Two Unrelated 8-Vinyl Reductases Ensure Production of Mature Chlorophylls in Acaryochloris marina

The major photopigment of the cyanobacterium Acaryochloris marina is chlorophyll d , while its direct biosynthetic precursor, chlorophyll a , is also present in the cell. These pigments, along with the majority of chlorophylls utilized by oxygenic pho- totrophs, carry an ethyl group at the C-8 position of the molecule, having undergone reduction of a vinyl group during biosyn- thesis. Two unrelated classes of 8-vinyl reductase involved in the biosynthesis of chlorophylls are known to exist, BciA and BciB. The genome of Acaryochloris marina contains open reading frames (ORFs) encoding proteins displaying high sequence similarity to BciA or BciB, although they are annotated as genes involved in transcriptional control ( nmrA ) and methanogenesis ( frhB ), respectively. These genes were introduced into an 8-vinyl chlorophyll a -producing delta bciB strain of Synechocystis sp. strain PCC 6803, and both were shown to restore synthesis of the pigment with an ethyl group at C-8, demonstrating their activities as 8-vinyl reductases. We propose that nmrA and frhB be reassigned as bciA and bciB , respectively; transcript and proteomic analysis of Acaryochloris marina reveal that both bciA and bciB are expressed and their encoded proteins are present in the cell, possibly in order to ensure that all synthesized chlorophyll pigment carries an ethyl group at C-8. Potential reasons for the presence of two 8-vinyl reductases in this strain, which is unique for cyanobacteria, are discussed

The ChlD subunit links the motor and porphyrin binding subunits of magnesium chelatase

Magnesium chelatase initiates chlorophyll biosynthesis, catalysing the MgATP2- dependent insertion of a Mg2+ ion into protoporphyin IX. The catalytic core of this large enzyme complex consists of three subunits: Bch/ChlI, Bch/ChlD and Bch/ChlH (in bacteriochlorophyll and chlorophyll producing species respectively). The D and I subunits are members of the AAA+ (ATPases associated with various cellular activities) superfamily of enzymes, and they form a complex that binds to H, the site of metal ion insertion. In order to investigate the physical coupling between ChlID and ChlH in vivo and in vitro , ChlD was FLAG-tagged in the cyanobacterium Synechocystis sp. PCC 6803 and co-immunoprecipitation experiments showed interactions with both ChlI and ChlH. Co-production of recombinant ChlD and ChlH in Escherichia coli yielded a ChlDH. Quantitative analysis using microscale thermophoresis (MST) showed magnesium-dependent binding ( K d 331 ± 58 nM) between ChlD and H. The physical basis for a ChlD-H interaction was investigated using chemical crosslinking coupled with mass spectrometry (XL-MS), together with modifications that either truncate ChlD or modify single residues. We found that the C-terminal integrin I domain of ChlD governs association with ChlH, the Mg2+ dependence of which also mediates the cooperative response of the Synechocystis chelatase to magnesium. Our work, showing the interaction site between the AAA+ motor and the chelatase domain of magnesium chelatase, will be essential for understanding how free energy from the hydrolysis of ATP on the AAA+ ChlI subunit is transmitted via the bridging subunit ChlD to the active site on ChlH

Absolute quantification of cellular levels of photosynthesis-related proteins in Synechocystis sp. PCC 6803

Quantifying cellular components is a basic and important step for understanding how a cell works, how it responds to environmental changes, and for re-engineering cells to produce valuable metabolites and increased biomass. We quantified proteins in the model cyanobacterium Synechocystis sp. PCC 6803 given the general importance of cyanobacteria for global photosynthesis, for synthetic biology and biotechnology research, and their ancestral relationship to the chloroplasts of plants. Four mass spectrometry methods were used to quantify cellular components involved in the biosynthesis of chlorophyll, carotenoid and bilin pigments, membrane assembly, the light reactions of photosynthesis, fixation of carbon dioxide and nitrogen, and hydrogen and sulfur metabolism. Components of biosynthetic pathways, such as those for chlorophyll or for photosystem II assembly, range between 1000 and 10,000 copies per cell, but can be tenfold higher for CO2 fixation enzymes. The most abundant subunits are those for photosystem I, with around 100,000 copies per cell, approximately 2 to fivefold higher than for photosystem II and ATP synthase, and 5–20 fold more than for the cytochrome b6f complex. Disparities between numbers of pathway enzymes, between components of electron transfer chains, and between subunits within complexes indicate possible control points for biosynthetic processes, bioenergetic reactions and for the assembly of multisubunit complexes

