Norifumi Muraki

Chlorophyll biosynthesis: spotlight on protochlorophyllide reduction

Photosynthetic organisms require chlorophyll or bacteriochlorophyll for their light trapping and energy transduction activities. The biosynthetic pathways of chlorophyll and bacteriochlorophyll are similar in most of their early steps, except for the reduction of protochlorophyllide (Pchlide) to chlorophyllide. Whereas angiosperms make use of a light-dependent enzyme, cyanobacteria, algae, bryophytes, pteridophytes and gymnosperms contain an additional, light-independent enzyme dubbed dark-operative Pchlide oxidoreductase (DPOR). Anoxygenic photosynthetic bacteria such as Rhodobacter capsulatus and Rhodobacter sphaeroides rely solely on DPOR. Recent atomic resolution of reductase and catalytic components of DPOR from R. sphaeroides and R. capsulatus, respectively, have revealed their similarity to nitrogenase components. In this review, we discuss the two fundamentally different mechanisms of Pchlide reduction in photosynthetic organisms.

Structures of cyanobacteriochromes from phototaxis regulators AnPixJ and TePixJ reveal general and specific photoconversion mechanism

Cyanobacteriochromes are cyanobacterial tetrapyrrole-binding photoreceptors that share a bilin-binding GAF domain with photoreceptors of the phytochrome family. Cyanobacteriochromes are divided into many subclasses with distinct spectral properties. Among them, putative phototaxis regulators PixJs of Anabaena sp. PCC 7120 and Thermosynechococcus elongatus BP-1 (denoted as AnPixJ and TePixJ, respectively) are representative of subclasses showing red-green-type and blue/green-type reversible photoconversion, respectively. Here, we determined crystal structures for the AnPixJ GAF domain in its red-absorbing 15Z state (Pr) and the TePixJ GAF domain in its green-absorbing 15E state (Pg). The overall structure of these proteins is similar to each other and also similar to known phytochromes. Critical differences found are as follows: (i) the chromophore of AnPixJ Pr is phycocyanobilin in a C5-Z,syn/C10-Z,syn/C15-Z,anti configuration and that of TePixJ Pg is phycoviolobilin in a C10-Z,syn/C15-E,anti configuration, (ii) a side chain of the key aspartic acid is hydrogen bonded to the tetrapyrrole rings A, B and C in AnPixJ Pr and to the pyrrole ring D in TePixJ Pg, (iii) additional protein-chromophore interactions are provided by subclass-specific residues including tryptophan in AnPixJ and cysteine in TePixJ. Possible structural changes following the photoisomerization of the chromophore between C15-Z and C15-E are discussed based on the X-ray structures at 1.8 and 2.0-Å resolution, respectively, in two distinct configurations.

Crystal Structures of Copper-depleted and Copper-bound Fungal Pro-tyrosinase INSIGHTS INTO ENDOGENOUS CYSTEINE-DEPENDENT COPPER INCORPORATION

Background: Fungal tyrosinase maturation involves multiple processes of the dinuclear copper assembly and proteolytic activation. Results: Structural examinations and mutational studies of the pro-tyrosinases revealed that three endogenous cysteines contribute to the copper incorporation. Conclusion: The three highly flexible cysteines are essential for assembly of the active site across the protein shell. Significance: Elucidation of such a copper incorporation process provides useful insights into metal homeostasis. Tyrosinase, a dinuclear copper monooxygenase/oxidase, plays a crucial role in the melanin pigment biosynthesis. The structure and functions of tyrosinase have so far been studied extensively, but the post-translational maturation process from the pro-form to the active form has been less explored. In this study, we provide the crystal structures of Aspergillus oryzae full-length pro-tyrosinase in the holo- and the apo-forms at 1.39 and 2.05 Å resolution, respectively, revealing that Phe513 on the C-terminal domain is accommodated in the substrate-binding site as a substrate analog to protect the dicopper active site from substrate access (proteolytic cleavage of the C-terminal domain or deformation of the C-terminal domain by acid treatment transforms the pro-tyrosinase to the active enzyme (Fujieda, N., Murata, M., Yabuta, S., Ikeda, T., Shimokawa, C., Nakamura, Y., Hata, Y., and Itoh, S. (2012) ChemBioChem. 13, 193–201 and Fujieda, N., Murata, M., Yabuta, S., Ikeda, T., Shimokawa, C., Nakamura, Y., Hata, Yl, and Itoh, S. (2013) J. Biol. Inorg. Chem. 18, 19–26). Detailed crystallographic analysis and structure-based mutational studies have shown that the copper incorporation into the active site is governed by three cysteines as follows: Cys92, which is covalently bound to His94 via an unusual thioether linkage in the holo-form, and Cys522 and Cys525 of the CXXC motif located on the C-terminal domain. Molecular mechanisms of the maturation processes of fungal tyrosinase involving the accommodation of the dinuclear copper unit, the post-translational His-Cys thioether cross-linkage formation, and the proteolytic C-terminal cleavage to produce the active tyrosinase have been discussed on the basis of the detailed structural information.

