Structure of biological solar energy converters – further revelations

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genes in the tocopherol biosynthetic pathway are conserved between cyanobacteria and plants has yet to be determined.

metabolic engineering can be used to have a positive impact on human nutrition and health on a global scale.

Engineering tocopherol composition with methyl transferases A major goal of dissecting tocopherol biosynthesis in model photosynthetic organisms such as Arabidopsis and Synechocystis PCC6803 is to use the knowledge and genes obtained to engineer the pathway, first in model organisms and ultimately in agricultural crops. Various groups have reported engineering tocopherol content composition in Arabidopsis leaves and seeds by overexpression of various pathway enzymes [6,8,10,15–19]. Alison Van Eennemaan et al. have now applied the genes and information gleaned from studies of the Arabidopsis VTE3 and VTE4 to engineer the a-tocopherol composition of a major oilseed crop, soybean. Seed-specific expression of Arabidopsis VTE3 and VTE4 alone or together did not significantly alter the total level of tocopherols in transgenic soybean seed but had a dramatic impact on tocopherol composition [19]. Overexpression of VTE3 alone increases soybean seed g- and a-tocopherol levels and correspondingly reduces the levels of d- and b-tocopherols. Like overexpression of VTE4 in Arabidopsis seed [8], VTE4 overexpression in soybean seed converts g-tocopherol almost completely to a-tocopherol, a sevenfold increase to 75% of total, and d-tocopherol almost completely to b-tocopherol, a tenfold increase to 25% of total. Overexpression of VTE3 and VTE4 together shifted the tocopherol composition of soybean seeds from only 10% of a-tocopherol to w90% a-tocopherol. Because the Vitamin E activity of a-tocopherol is 2-, 10- and 33-fold that of b-, g-, d-tocopherols, respectively, the total Vitamin E activity of VTE3 and VTE4 overexpressors increased approximately fivefold relative to wild-type soybean without an increase in total tocopherol levels. The results of Van Eennemaan et al. and others [6,8,14,18,19] exemplify the potential for using model photosynthetic organism to dissect plant metabolic pathways of significance for human nutrition and then using the genes and knowledge obtained to engineer metabolism to improve human nutrition in a crop species. If one extends this logic, the stacking of VTE3 and VTE4 overexpression with overexpression of other transgenes (HPPD, VTE2) previously shown to increase total tocopherol content in Arabidopsis seed [10,14,8] is likely to lead to even higher levels of a-tocopherol in soybean seed. Regardless of the outcome, the demonstration that data obtained from engineering tocopherol synthesis in model systems can be readily transferred to crop plants indicates that we are on the cusp of an exciting era in which plant

References

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1 Traber, M.G. and Sies, H. (1996) Vitamin E in humans: demand and delivery. Annu. Rev. Nutr. 16, 321–347 2 Winklhofer-Roob, B.M. et al. (2003) Effects of vitamin E and carotenoid status on oxidative stress in health and disease. Evidence obtained from human intervention studies. Mol. Aspects Med. 24, 391–402 3 Girotti, A.W. (1998) Lipid hydroperoxide generation, turnover, and effector action in biological systems. J. Lipid Res. 39, 1529–1542 4 Brigelius-Flohe, R. and Traber, M. (1999) Vitamin E: function and metabolism. FASEB J. 13, 1145–1155 5 Azzi, A. et al. (2002) Non-antioxidant molecular functions of alphatocopherol (vitamin E). FEBS Lett. 519, 8–10 6 Savidge, B. et al. (2002) Isolation and characterization of homogentisate phytyltransferase genes from Synechocystis sp. PCC 6803 and Arabidopsis. Plant Physiol. 129, 321–332 7 Norris, S.R. et al. (1995) Genetic dissection of carotenoid synthesis in Arabidopsis defines plastoquinone as an essential component of phytoene desaturation. Plant Cell 7, 2139–2149 8 Shintani, D. and DellaPenna, D. (1998) Elevating the vitamin E content of plants through metabolic engineering. Science 282, 2098–2100 9 Schledz, M. et al. (2001) A novel phytyltransferase from Synechocystis sp. PCC 6803 involved in tocopherol biosynthesis. FEBS Lett. 499, 15–20 10 Collakova, E. and DellaPenna, D. (2001) Isolation and functional analysis of homogentisate phytyltransferase from Synechocystis sp. PCC 6803 and Arabidopsis. Plant Physiol. 127, 1113–1124 11 Porfirova, S. et al. (2002) Isolation of an Arabidopsis mutant lacking vitamin E and identification of a cyclase essential for all tocopherol biosynthesis. Proc. Natl. Acad. Sci. U. S. A. 99, 12495–12500 12 Cheng, Z. et al. (2003) Highly divergent methyltransferases catalyze a conserved reaction in tocopherol and plastoquinone synthesis in cyanobacteria and photosynthetic eukaryotes. Plant Cell 15, 2343–2356 13 Sattler, S.E. et al. (2003) Characterization of tocopherol cyclases from higher plants and cyanobacteria. Evolutionary implications for tocopherol synthesis and function. Plant Physiol. 132, 2184–2195 14 Collakova, E. and DellaPenna, D. (2003) Homogentisate phytyltransferase activity is limiting for tocopherol biosynthesis in Arabidopsis. Plant Physiol. 131, 632–642 15 Cahoon, E.B. et al. (2003) Metabolic redesign of vitamin E biosynthesis in plants for tocotrienol production and increased antioxidant content. Nat. Biotechnol. 21, 1082–1087 16 Rippert, P. et al. (2004) Engineering plant shikimate pathway for production of tocotrienol and improving herbicide resistance. Plant Physiol. 134, 92–100 17 Tsegaye, Y. et al. (2002) Over-expression of the enzyme p-hydoxyphenolpyruvate dioxygenase in Arabidopsis and its relation to tocopherol biosynthesis. Plant Physiol. Biochem. 40, 913–920 18 Van Eenennaam, A.L. et al. (2003) Engineering vitamin E content: from Arabidopsis mutant to soy oil. Plant Cell 15, 3007–3019 19 Rocheford, T.R. et al. (2002) Enhancement of vitamin E levels in corn. J. Am. Coll. Nutr. 21, 191S–198S 1360-1385/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2004.06.005