Membrane organization of photosystem I complexes in the most abundant phototroph on Earth

Prochlorococcus is a major contributor to primary production, and globally the most abundant photosynthetic genus of picocyanobacteria because it can adapt to highly stratified low-nutrient conditions that are characteristic of the surface ocean. Here, we examine the structural adaptations of the photosynthetic thylakoid membrane that enable different Prochlorococcus ecotypes to occupy high-light, low-light and nutrient-poor ecological niches. We used atomic force microscopy to image the different photosystem I (PSI) membrane architectures of the MED4 (high-light) Prochlorococcus ecotype grown under high-light and low-light conditions in addition to the MIT9313 (low-light) and SS120 (low-light) Prochlorococcus ecotypes grown under low-light conditions. Mass spectrometry quantified the relative abundance of PSI, photosystem II (PSII) and cytochrome b6f complexes and the various Pcb proteins in the thylakoid membrane. Atomic force microscopy topographs and structural modelling revealed a series of specialized PSI configurations, each adapted to the environmental niche occupied by a particular ecotype. MED4 PSI domains were loosely packed in the thylakoid membrane, whereas PSI in the low-light MIT9313 is organized into a tightly packed pseudo-hexagonal lattice that maximizes harvesting and trapping of light. There are approximately equal levels of PSI and PSII in MED4 and MIT9313, but nearly twofold more PSII than PSI in SS120, which also has a lower content of cytochrome b6f complexes. SS120 has a different tactic to cope with low-light levels, and SS120 thylakoids contained hundreds of closely packed Pcb–PSI supercomplexes that economize on the extra iron and nitrogen required to assemble PSI-only domains. Thus, the abundance and widespread distribution of Prochlorococcus reflect the strategies that various ecotypes employ for adapting to limitations in light and nutrient levels

Identification of protein W, the elusive sixth subunit of the Rhodopseudomonas palustris reaction center-light harvesting 1 core complex

The X-ray crystal structure of the Rhodopseudomonas (Rps.) palustris reaction center-light harvesting 1 (RC-LH1) core complex revealed the presence of a sixth protein component, variably referred to in the literature as helix W, subunit W or protein W. The position of this protein prevents closure of the LH1 ring, possibly to allow diffusion of ubiquinone/ubiquinol between the RC and the cytochrome bc1 complex in analogous fashion to the well-studied PufX protein from Rhodobacter sphaeroides. The identity and function of helix W have remained unknown for over 13 years; here we use a combination of biochemistry, mass spectrometry, molecular genetics and electron microscopy to identify this protein as RPA4402 in Rps. palustris CGA009. Protein W shares key conserved sequence features with PufX homologs, and although a deletion mutant was able to grow under photosynthetic conditions with no discernible phenotype, we show that a tagged version of protein W pulls down the RC-LH1 complex. Protein W is not encoded in the photosynthesis gene cluster and our data indicate that only approximately 10% of wild-type Rps. palustris core complexes contain this non-essential subunit; functional and evolutionary consequences of this observation are discussed. The ability to purify uniform RC-LH1 and RC-LH1-protein W preparations will also be beneficial for future structural studies of these bacterial core complexes

Structures of Rhodopseudomonas palustris RC-LH1 complexes with open or closed quinone channels

The reaction-center light-harvesting complex 1 (RC-LH1) is the core photosynthetic component in purple phototrophic bacteria. We present two cryo–electron microscopy structures of RC-LH1 complexes from Rhodopseudomonas palustris. A 2.65-Å resolution structure of the RC-LH114-W complex consists of an open 14-subunit LH1 ring surrounding the RC interrupted by protein-W, whereas the complex without protein-W at 2.80-Å resolution comprises an RC completely encircled by a closed, 16-subunit LH1 ring. Comparison of these structures provides insights into quinone dynamics within RC-LH1 complexes, including a previously unidentified conformational change upon quinone binding at the RC QB site, and the locations of accessory quinone binding sites that aid their delivery to the RC. The structurally unique protein-W prevents LH1 ring closure, creating a channel for accelerated quinone/quinol exchange

