Jiro Harada

Background/Objectives: Carotenoids play essential roles in light harvesting, photoprotection, and oxidative stress defense in photosynthetic organisms. The filamentous anoxygenic phototroph Chloroflexus aurantiacus synthesizes diverse carotenoids; however, the biosynthetic basis and regulation of these pigments are unclear. Methods: Carotenoid compositions of Cfx. aurantiacus grown under various conditions were analyzed by HPLC. Furthermore, candidate carotenoid biosynthesis genes from Cfx. aurantiacus were heterologously expressed in Escherichia coli to analyze their functions. Results: We examined carotenoid composition in cells grown under anaerobic light, aerobic dark, and aerobic light conditions, and found that the major carotenoid species changed markedly in response to oxygen availability. Under anaerobic photosynthetic conditions, γ-carotene, β-carotene, and hydroxylated γ-carotene derivatives were predominant, whereas aerobic growth induced the accumulation of keto-carotenoids, including canthaxanthin and echinenone-related compounds. Genome mining revealed candidate carotenoid biosynthesis genes, including three crtI paralogs and genes encoding enzymes involved in cyclization, hydroxylation, glucosylation, esterification, and ketolation. Heterologous expression in E. coli revealed that CrtI1 acts as a phytoene desaturase, CrtY catalyzes cyclization of lycopene to β-carotene via γ-carotene, CruF functions as a 1,2-hydratase, CruC as a carotenoid glucosyltransferase, CruD as an acyltransferase involved in esterification, and CrtO as a β-carotene ketolase. Conclusions: These results establish a carotenoid biosynthetic pathway in Cfx. aurantiacus and show that oxygen-dependent transcriptional regulation, particularly of CrtO, contributes to the selective production of keto-carotenoids under aerobic conditions. Our findings provide new insight into carotenoid metabolism in filamentous anoxygenic phototrophs and the adaptive physiological strategies that support growth under contrasting environmental conditions.

Chlorosomes are the largest known photosynthetic light-harvesting antennas, yet unlike protein-based antennas, they lack protein scaffolds that organize pigment molecules and instead contain self-assembled tubular and lamellar bacteriochlorophyll aggregates. How these antennas achieve directional and efficient energy transfer has remained unresolved. By applying ultrafast transient absorption spectroscopy to individual wild-type and mutant chlorosomes, we resolved six kinetic components obscured by ensemble averaging and assigned each to either lamellar or tubular aggregates. Lamellar aggregates expand light-harvesting capacity, whereas tubular aggregates serve as the primary energy donors to the baseplate. Such structural heterogeneity is therefore not merely suppressed but tuned to balance light-harvesting capacity with robust energy delivery. These findings reveal a division-of-labor strategy among pigment aggregates for efficient light harvesting without protein scaffolds.

Light-harvesting complexes (LHCs) play crucial roles in efficient photoenergy conversion and photoprotection of photosynthetic systems. In LHCs, functional pigments such as chlorophylls (Chls), bacteriochlorophylls (BChls), and carotenoids are sophisticatedly assembled with the help of polypeptides. The pigment assemblies in LHCs control the site-energy of each pigment, excitonic interactions among pigments, and excitation energy gradient in the protein matrix, as well as the formation and stability of the protein structure. In vitro reconstitution of LHCs is promising in understanding these structural and functional mechanisms of LHCs. In this review, we summarize two strategies of pigment reconstitution of LHCs; one is the formation of LHCs from a mixture of photosynthetic pigments and denatured polypeptides by their self-assembly, and the other is pigment substitution by the insertion of exogenous pigments into apoproteins partially lacking bound pigments. Next, we overview reconstitution studies of major LHC II derived from oxygenic photosynthetic organisms and core and peripheral antenna proteins of purple photosynthetic bacteria. Here, we focus on substituting Chls and BChls, key pigments in photosynthesis, in LHCs by the reconstitution. (B)Chl reconstitution of LHCs has allowed us to change essential parameters for the pigment-protein interactions and photofunctions, deepening our understanding of the molecular basis of the efficient light-harvesting functions. Reconstitution of LHCs will also be helpful for the modification and design of pigment-protein complexes toward utilization of sunlight energy for global problems on agricultural productivity and bioenergy production.

Bacteriochlorophylls (BChls) c and e are responsible for the main part of the light-harvesting process in chlorosome antenna systems of green sulfur bacteria, and contain a methyl group at the peripheral C-20 position of their core chlorin rings. This study performed in vitro and in vivo analysis of the C-20 methyltransferase BchU derived from the green sulfur bacterium Chlorobaculum tepidum, which synthesizes BChl c, to clarify the role of this enzyme in the biosynthetic pathway. Although the reaction step of BchU in the biosynthesis could not be determined by genetic analysis, enzymatic assays using various substrates showed that BchU reacts primarily with substrates after hydration of BchF and BchV at the C-3 position. The results in this study allow the proposition of a biosynthetic pathway for BChl c and e involving this enzyme.

