The \u3cem\u3eChlamydomonas\u3c/em\u3e Genome Reveals the Evolution of Key Animal and Plant Functions

Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the ∼120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella

Do sugar-sweetened beverages cause adverse health outcomes in adults? A systematic review protocol

Background: Chronic diseases, such as cardiovascular disease and type 2 diabetes, impose significant burden to public health. Most chronic diseases are associated with underlying preventable risk factors, such as elevated blood pressure, blood glucose, and lipids, physical inactivity, excessive sedentary behaviours, overweight and obesity, and tobacco usage. Sugar-sweetened beverages are known to be significant sources of additional caloric intake, and given recent attention to their contribution in the development of chronic diseases, a systematic review is warranted. We will assess whether the consumption of sugar-sweetened beverages in adults is associated with adverse health outcomes and what the potential moderating factors are. Methods/Design: Of interest are studies addressing sugar-sweetened beverage consumption, taking a broad perspective. Both direct consumption studies as well as those evaluating interventions that influence consumption (e.g. school policy, educational) will be relevant. Non-specific or multi-faceted behavioural, educational, or policy interventions may also be included subject to the level of evidence that exists for the other interventions/exposures. Comparisons of interest and endpoints of interest are pre-specified. We will include randomized controlled trials, controlled clinical trials, interrupted time series studies, controlled before-after studies, prospective and retrospective comparative cohort studies, case-control studies, and nested case-control designs. The MEDLINE®, Embase, The Cochrane Library, CINAHL, ERIC, and PsycINFO® databases

Chlorophyll synthesis

Chlorophyll is the dominant pigment in a mature plant cell, whether in the leaf of a plant or in the abundant algal species. Chlorophyll is synthesized within the chloroplast from a plentiful precursor, the amino acid glutamate. From glutamate to the tetrapyrrole protoporphyrin IX, at which the pathway branches between chlorophyll and heme, the reactions occur in the plastid stroma and are catalyzed by soluble enzymes. The latter steps to chlorophyll, the first being the insertion of the central magnesium atom, occur with enzyme complexes that are at least partially if not entirely localized on membranes. Magnesium chelatase, the key enzyme in this pathway, is a complex of proteins that includes soluble and membrane-bound subunits. Subsequent reactions occur primarily on membranes and involve modification of structural groups on the periphery of the molecule. The pentultimate precursor of chlorophyll, protochlorophyllide, is reduced by NADPH to chlorophyllide in the only reaction in the pathway that requires light. This reaction, which in angiosperms is catalyzed by light-dependent NADPH:protochlorophyllide oxidoreductase, dramatically changes the property of the molecule and allows the product chlorophyllide, and its esterified product, chlorophyll, to interact with proteins. These chlorophyll-protein complexes become the building blocks of the photosynthetic apparatus. The biosynthetic pathway is tightly regulated, particularly at the key reactions that generate 5-aminolevulinic acid, magnesium-protoporphyrin IX and chlorophyllide. Expression of genes encoding critical enzymes is usually regulated markedly by light, and the activities of the enzymes are also regulated by end-products in typical feedback inhibition.19 page(s

Kinetic Analyses of the Magnesium Chelatase Provide Insights into the Mechanism, Structure, and Formation of the Complex

9 page(s

BchJ and BchM interact in a 1 : 1 ratio with the magnesium chelatase BchH subunit of Rhodobacter capsulatus

Substrate channeling between the enzymatic steps in the (bacterio)chlorophyll biosynthetic pathway catalyzed by magnesium chelatase (BchI/ChlI, BchD/ChlD and BchH/ChlH subunits) and S-adenosyl-l-methionine:magnesium-protoporphyrin IX O-methyltransferase (BchM/ChlM) has been suggested. This involves delivery of magnesium-protoporphyrin IX from the BchH/ChlH subunit of magnesium chelatase to BchM/ChlM. Stimulation of BchM/ChlM activity by BchH/ChlH has previously been shown, and physical interaction of the two proteins has been demonstrated. In plants and cyanobacteria, there is an added layer of complexity, as Gun4 serves as a porphyrin (protoporphyrin IX and magnesium-protoporphyrin IX) carrier, but this protein does not exist in anoxygenic photosynthetic bacteria. BchJ may play a similar role to Gun4 in Rhodobacter, as it has no currently assigned function in the established pathway. Purified recombinant Rhodobacter capsulatus BchJ and BchM were found to cause a shift in the equilibrium amount of Mg-protoporphyrin IX formed in a magnesium chelatase assay. Analysis of this shift revealed that it was always in a 1 : 1 ratio with either of these proteins and the BchH subunit of the magnesium chelatase. The establishment of the new equilibrium was faster with BchM than with BchJ in a coupled magnesium chelatase assay. BchJ bound magnesium-protoporphyrin IX or formed a ternary complex with BchH and magnesium-protoporphyrin IX. These results suggest that BchJ may play a role as a general magnesium porphyrin carrier, similar to one of the roles of GUN4 in oxygenic organisms.13 page(s