Changes in supramolecular organization of cyanobacterial thylakoid membrane complexes in response to far-red light photoacclimation

Cyanobacteria are ubiquitous in nature and have developed numerous strategies that allow them to live in a diverse range of environments. Certain cyanobacteria synthesize chlorophylls d and f to acclimate to niches enriched in far-red light (FRL) and incorporate paralogous photosynthetic proteins into their photosynthetic apparatus in a process called FRL-induced photoacclimation (FaRLiP). We characterized the macromolecular changes involved in FRL-driven photosynthesis and used atomic force microscopy to examine the supramolecular organization of photosystem I associated with FaRLiP in three cyanobacterial species. Mass spectrometry showed the changes in the proteome of Chroococcidiopsis thermalis PCC 7203 that accompany FaRLiP. Fluorescence lifetime imaging microscopy and electron microscopy reveal an altered cellular distribution of photosystem complexes and illustrate the cell-to-cell variability of the FaRLiP response

Probing the quality control mechanism of theEscherichia colitwin-arginine translocase with folding variants of ade novo-designed heme protein

Protein transport across the cytoplasmic membrane of bacterial cells is mediated by either the general secretion (Sec) system or the twin arginine translocase (Tat). The Tat machinery exports folded and cofactor containing proteins from the cytoplasm to the periplasm by using the transmembrane proton motive force as a source of energy. The Tat apparatus apparently senses the folded state of its protein substrates, a quality control mechanism that prevents premature export of nascent unfolded or misfolded polypeptides, but its mechanistic basis has not yet been determined. Here, we investigated the innate ability of the model Escherichia coli Tat system to recognize and translocate de novo-designed protein substrates with experimentally determined differences in the extent of folding. Water-soluble, four-helix bundle maquette proteins were engineered to bind two, one or no heme b cofactors, resulting in a concomitant reduction in the extent of their folding, assessed with temperature-dependent CD spectroscopy and one-dimensional 1H NMR spectroscopy. Fusion of the archetypal N-terminal Tat signal peptide of the E. coli trimethylamine-N-oxide (TMAO) reductase (TorA) to the N-terminus of the protein maquettes was sufficient for the Tat system to recognize them as substrates. The clear correlation between the level of Tat-dependent export and the degree of heme b-induced folding of the maquette protein suggested that the membrane-bound Tat machinery can sense the extent of folding and conformational flexibility of its substrates. We propose that these artificial proteins are ideal substrates for future investigations of the Tat system’s quality control mechanism

Precompetitive consensus building to facilitate the use of digital health technologies to support Parkinson Disease drug development through regulatory science

Innovative tools are urgently needed to accelerate the evaluation and subsequent approval of novel treatments that may slow, halt, or reverse the relentless progression of Parkinson disease (PD). Therapies that intervene early in the disease continuum are a priority for the many candidates in the drug development pipeline. There is a paucity of sensitive and objective, yet clinically interpretable, measures that can capture meaningful aspects of the disease. This poses a major challenge for the development of new therapies and is compounded by the considerable heterogeneity in clinical manifestations across patients and the fluctuating nature of many signs and symptoms of PD. Digital health technologies (DHT), such as smartphone applications, wearable sensors, and digital diaries, have the potential to address many of these gaps by enabling the objective, remote, and frequent measurement of PD signs and symptoms in natural living environments. The current climate of the COVID-19 pandemic creates a heightened sense of urgency for effective implementation of such strategies. In order for these technologies to be adopted in drug development studies, a regulatory-aligned consensus on best practices in implementing appropriate technologies, including the collection, processing, and interpretation of digital sensor data, is required. A growing number of collaborative initiatives are being launched to identify effective ways to advance the use of DHT in PD clinical trials. The Critical Path for Parkinson’s Consortium of the Critical Path Institute is highlighted as a case example where stakeholders collectively engaged regulatory agencies on the effective use of DHT in PD clinical trials. Global regulatory agencies, including the US Food and Drug Administration and the European Medicines Agency, are encouraging the efficiencies of data-driven engagements through multistakeholder consortia. To this end, we review how the advancement of DHT can be most effectively achieved by aligning knowledge, expertise, and data sharing in ways that maximize efficiencies