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Structure of biological solar energy converters – further revelations Jonathan H.A. Nugent and Mike C.W. Evans Department of Biology, University College London, Gower St, London, UK WC1E 6BT

Photosynthetic organisms harvest solar energy by absorbing light and ultimately transferring energy through a cascade of chemical reactions to power all cellular processes. Core components initiating this reaction cascade are the photosynthetic reaction centres Photosystem I and Photosystem II. Two recent publications on the structure of the reaction centres by Adam Ben-Shem et al. and Kristina Ferreira et al. represent a big step towards understanding the evolutionary development of the core energy conversion process and identifying the site of the water oxidation process, the source of atmospheric oxygen. In the initial stages of photosynthesis, light energy is converted efficiently to electrical energy, first by photochemical reactions and then by electron flow, which separates the negative and positive charges. These processes occur in multi-subunit membrane-spanning complexes termed reaction centres, which contain many cofactors and pigments. Oxygen-evolving photosynthetic organisms (cyanobacteria, algae and plants) contain two types of photosynthetic reaction centre, called Photosystem I (PSI) and Photosystem II (PSII). PSI and PSII provide environments in the thylakoid membrane in which several cofactors are placed at optimum distance and orientation, ensuring rapid efficient trapping and conversion of light energy. The cofactors responsible for the initial photochemistry are arranged in two almost symmetrical branches that span the membrane. The pseudo twofold symmetry extends to the polypeptides surrounding the core. The details of the cofactors and the relationship between reaction centres are discussed in Ref. [1]. New 3D X-ray structures are now available, which give further insight into these complex and fascinating systems [2,3]. Wider view of Photosystem I Prior to the work of Adam Ben-Shem et al. [3], all structures were of reaction centres isolated from prokaryotic organisms. The new eukaryotic structure for PSI illustrates both the extreme conservation of the basic energy conversion process and the variability of the light harvesting structures. Although at a significantly lower resolution than the cyanobacterial PSI structure of Jordan ˚ compared with 2.8 A ˚ , the almost total et al. [4], 4.4 A conservation of the protein and cofactor structure of the heterodimeric reaction centre core is clearly Corresponding author: Mike C.W. Evans ([email protected]). Available online 17 July 2004 www.sciencedirect.com