Geranylgeranyl reductase (GGR) encoded by the bchP gene catalyzes the reductions of three unsaturated C = C double bonds (C6 = C7, C10 = C11, and C14 = C15) in a geranylgeranyl (GG) group of the esterifying moiety in 17-propionate residue of bacteriochlorophyll (BChl) molecules. It was recently reported that GGR in Halorhodospira halochloris potentially catalyzes two hydrogenations, yielding BChl with a tetrahydrogeranylgeranyl (THGG) tail. Furthermore, its engineered GGR, in which N-terminal insertion peptides characteristic for H. halochloris were deleted, performed single hydrogenation, producing BChl with a dihydrogeranylgeranyl (DHGG) tail. In some of these enzymatic reactions, it remained unclear in which order the C = C double bond in a GG group was first reduced. In this study, we demonstrated that the (variant) GGR from H. halochloris catalyzed an initial reduction of the C6 = C7 double bond to yield a 6,7-DHGG tail. The intact GGR of H. halochloris catalyzed the further hydrogenation of the C14 = C15 double bonds to give a 6,7,14,15-THGG group, whereas deleting the characteristic peptide region from the GGR suppressed the C14 = C15 reduction. We also verified that in a model bacterium, Blastochloris viridis producing standard BChl-b, the reduction of a GG to phytyl group occurred via 10,11-DHGG and 6,7,10,11-THGG. The high-performance liquid chromatographic elution profiles of BChls-a/b employed in this study are essential for identifying the regioisomeric diterpenoid tails in the BChls of phototrophic bacteria distributed in nature and elucidating GGR enzymatic reactions.

Chlorosomes in green photosynthetic bacteria are the largest and most efficient light-harvesting antenna systems of all phototrophs. The core part of chlorosomes consists of bacteriochlorophyll c, d, or e molecules. In their biosynthetic pathway, a BciC enzyme catalyzes the removal of the C13²-methoxycarbonyl group of chlorophyllide a. In this study, the in vitro enzymatic reactions of chlorophyllide a analogues, C13²-methylene- and ethylene-inserted zinc complexes, were examined using a BciC protein from Chlorobaculum tepidum. As the products, their hydrolyzed free carboxylic acids were observed without the corresponding demethoxycarbonylated compounds. The results showed that the in vivo demethoxycarbonylation of chlorophyllide a by an action of the BciC enzyme would occur via two steps: (1) an enzymatic hydrolysis of a methyl ester at the C13²-position, followed by (2) a spontaneous (nonenzymatic) decarboxylation in the resulting carboxylic acid.

Green sulfur bacteria have light‐harvesting antenna systems, called chlorosomes, which usually contain one of bacteriochlorophyll (BChl) c, d, and e molecules depending on the bacterial strain. Additionally, BChl f has never been found in nature but was observed in chlorosomes of the constructed mutant. In this study, we used the brown‐colored green sulfur bacterium Chlorobaculum limnaeum RK‐j‐1 strain possessing BChl e, and constructed its mutants accumulating only either BChl c, d, or f. In the mutant cells, these pigments showed different electronic absorption spectra, and their Qy peaks shifted hypsochromically in the order of BChls c, d, e, and f. The energy transfer from the chlorosomal aggregates to BChl a in these mutant cells was observed at 77 K by using picosecond time‐resolved fluorescence measurements. According to the Förster energy‐transfer mechanism, the energy‐transfer efficiency from the chlorosomal aggregates to BChl a decreased in the order of BChl c, BChl d, BChl e to BChl f.

A BciC enzyme is related to the removal of the C13²-methoxycarbonyl group in biosynthesis of bacteriochlorophylls (BChls) c, d and e functioning in green sulfur bacteria, filamentous anoxygenic phototrophs and phototrophic acidobacteria. These photosynthetic bacteria have the largest and the most efficient light-harvesting antenna systems, called chlorosomes, containing unique self-aggregates of BChl c, d or e pigments, that lack the C13²-methoxycarbonyl group which disturbs chlorosomal self-aggregation. In this study, we characterized the BciC derived from the green sulfur bacterium Chlorobaculum tepidum, and examined the in vitro enzymatic activities of its recombinant protein. The BciC-catalyzing reactions of various substrates showed that the enzyme recognized chlorophyllide (Chlide) a and 3,8-divinyl(DV)-Chlide a as chlorin substrates to give 3-vinyl-bacteriochlorophyllide (3V-BChlide) d and DV-BChlide d, respectively. Since the BciC afforded a higher activity with Chlide a than that with DV-Chlide a and no activity with (DV-)protoChlides a (porphyrin substrates) and 3V-BChlide a (a bacteriochlorin substrate), this enzyme was effective for diverting the chlorosomal pigment biosynthetic pathway at the stage of Chlide a away from syntheses of other pigments such as BChl a and Chl a. The addition of methanol to the reaction mixture did not prevent the BciC activity, and we identified this enzyme as Chlide a demethoxycarbonylase, not methylesterase.

Summary: Photosynthetic green sulfur bacteria inhabit anaerobic environments with very low‐light conditions. To adapt to such environments, these bacteria have evolved efficient light‐harvesting antenna complexes called as chlorosomes, which comprise self‐aggregated bacteriochlorophyll c in the model green sulfur, bacterium Chlorobaculum tepidum. The pigment possess a hydroxy group at the C3¹ position that produces a chiral center with R‐ or S‐stereochemistry and the C3¹‐hydroxy group serves as a connecting moiety for the self‐aggregation. Chlorobaculum tepidum carries the two possible homologous genes for C3‐vinyl hydratase, bchF and bchV. In the present study, we constructed deletion mutants of each of these genes. Pigment analyses of the bchF‐inactivated mutant, which still has BchV as a sole hydratase, showed higher ratios of S‐epimeric bacteriochlorophyll c than the wild‐type strain. The heightened prevalence of S‐stereoisomers in the mutant was more remarkable at lower light intensities and caused a red shift of the chlorosomal Q_y absorption band leading to advantages for light‐energy transfer. In contrast, the bchV‐mutant possessing only BchF showed a significant decrease of the S‐epimers and accumulations of C3‐vinyl BChl c species. As trans‐ criptional level of bchV was upregulated at lower light intensity, the Chlorobaculum tepidum adapted to low‐light environments by control of the bchV transcription.