Mobility of four structural regions drives isoform-specific properties of photoenzyme LPOR in plants

Abstract

Light-dependent protochlorophyllide oxidoreductase (LPOR) is a photocatalytic enzyme in the chlorophyll 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, having diverse phylogenetic backgrounds, 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 chlorophyll 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.

Keywords: in vitro; light-dependent protochlorophyllide oxidoreductase; photoenzyme; photomorphogenesis; recombinant proteins.

Figure 1

Phylogeny ofLPOR isoforms in selected plant species. The colors emphasize distinct clades of LPOR, reflecting their phylogenetic relationships. A, rooted phylogenetic tree of seed plants. B, the simplified phylogenetic tree showing the origin of multiple isoforms in selected plant species. LPOR, light-dependent protochlorophyllide oxidoreductase.

Figure 2

The composition of the reaction mixture affects the emission maxima of Pchlide and Chlide.A and C, normalized fluorescence emission spectra of reaction mixtures with Pchlide (A) and Chlide (C), along with their emission maxima. Shaded areas represent the SD between replicates. B and D, fluorescence emission maxima of complexes with Pchlide (B) and Chlide (E) in the presence of NADP+ (left panels) or NADPH (right panels) for 13 plant LPOR isoforms, measured in a lipid-free buffer, with PG, or with OPT lipids. E and F, distribution of the emission maxima of Pchlide (C) and Chlide (F) shown in panels B and E. The overall distribution of data points for each pair of pigment and dinucleotide is shown in gray. A–F, the reaction mixtures contained 15 μM LPOR, 5 μM pigment, 200 μM dinucleotide, and 400 μM lipids in various combinations. See Materials and methods for details on the sample preparation. LPOR, light-dependent protochlorophyllide oxidoreductase; Pchlide, protochlorophyllide; PG, phosphatidyl glycerol; Chlide, chlorophyllide.

Figure 3

Low-temperature fluorescence emission spectra enable monitoring of NADPH binding and product formation.A, normalized spectra of reaction mixtures of HaPOR3 containing 5 μM Pchlide, 15 μM HaPOR3, with or without the addition of OPT lipids, and varying NADPH concentrations (indicated next to the spectra), measured before (dark) and after illumination (illuminated). Shaded areas represent the SD between replicates. B and C, relationship between NADPH concentration and: (B) the intensity ratio of 648/635, (C) the relative intensities of the generated product (F Chlide), and the remaining substrate (F Pchlide). A fit of a modified Michaelis–Menten equation is shown, along with the graphical representation of K_M^D (B) or K_M^L (C). Spectra corresponding to the given F648/F635 values (B) or F Chlide/F Pchlide values (C) are shown on the right side of the plot. D, relationship between K_M^D and K_M^L for investigated isoforms. The overall distribution of data points is shown in gray. Error bars represent the uncertainty associated with the fitted constant K_M^x. Closed symbols represent constants determined for samples not supplemented with lipids, while open symbols these determined in the presence of 400 μM OPT lipids. The values of the constants are presented in Table S1. LPOR, light-dependent protochlorophyllide oxidoreductase; Pchlide, protochlorophyllide; Chlide, chlorophyllide.

Figure 4

Lipids affect the spectrum of the LPOR ternary complex.A, normalized spectra of reaction mixtures containing 5 μM Pchlide, 15 μM HaPOR3, 200 μM NADPH, and varying concentrations of OPT (indicated next to the spectra). B, relationship between the base-10 logarithm of the intensity ratio (658/631) and OPT concentration. Spectra corresponding to the given log (F658/F631) values are shown on the right side of the plot. Shaded areas represent the SD between replicates. The overall distribution of data points is shown in gray. C, the relationship between OPT lipid concentration and the relative fluorescence intensity of PinPOR complexes with 200 μM NADPH (658/631, green series, left axis) or with NADP+ (647/631, purple series, right axis). A gray horizontal line labeled “Pchlide” represents the 658/631 and 647/631 ratios for pure Pchlide in buffer. Shaded areas represent the SD between replicates. D, normalized spectra of reaction mixtures containing 5 μM Pchlide, 15 μM AtPORB, 200 μM NADPH, and either 100 μM OPT lipids or a lipid mixture with low MGDG concentration (lowMGDG: 2.5 mol% MGDG, 47.5 mol% PG, 50 mol% DGDG). E and F, negative-stain transmission electron microscopy micrographs of reaction mixtures containing 5 μM Pchlide, 15 μM AtPORB, 200 μM NADPH, and either 100 μM lowMGDG lipid mixture (E) or OPT lipids (F). The presented structures are typical and frequently observed for each composition of the reaction mixture. DGDG, digalactosyl diacylglycerol; LPOR, light-dependent protochlorophyllide oxidoreductase; MGDG, monogalactosyl diacylglycerol; Pchlide, protochlorophyllide; PG, phosphatidyl glycerol.

Figure 5

Relationship between emission maxima of Pchlide and Chlide generated after illumination of the reaction mixture.A, emission maxima of samples ([NADPH] of at least 1 μM), measured before and after 20 s of illumination, for 13 different LPOR isoforms. Marker color and style correspond to those used in Figure 3. The distribution of data points is shown on the top and right axes. B, distribution of emission maxima for Pchlide (left panels, yellow bars) and the corresponding maxima of Chlide generated after 20 s of illumination (right panels, yellow bars). Three subsets of Pchlide emissions were selected based on maxima at 637, 648, and 655 nm (±2.5 nm). The overall distribution of data points is shown in gray. C and D, distribution of emission maxima for Pchlide (C) and Chlide generated after 20 s of illumination (D), determined for the samples ([NADPH] of at least 1 μM) presented in Figures 3 and 4, for each analyzed isoform. The arrangement of the isoforms in (D) is the same as in (C). The maxima of the reaction mixtures containing lipids are shown in the darker shade. Chlide, chlorophyllide; LPOR, light-dependent protochlorophyllide oxidoreductase; Pchlide, protochlorophyllide.

Figure 6

LPOR in plants has flexible regions.A, root mean square fluctuation of alpha carbon atoms (flexibility) of the AtPORB predicted ensemble, calculated from 480 independent AF2 predictions, after alpha carbon alignment with the reference structure of LPOR (top panel). Variability was calculated using the previously published dataset (8) of 194 LPOR sequences of seed plants and it represents the percentage of sequences that do not share the most common residue at a given position. The four identified flexible regions are highlighted in pink: Pchlide loop (230–242), oligomerization interface I (256–263), helix α10 (316–338), and oligomerization interface II (367–375). Residues involved in dimer formation are highlighted in blue. B and C, four flexible regions of LPOR (pink) highlighted on the LPOR structure of a monomer (B) and dimer (C), extracted from the cryo-EM structure of the LPOR oligomer. Residues involved in dimer formation are highlighted in blue. LPOR, light-dependent protochlorophyllide oxidoreductase; Pchlide, protochlorophyllide.

Figure 7

LPOR isoforms differ in the conformations that the flexible regions can adopt.A, representative conformations determined for AtPORB. The reference structure (black) was extracted from the cryo-EM structure of the LPOR oligomer, while the alterative conformations were predicted with AF2. B, distribution of RMSD calculated for the alpha carbon atoms of four flexible regions between the predicted conformations and the reference structure of AtPORB (PDB: 7JK9). Median and the range of the first and third quadruple are marked with a white dot and white lines. AF2, AlphaFold2; LPOR, light-dependent protochlorophyllide oxidoreductase.

Figure 8

The model of conformational changes of LPOR leading to the oligomerization. See the main text for a detailed explanation. LPOR, light-dependent protochlorophyllide oxidoreductase.