demonstrated. This result is not unexpected in view of the identity of the mechanism found in all PSI systems by spectroscopic analysis [5]. Outside this conserved region, two subunits, X and M, are found in cyanobacteria but not in plants, and two, H and G, of the four uniquely eukaryotic subunits have been resolved [3]. The overall structure is also different; the plant PSI is monomeric, in contrast with the normally trimeric cyanobacterial PSI. The crystallization of the plant complex with the light harvesting complex, LHC1, a standard component of eukaryotic systems that is lacking in cyanobacteria, is a major achievement, providing the first structure of LHC1. Four LHC1 subunits in two dimeric structures form a half moon belt on the sub-unit F side of the reaction centre (Figure 1). The LHC1 organization has no resemblance to the ring systems formed by purple bacterial light harvesting complexes, but is to some extent reminiscent of the ring structures formed by the IsaiA or Pcb light harvesting proteins in Prochlorophyta and iron-stressed cyanobacteria [6,7]. Some ‘bridging’ chlorophylls are located on LHC1 in the cleft between the light harvesting complexes and the PSI core suggesting a role in energy transfer and giving clear ‘routes’ for energy transfer. The new structure provides a strong basis for investigating the evolutionary development of light harvesting systems and energy transfer and indicates the remarkable conservation of the core energy conversion process. Clearer view of Photosystem II The PSII reaction centre is unique because it uses a Mn–Ca complex and light energy to oxidize water to protons and oxygen. Three laboratories have reported crystal structures of cyanobacterial PSII in the past three ˚ in 2001 [8], 3.7 A ˚ in 2003 [9] and now 3.5 A ˚ years, at 3.8 A in 2004 [2]. Although only a medium resolution has been obtained so far, the sequences of the polypeptide chains are known and are aiding model building. The latest structure from Kristina Ferreira and colleagues [2] represents a much bigger advance than the slight increase in resolution implies because the R-factor is much improved compared with the earlier structures. Most of the amino acids have also been assigned, leading to new positions for some of the smaller polypeptides and assigning others not resolved in the earlier structures. Most significantly, the resolution of ligands to cofactors, particularly Mn and Ca, has been improved, the position of Ca has been identified and a model structure has been proposed for the Mn–Ca cluster in the water-oxidizing complex (WOC). This represents

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˚ represented as a Figure 1. The structural model of plant Photosystem I (PSI) at 4.4 A Ca backbone. The four light-harvesting proteins are in green (Lhca1–Lhca4). Novel structural elements within the reaction centre that are not present in the cyanobacterial counterpart are coloured red; conserved features of the reaction centre are in grey. The three Fe4–S4 clusters are depicted as red (Fe) and green (S) balls. (a) View from the stromal side of the thylakoid membrane.

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of X-ray techniques places volumes of likely Mn and Ca density, allowing better modelling of 3 Mn and 1 Ca into the core of the cluster and a single Mn extending from it. The proposed Mn–Ca model (Figure 2) is a distorted cubane of 3 Mn and 1 Ca linked by oxo bridges, with a fourth Mn linked through oxygen to the Mn3–Ca. Many metal–amino acid ligands are identified, confirming proposals from mutagenesis studies. There are also some surprises, such as a ligand from the CP43 chlorophyll binding subunit. Ferreira et al. also propose a hydrophilic pathway from the thylakoid lumenal surface to the complex. As expected, TyrZ (D1 Tyr161), the cofactor transferring electrons into the reaction centre from the WOC, is close to the proposed Mn–Ca complex and hydrogen bonding to D1 His190 has been indicated. Although no substrate (water) is seen at this resolution, several ligands to the complex are still free as possible binding sites. The suggestion that a small unidentified ligand to the Mn–Ca cluster is bicarbonate brings interesting new possibilities. Bicarbonate has been proposed to act in the WOC [12] but evidence has until recently been limited. The WOC bicarbonate is proposed to be between Ca and the fourth Mn, with TyrZ nearby (Figure 2), making this region a candidate for the site of water oxidation. The possible roles of bicarbonate are intriguing. It might be a tridentate ligand that occupies substrate sites during some stages of WOC turnover [2]. Because bicarbonate is CO2 C water and can also act as a proton sink, source or carrier, it could be involved in transporting water to the site and in the mechanism of water oxidation itself. A glutamine, D1 Gln165, and arginine, CP43 Arg357, are nearby. Bicarbonate can covalently modify amine groups, releasing water, the carbamino group forming a bidentate ligand. Therefore bicarbonate could be important in water oxidation. Although the mechanism of water oxidation is far from understood, the models now have a clearer structural

Subunits F, G, H and K of the reaction centre are indicated. The assignment of the four different Lhca proteins is shown. (b) A view from the LHCI side. Subunits F, G and D are indicated. The helix–loop–helix N-terminal domain of subunit F and the Nterminus of subunit D that are unique to plant PSI are coloured red. Figure and legend reproduced, with permission, from Ref. [3].big step towards understanding how water oxidation a

can occur. Water oxidation-catalytic site revealed The Mn–Ca cluster appears to act as a device for the accumulation of oxidizing equivalents and as the site of water oxidation. PSII has been bombarded with a variety of spectroscopic techniques for many years, yielding a variety of possible models for the Mn–Ca cluster and its mechanism of action [10,11]. Now we have an improved structure from which to develop the mechanistic detail better. In the initial X-ray structure [8], the Mn–Ca region was only a blob of density thought to contain 3 to 4 Mn. The second structure [9] improved on this by resolving some ligands. Ferreira and colleagues [2] application of a range www.sciencedirect.com

Figure 2. Model of the water oxidizing complex (WOC) with side chain ligands and possible catalytically important side chain amino acid residues. Mn in magenta, Ca in green, oxygen in red and unidentified ligand, possibly bicarbonate, in blue. Figure kindly supplied by Jim Barber (Imperial College London, UK).

