Dual role of the active site ‘lid’ regions of protochlorophyllide oxidoreductase in photocatalysis and plant development

Abstract

Protochlorophyllide oxidoreductase (POR) catalyses reduction of protochlorophyllide (Pchlide) to chlorophyllide, a light-dependent reaction of chlorophyll biosynthesis. POR is also important in plant development as it is the main constituent of prolamellar bodies in etioplast membranes. Prolamellar bodies are highly organised, paracrystalline structures comprising aggregated oligomeric structures of POR-Pchlide-NADPH complexes. How these oligomeric structures are formed and the role of Pchlide in oligomerisation remains unclear. POR crystal structures highlight two peptide regions that form a 'lid' to the active site, and undergo conformational change on binding Pchlide. Here, we show that Pchlide binding triggers formation of large oligomers of POR using size exclusion chromatography. A POR 'octamer' has been isolated and its structure investigated by cryoelectron microscopy at 7.7Å resolution. This structure shows that oligomer formation is most likely driven by the interaction of amino acid residues in the highly conserved lid regions. Computational modelling indicates that Pchlide binding stabilises exposure of hydrophobic surfaces formed by the lid regions, which supports POR dimerisation and ultimately oligomer formation. Studies with variant PORs demonstrate that lid residues are involved in substrate binding and photocatalysis. These highly conserved lid regions therefore have a dual function. The lid residues position Pchlide optimally to enable photocatalysis. Following Pchlide binding, they also enable POR oligomerisationa process that is reversed through subsequent photocatalysis in the early stages of chloroplast development.

Molecular landscape of etioplast inner membranes in higher plants

“…5e). In cyanobacterial LPOR, the corresponding residues were suggested to be part of a large hydrophobic patch that plays a role in catalytic activity, protochlorophyllide binding and the formation of higher-order assemblies 48 .…”

“…Policy information about availability of computer code Data collection SerialEM v3.6 (described in: Mastronarde, D. N. 2005. J Struct Biol, 152: [36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51].…”

Photocatalysis as the ‘master switch’ of photomorphogenesis in early plant development

“…The size, shape and architecture of PLBs is known to be determined by the structural properties of the LPOR protein. These include its ability to form oligomers, 62,[65][66][67][68][69][70][71] the association of LPOR with the membrane 71,72 and the interaction of the enzyme with lipids. 44,59,71 The propensity of LPOR to form large, substrate-bound, PLB aggregates in the etioplastand potentially during the greening processwas first observed over 30 years ago 65 and has generated much interest in understanding this process in vitro.…”

“…44,59,71 The propensity of LPOR to form large, substrate-bound, PLB aggregates in the etioplastand potentially during the greening processwas first observed over 30 years ago 65 and has generated much interest in understanding this process in vitro. 62,[66][67][68][69][70][71] LPORs from different organisms can adopt a variety of soluble oligomeric forms in the absence of substrates, ranging from monomers in cyanobacterial LPORs 66,67,70 through to higher order oligomers in plant enzymes. 62,68,69 In all cases, formation of a LPOR-Pchlide-NADPH ternary complex leads to formation of larger multimeric complexes (Figure 3).…”

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 ).…”