N-Terminal Structure of Maize Ferredoxin:NADP + Reductase Determines Recruitment into Different Thylakoid Membrane Complexes

Maize chloroplasts conducting either cyclic or linear electron flow vary in their ferredoxin:NADP+ reductase (FNR) composition. By comparing FNR crystal structures and introducing modified FNRs back into plants, we show that N-terminal structure determines recruitment to different thylakoid complexes. Furthermore, electron flow is analyzed in plants enriched in FNR at alternative locations. To adapt to different light intensities, photosynthetic organisms manipulate the flow of electrons through several alternative pathways at the thylakoid membrane. The enzyme ferredoxin:NADP+ reductase (FNR) has the potential to regulate this electron partitioning because it is integral to most of these electron cascades and can associate with several different membrane complexes. However, the factors controlling relative localization of FNR to different membrane complexes have not yet been established. Maize (Zea mays) contains three chloroplast FNR proteins with totally different membrane association, and we found that these proteins have variable distribution between cells conducting predominantly cyclic electron transport (bundle sheath) and linear electron transport (mesophyll). Here, the crystal structures of all three enzymes were solved, revealing major structural differences at the N-terminal domain and dimer interface. Expression in Arabidopsis thaliana of maize FNRs as chimeras and truncated proteins showed the N-terminal determines recruitment of FNR to different membrane complexes. In addition, the different maize FNR proteins localized to different thylakoid membrane complexes on expression in Arabidopsis, and analysis of chlorophyll fluorescence and photosystem I absorbance demonstrates the impact of FNR location on photosynthetic electron flow.

X-ray Structure and Nuclear Magnetic Resonance Analysis of the Interaction Sites of the Ga-Substituted Cyanobacterial Ferredoxin

In chloroplasts, ferredoxin (Fd) is reduced by Photosystem I (PSI) and oxidized by Fd-NADP(+) reductase (FNR) that is involved in NADP(+) reduction. To understand the structural basis for the dynamics and efficiency of the electron transfer reaction via Fd, we complementary used X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. In the NMR analysis of the formed electron transfer complex with Fd, the paramagnetic effect of the [2Fe-2S] cluster of Fd prevented us from detecting the NMR signals around the cluster. To solve this problem, the paramagnetic iron-sulfur cluster was replaced with a diamagnetic metal cluster. We determined the crystal structure of the Ga-substituted Fd (GaFd) from Synechocystis sp. PCC6803 at 1.62 Å resolution and verified its functional complementation using affinity chromatography. NMR analysis of the interaction sites on GaFd with PSI (molecular mass of ∼1 MDa) and FNR from Thermosynechococcus elongatus was achieved with high-field NMR spectroscopy. With reference to the interaction sites with FNR of Anabaena sp. PCC 7119 from the published crystal data, the interaction sites of Fd with FNR and PSI in solution can be classified into two types: (1) the core hydrophobic residues in the proximity of the metal center and (2) the hydrophilic residues surrounding the core. The former sites are shared in the Fd:FNR and Fd:PSI complex, while the latter ones are target-specific and not conserved on the residual level.

Concentration-dependent oligomerization of cross-linked complexes between ferredoxin and ferredoxin–NADP+ reductase