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constraint [13]. We have a model for the Mn–Ca cluster and confirmation that TyrZ is deep in the WOC cavity, making some mechanistic models less likely. Mechanistic models where the charge on the oxidized TyrZ site cause proton release from the Mn complex are becoming more likely [10]. However, there is still a long way to go and more excitement to come. We have a snapshot of the PSII structure. We now need to know where the water is, what the Mn oxidation states are, the structure in each of the oxidation states, and how the cluster changes during turnover. Improved resolution of the PSII structure and new structures from different states should help. The challenge is now on for the spectroscopists to put the remaining pieces of this jigsaw in place. Using spectroscopy and molecular biology we should be able to understand the process, recreate this primary biological function and then develop biomimetic catalysts of water oxidation. References 1 Heathcote, P. et al. (2002) Reaction centres: the structure and evolution of biological solar power. Trends Biochem. Sci. 27, 79–87 2 Ferreira, K.N. et al. (2004) Architecture of the photosynthetic oxygenevolving centre. Science 303, 1831–1838 3 Ben-Shem, A. et al. (2003) Crystal structure of plant photosystem I. Nature 426, 630–635

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4 Jordan, P. et al. (2001) Three dimensional structure of cyanobacterial ˚ resolution. Nature 411, 909–917 photosystem I at 2.5 A 5 Heathcote, P., ed. (2001) Type 1 photosynthetic reaction centres. Biochim. Biophys. Acta 1507, 1–312 6 Bibby, T.S. et al. (2001) Iron deficiency induces the formation of an antenna ring around trimeric photosystem I in cyanobacteria. Nature 412, 743–745 7 Boekema, E.J. et al. (2001) A giant chlorophyll–protein complex induced by iron deficiency in cyanobacteria. Nature 412, 745–748 8 Zouni, A. et al. (2001) Crystal structure of photosystem II from ˚ resolution. Nature 409, 739–743 Synechococcus elongatus at 3.8 A 9 Kamiya, N. and Shen, J-R. (2003) Crystal structure of oxygen-evolving ˚ resoluphotosystem II from Thermosynechococcus vulcanus at 3.7 A tion. Proc. Natl. Acad. Sci. U. S. A. 100, 98–103 10 Nugent, J.H.A., ed. (2001) Photosynthetic water oxidation. Biochim. Biophys. Acta 1503, 1–259 11 Debus, R.J. (1999) The polypeptides of photosystem II and their influence on manganotyrosyl-based oxygen evolution. Manganese and its role in biological processes. In Metal Ions in Biological Systems (Sigel, A. and Sigel, H. ed.), pp. 657–710, Marcel Dekker 12 Klimov, V.V. and Baranov, S.V. (2001) Bicarbonate requirement for the water-oxidizing complex of photosystem II. Biochim. Biophys. Acta 1503, 187–196 13 Rutherford, A.W. and Faller, P. (2001) The heart of photosynthesis in glorious 3D. Trends Biochem. Sci. 26, 341–344 1360-1385/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2004.06.005

Abiotic stress in plants Over the next few months, stretching into the early issues of 2005, we will be featuring a series of articles focusing on various aspects of abiotic stress in plants. We begin with two articles in this issue. The first article examines the effects of cold stress on plants by Marilyn Griffith and Mahmoud W.F. Yaish and the second article highlights the role of aldehyde dehydrogenases in abiotic stress tolerance by Andrew Wood and colleagues. Other review topics that will appear in forthcoming issues include: Plant responses to hypoxia – is survival a balancing act? Takeshi Fukao and Julia Bailey-Serres The reactive oxygen gene network of plants, Ron Mittler et al. Networks of transcription factors with roles in environmental stress responses, Tong Zhu Salt tolerant crops: physiology, genetics and molecular biology, Eduardo Blumwald and Emanuel Epstein Stress-activated phopholipid signaling, Teun Munnik Functional genomics of root growth and root signaling under drought, Robert Sharp Drought stress and ABA signaling, Kazuo Shinozaki

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