Structure of chlorophyll synthase in complex with the LHC-like protein HliD

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Questions to the authors

1. Throughout your paper you mention how the sequence and structure of the Hlips (high-light-inducible proteins) – in cyanobacteria – that you have solved is similar to the light-harvesting complexes of algae and plants. Can you comment on this from an evolutionary and biochemical point of view – why are there such similarities?

Plant and algal light-harvesting complex (LHC) proteins are used for the collection of light; however, they are also responsible for energy dissipation to protect the photosynthetic apparatus from damage under stress conditions. Evolutionarily, LHC proteins are thought to be descendants of cyanobacterial Hlips. Structurally, the single transmembrane (TM) helix of Hlips corresponds to the first and third TM helices of LHC proteins, suggesting that LHC proteins may have arisen from Hlips through gene duplication or fusion.

Hlips, like LHC proteins, contain a conserved ExxH/NxR-type chlorophyll a (Chl a)-interaction motif and bind a carotenoid quencher, positioning the chlorophylls and carotenoids in a characteristic geometry. This shared arrangement suggests a common mechanism of Chl a quenching by carotenoids in Hlips and LHC proteins (Staleva et al., Nat. Chem. Biol., 2015, 11:287–291). Interestingly, the identity of the carotenoid quencher can vary among different LHC proteins and among Hlips, as well as the orientation of Chl a2 in the HliD dimer is flipped and rotated relative to the corresponding Chl 603/612 in the LHC-II protein. Future studies will elucidate the specific structural determinants of Chl a quenching by Hlips, as well as the specific roles of different Hlip species in cyanobacteria.

2.You have identified various molecules bound to your structure including Zeaxanthin and Myxoxanthophyll. It was stated that the role of Zeaxanthin was to provide structural support and to act as a ‘glue’, but the role of Myxoxanthophyll is not clear. Based on the structural information you have acquired, why is this binding partner essential and what further steps need to be taken to uncover its role?

Although myxoxanthophylls are major carotenoid glycosides present in cyanobacteria, their role remains largely speculative. In Synechocystis sp. PCC 6803, myxoxanthophyll is present in the thylakoid membrane and has also been linked to the biogenesis of the outer cell wall. It is generally believed that this carotenoid, which accumulates under stress conditions, acts as an antioxidant and can shield the cell by absorbing light in the outer membrane. Interestingly, myxoxanthophyll has never previously been found as a protein cofactor; our structure represents the first such case.

Based on our previously published data (Proctor et al., Biochem. J., 2020, 477:4021), we know that myxoxanthophyll is not essential for the function of chlorophyll synthase as chlorophyll biosynthesis apparently works in its absence. We speculate that the role of myxoxanthophyll in the chlorophyll synthase complex is to quench a potential triplet state of newly synthetized chlorophyll immediately after its release from the catalytic site. The next step is to disrupt the interaction between chlorophyll synthase and myxoxanthophyll and analyse the phenotype of the resulting strain.

3. The work presented in your paper adds to the knowledge of how organisms are able to produce chlorophyll, which as stated is the major-light absorbing pigment on Earth. Can this knowledge be applied to harvest energy from the sun more efficiently?

Understanding the mechanism of chlorophyll biosynthesis, and its coupling to photoprotection by HliD, has several biotechnological applications relevant to harvesting light energy. First, our structural data can guide the engineering of ChlG variants with altered activity that not only synthesise chlorophyll but also bacteriochlorophyll pigments absorbing near-infrared wavelengths. This would be a crucial step for the engineering of infrared light-driven photosynthesis in eukaryotes. In addition, the structure of HliD is very important for our future studies of energy dissipation in LHC complexes. This paves the way for the design of more durable, photoprotected pigment-protein systems that can better resist photodamage under high-light conditions. Finally, since ChlG is essential for chlorophyll production in all oxygenic phototrophs, the active-site residues we identified as required for substrate binding and catalysis are attractive targets for the design of new herbicides. Such specific herbicides could potentially help combat harmful algal blooms, which disrupt aquatic ecosystems.

References

1. Staleva, H., Komenda, J., Shukla, M. et al. Mechanism of photoprotection in the cyanobacterial ancestor of plant antenna proteins. Nat Chem Biol 11, 287–291 (2015). https://doi.org/10.1038/nchembio.1755

2.Matthew S. Proctor, Marek Pazderník, Philip J. Jackson, Jan Pilný, Elizabeth C. Martin, Mark J. Dickman, Daniel P. Canniffe, Matthew P. Johnson, C. Neil Hunter, Roman Sobotka, Andrew Hitchcock; Xanthophyll carotenoids stabilise the association of cyanobacterial chlorophyll synthase with the LHC-like protein HliD. Biochem J 30 October 2020; 477 (20): 4021–4036. doi: https://doi.org/10.1042/BCJ20200561