Structural and Functional Analysis of Nonheme Iron Enzymes BCMO-1 and BCMO-2 from Caenorhabditis elegans.

Carotenoid metabolism is critical for diverse physiological processes. The nematode Caenorhabditis elegans has two genes that are annotated as ß-carotene 15,15'-monooxygenase (BCMO) and are 17 centimorgan apart on chromosome II, but the function of BCMO-1 and BCMO-2 remains uncharacterized. Sequence homology indicates that the two enzymes belong to the carotenoid cleavage dioxygenase family that share a seven-bladed ß-propeller fold with a nonheme iron center. Here we determined crystal structures of BCMO-1 and BCMO-2 at resolutions of 1.8 and 1.9 Å, respectively. Structural analysis reveals that BCMO-1 and BCMO-2 are strikingly similar to each other. We also characterized their ß-carotene cleavage activity, but the results suggest that they may not act as ß-carotene 15,15'-oxygenases.

Crystal structure of the large subunit of cobaltochelatase from Mycobacterium tuberculosis.

Cobaltochelatase in aerobic cobalamin biosynthesis is a complex composed of three subunits. The large subunit CobN is a 140-kDa protein and is homologous to the ChlH subunit of magnesium chelatase. Previously we have reported the 2.5-Å structure of a cyanobacterial ChlH. Here we present the 1.8-Å structure of CobN from Mycobacterium tuberculosis. The overall structure of CobN and ChlH is similar, but significant difference occurs in the head domain. Structural comparison of domains between the two proteins unravels candidate regions for substrate binding and helps to locate a triad of residues that may be essential for metal ion binding.

Unique organization of photosystem I-light-harvesting supercomplex revealed by cryo-EM from a red alga.

Photosystem I (PSI) is one of the two photosystems present in oxygenic photosynthetic organisms and functions to harvest and convert light energy into chemical energy in photosynthesis. In eukaryotic algae and higher plants, PSI consists of a core surrounded by variable species and numbers of light-harvesting complex (LHC)I proteins, forming a PSI-LHCI supercomplex. Here, we report cryo-EM structures of PSI-LHCR from the red alga Cyanidioschyzon merolae in two forms, one with three Lhcr subunits attached to the side, similar to that of higher plants, and the other with two additional Lhcr subunits attached to the opposite side, indicating an ancient form of PSI-LHCI. Furthermore, the red algal PSI core showed features of both cyanobacterial and higher plant PSI, suggesting an intermediate type during evolution from prokaryotes to eukaryotes. The structure of PsaO, existing in eukaryotic organisms, was identified in the PSI core and binds three chlorophylls a and may be important in harvesting energy and in mediating energy transfer from LHCII to the PSI core under state-2 conditions. Individual attaching sites of LHCRs with the core subunits were identified, and each Lhcr was found to contain 11 to 13 chlorophylls a and 5 zeaxanthins, which are apparently different from those of LHCs in plant PSI-LHCI. Together, our results reveal unique energy transfer pathways different from those of higher plant PSI-LHCI, its adaptation to the changing environment, and the possible changes of PSI-LHCI during evolution from prokaryotes to eukaryotes.

The Non-canonical Tetratricopeptide Repeat (TPR) Domain of Fluorescent (FLU) Mediates Complex Formation with Glutamyl-tRNA Reductase.

The tetratricopeptide repeat (TPR)-containing protein FLU is a negative regulator of chlorophyll biosynthesis in plants. It directly interacts through its TPR domain with glutamyl-tRNA reductase (GluTR), the rate-limiting enzyme in the formation of δ-aminolevulinic acid (ALA). Delineation of how FLU binds to GluTR is important for understanding the molecular basis for FLU-mediated repression of synthesis of ALA, the universal tetrapyrrole precursor. Here, we characterize the FLU-GluTR interaction by solving the crystal structures of the uncomplexed TPR domain of FLU (FLU(TPR)) at 1.45-Šresolution and the complex of the dimeric domain of GluTR bound to FLU(TPR) at 2.4-Šresolution. Three non-canonical TPR motifs of each FLU(TPR) form a concave surface and clamp the helix bundle in the C-terminal dimeric domain of GluTR. We demonstrate that a 2:2 FLU(TPR)-GluTR complex is the functional unit for FLU-mediated GluTR regulation and suggest that the formation of the FLU-GluTR complex prevents glutamyl-tRNA, the GluTR substrate, from binding with this enzyme. These results also provide insights into the spatial regulation of ALA synthesis by the membrane-located FLU protein.

Crystal structure of the catalytic subunit of magnesium chelatase.

Tetrapyrroles, including haem and chlorophyll, play vital roles for various biological processes, such as respiration and photosynthesis, and their biosynthesis is critical for virtually all organisms. In photosynthetic organisms, magnesium chelatase (MgCh) catalyses insertion of magnesium into the centre of protoporphyrin IX, the branch-point precursor for both haem and chlorophyll, leading tetrapyrrole biosynthesis into the magnesium branch(1,2). This reaction needs a cooperated action of the three subunits of MgCh: the catalytic subunit ChlH and two AAA(+) subunits, ChlI and ChlD (refs 3-5). To date, the mechanism of MgCh awaits further elucidation due to a lack of high-resolution structures, especially for the ∼150 kDa catalytic subunit. Here we report the crystal structure of ChlH from the photosynthetic cyanobacterium Synechocystis PCC 6803, solved at 2.5 Šresolution. The active site is buried deeply inside the protein interior, and the surrounding residues are conserved throughout evolution. This structure helps to explain the loss of function reported for the cch and gun5 mutations of the ChlH subunit, and to provide the molecular basis of substrate channelling during the magnesium-chelating process. The structure advances our understanding of the holoenzyme of MgCh, a metal chelating enzyme other than ferrochelatase.