Ferredoxin-NADP(+) reductase (FNR) forms a 1:1 complex with ferredoxin (Fd), and catalyzes the electron transfer between Fd and NADP(+). In our previous study, we prepared a series of site-specifically cross-linked complexes of Fd and FNR, which showed diverse electron transfer properties. Here, we show that X-ray crystal structures of the two different Fd-FNR cross-linked complexes form oligomers by swapping Fd and FNR moieties across the molecules; one complex is a dimer from, and the other is a successive multimeric form. In order to verify whether these oligomeric structures are formed only in crystal, we investigated the possibility of the oligomerization of these complexes in solution. The mean values of the particle size of these cross-linked complexes were shown to increase with the rise of protein concentration at sub-milimolar order, whereas the size of dissociable wild-type Fd:FNR complex was unchanged as analyzed by dynamic light scattering measurement. The oligomerization products were detected by SDS-PAGE after chemical cross-linking of these complexes at the sub-milimolar concentrations. The extent and concentration-dependent profile of the oligomerizaion were differentiated between the two cross-linked complexes. These results show that these Fd-FNR cross-linked complexes exhibit concentration-dependent oligomerization, possibly through swapping of Fd and FNR moieties also in solution. These findings lead to the possibility that some native multi-domain proteins may present similar phenomenon in vivo.

Crystallization and preliminary X-ray studies of an electron-transfer complex of ferredoxin and ferredoxin-dependent glutamate synthase from the cyanobacterium Leptolyngbya boryana

Ferredoxin (Fd) dependent glutamate synthase (Fd-GOGAT) is a key enzyme involved in nitrogen assimilation that catalyzes the two-electron reductive conversion of Gln and 2-oxoglutarate to two molecules of Glu. Fd serves as an electron donor for Fd-GOGAT and the two proteins form a transient electron-transfer complex. In this study, these two proteins were cocrystallized using the hanging-drop vapour-diffusion method. Diffraction data were collected and processed at 2.65 Å resolution. The crystals belonged to space group P4(3), with unit-cell parameters a = b = 84.95, c = 476.31 Å.

Cloning, expression, crystallization and preliminary X-ray studies of the ferredoxin-NAD(P)+ reductase from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1.

Ferredoxin-NADP(+) reductase (FNR) is a flavoenzyme that catalyses the reduction of NADP(+) in the final step of the photosynthetic electron-transport chain. FNR from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (TeFNR) contains an additional 9 kDa domain at its N-terminus relative to chloroplastic FNRs and is more thermostable than those from mesophilic cyanobacteria. With the aim of understanding the structural basis of the thermostability of TeFNR and assigning a structural role to the small additional domain, the gene encoding TeFNR with and without an additional domain was engineered for heterologous expression and the recombinant proteins were purified and crystallized. Crystals of TeFNR without the additional domain belonged to space group P2(1), with unit-cell parameters a = 55.05, b = 71.66, c = 89.73 Å, α = 90, β = 98.21, γ = 90°.

Structure of protochlorophyllide reductase: a greening mechanism for plants in the dark

Chlorophyll (Chl) is a tetrapyrrole macrocycle containing Mg and phytol chain. The Chl biosynthetic pathway consists of the multi-enzymatic reactions. An asymmetric conjugated double bond system of Chl a, which is crucial for efficient light absorption, is formed in the penultimate step of biosynthesis, reducing protochlorophyllide (Pchlide) to form chlorophyllide a. Photosynthetic organisms adopt two different strategies for the reduction of Pchlide; one is the dark-operative (lightindependent) Pchlide oxidoreductase (DPOR) that operates even in the dark. The greening ability of plant in the dark is attributed to the activity of DPOR. DPOR is characterized to be a nitrogenase-like enzyme requiring ATP hydrolysis and electron supply from Ferredoxin. As same as nitrogenase which catalyzes reduction of nitrogen, DPOR consists of the electron transfer component, L-protein and the catalytic component, NB-protein. We show a crystal structure of the catalytic component, NBprotein, of the DPOR from Rhodobacter capsulatus at 2.3angstrom resolution. Overall structure with two copies of homologous BchN and BchB subunits is similar to that of nitrogenase MoFe-protein. Each catalytic BchN-BchB unit contains one Pchlide held without any axial ligations from amino acid residues and one [4Fe-4S] cluster (NB-cluster) at the subunit interface. A surprise of the structure is direct coordination of BchB-Asp36 to the cluster, instead of BchBCys95 anticipated to coordinate the cluster based on the sequence similarity. The orientation of bound Pchlide is mainly provided by hydrophobic interaction, keeping the reduction site of Pchlide away from the NB-cluster. The structure in the presence or absence of Pchlide has revealed the displacement of C-terminal helix of BchB when accommodating Pchlide. Intriguingly, NB-cluster and Pchlide are arranged spatially as almost identical to P-cluster and FeMo-cofactor in MoFe-protein of nitrogenase, illustrating a common architectire to reduce chemically stable multi-bonds such as porphyrin and dinitrogen[1].