopy (TEM) images of etiolated oat leaves.…”