Biosynthesis of bacteriochlorophylls in purple bacteria

The purple bacteria make bacteriochlorophylls for the photosynthetic mode of growth. These pigments are made from the simple precursors glycine and succinyl CoA and the initial steps in the pathway of bacteriochlorophyll biosynthesis are shared with the vitamin B12 and heme biosynthetic pathways. This chapter concentrates on the biochemical properties of the enzymes involved in each step of the pathway and the discovery and assignment of the genes encoding these enzymes. The characterization of purple bacterial enzymes involved in these steps has been crucial in understanding similar enzymes from other sources. The characterization of the early steps in the pathway within purple bacteria, such as δ-aminolevulinate synthase, contributed significantly to the understanding of the mammalian enzymes in the 1950s and 1960s. More recently the study of the purple bacterial enzymes toward the end of the pathway has been instrumental in identifying and characterizing the orthologous enzymes from cyanobacteria and plants. In this review we present the details of the properties of these enzymes from the purple bacteria, such as purification methods and kinetic analyses from the early literature, through to more recent studies using recombinant purple bacterial enzymes.23 page(s

In vitro conversion of vinyl to formyl groups in naturally occurring chlorophylls

The chemical structural differences distinguishing chlorophylls in oxygenic photosynthetic organisms are either formyl substitution (chlorophyll b, d, and f) or the degree of unsaturation (8-vinyl chlorophyll a and b) of a side chain of the macrocycle compared with chlorophyll a. We conducted an investigation of the conversion of vinyl to formyl groups among naturally occurring chlorophylls. We demonstrated the in vitro oxidative cleavage of vinyl side groups to yield formyl groups through the aid of a thiol-containing compound in aqueous reaction mixture at room temperature. Heme is required as a catalyst in aqueous solution but is not required in methanolic reaction mixture. The conversion of vinyl- to formyl- groups is independent of their position on the macrocycle, as we observed oxidative cleavages of both 3-vinyl and 8-vinyl side chains to yield formyl groups. Three new chlorophyll derivatives were synthesised using 8-vinyl chlorophyll a as substrate: 8-vinyl chlorophyll d, [8-formyl]-chlorophyll a, and [3,8-diformyl]-chlorophyll a. The structural and spectral properties will provide a signature that may aid in identification of the novel chlorophyll derivatives in natural systems. The ease of conversion of vinyl- to formyl- in chlorophylls demonstrated here has implications regarding the biosynthetic mechanism of chlorophyll d in vivo.9 page(s

Hydroxylation of the C13² and C18 carbons of chlorophylls by heme and molecular oxygen

Following extraction from photosynthetic organisms, chlorophylls are prone to reactions including demetalation, dephytylation and specific oxidations of the exocyclic ring E, termed allomerizations. Allomerization of chlorophylls has been well-characterized in methanol and to a lesser extent in aqueous solution. Here we detail novel allomerization-like reactions of chlorophyll a and chlorophyll b. In the presence of heme, detergent-solubilized chlorophyll a is hydroxylated at its C13² position in ring E and, surprisingly, the C18 position in ring D. Two major oxidation products are synthesized - a C13²−OH and a C13²−OH, C18−OH derivative of chlorophyll a. We track the origin of the oxygen atoms added in these hydroxylated chlorophylls using ¹⁸O₂ labeling and demonstrate that the additional oxygen atoms are derived from molecular oxygen. A similar heme-catalyzed reaction is also observed using chlorophyll b as a substrate. These results highlight the need for care when dealing with extracted chlorophylls and demonstrate an unusual hydroxylation of the C18 position of chlorophylls in the presence of heme.7 page(s

C-terminal residues of Oryza sativa GUN4 are required for the activation of the ChlH subunit of magnesium chelatase in chlorophyll synthesis

Oryza sativa GUN4 together with the magnesium chelatase subunits ChlI, ChlD, and ChlH have been heterologously expressed and purified to reconstitute magnesium chelatase activity in vitro. Maximum magnesium chelatase activity requires pre-activation of OsChlH with OsGUN4, Mg²⁺ and protoporphyrin-IX. OsGUN4 and OsChlH preincubated without protoporphyrin-IX yields magnesium chelatase activity similar to assays without OsGUN4, suggesting formation of a dead-end complex. Either 9 or 10 C-terminal amino acids of OsGUN4 are slowly hydrolyzed to yield a truncated OsGUN4. These truncated OsGUN4 still bind protoporphyrin-IX and Mg-protoporphyrin-IX but are unable to activate OsChlH. This suggests the mechanism of GUN4 activation of magnesium chelatase is different in eukaryotes compared to cyanobacteria as the orthologous cyanobacterial GUN4 proteins lack this C-terminal extension.6 page(s