Structural and catalytic characterization of Blastochloris viridis and Pseudomonas aeruginosa homospermidine synthases supports the essential role of cation–π interaction

Polyamines influence medically relevant processes in the opportunistic pathogen Pseudomonas aeruginosa, including virulence, biofilm formation and susceptibility to antibiotics. Although homospermidine synthase (HSS) is part of the polyamine metabolism in various strains of P. aeruginosa, neither its role nor its structure has been examined so far. The reaction mechanism of the nicotinamide adenine dinucleotide (NAD+)-dependent bacterial HSS has previously been characterized based on crystal structures of Blastochloris viridis HSS (BvHSS). This study presents the crystal structure of P. aeruginosa HSS (PaHSS) in complex with its substrate putrescine. A high structural similarity between PaHSS and BvHSS with conservation of the catalytically relevant residues is demonstrated, qualifying BvHSS as a model for mechanistic studies of PaHSS. Following this strategy, crystal structures of single-residue variants of BvHSS are presented together with activity assays of PaHSS, BvHSS and BvHSS variants. For efficient homospermidine production, acidic residues are required at the entrance to the binding pocket (`ionic slide') and near the active site (`inner amino site') to attract and bind the substrate putrescine via salt bridges. The tryptophan residue at the active site stabilizes cationic reaction components by cation–π interaction, as inferred from the interaction geometry between putrescine and the indole ring plane. Exchange of this tryptophan for other amino acids suggests a distinct catalytic requirement for an aromatic interaction partner with a highly negative electrostatic potential. These findings substantiate the structural and mechanistic knowledge on bacterial HSS, a potential target for antibiotic design.

Could photosynthesis function on Proxima Centauri b?

Could oxygenic and/or anoxygenic photosynthesis exist on planet Proxima Centauri b? Proxima Centauri (spectral type – M5.5 V, 3050 K) is a red dwarf, whereas the Sun is type G2 V (5780 K). The light regimes on Earth and Proxima Centauri b are compared with estimates of the planet's suitability for Chlorophylla(Chla) and Chld-based oxygenic photosynthesis and for bacteriochlorophyll (BChl)-based anoxygenic photosynthesis. Proxima Centauri b has low irradiance in the oxygenic photosynthesis range (400–749 nm: 64–132 µmol quanta m−2s−1). Much larger amounts of light would be available for BChl-based anoxygenic photosynthesis (350–1100 nm: 724–1538 µmol quanta m−2s−1). We estimated primary production under these light regimes. We used the oxygenic algaeSynechocystisPCC6803,Prochlorothrix hollandica,Acaryochloris marina,Chlorella vulgaris,Rhodomonassp. andPhaeodactylum tricornutumand the anoxygenic photosynthetic bacteriaRhodopseudomonas palustris(BChla),Afifella marina(BChla),Thermochromatium tepidum(BChla),Chlorobaculum tepidum(BChla + c) andBlastochloris viridis(BChlb) as representative photosynthetic organisms. Proxima Centauri b has only ≈3% of the PAR (400–700 nm) of Earth irradiance, but we found that potential gross photosynthesis (Pg) on Proxima Centauri b could be surprisingly high (oxygenic photosynthesis: earth ≈0.8 gC m−2h−1; Proxima Centauri b ≈0.14 gC m−2h−1). The proportion of PAR irradiance useable by oxygenic photosynthetic organisms (the sum of Blue + Red irradiance) is similar for the Earth and Proxima Centauri b. The oxygenic photic zone would be only ≈10 m deep in water compared with ≈200 m on Earth. ThePgof an anoxic Earth (gC m−2h−1) is ≈0.34–0.59 (land) and could be as high as ≈0.29–0.44 on Proxima Centauri b. 1 m of water does not affect oxygenic or anoxygenic photosynthesis on Earth, but on Proxima Centauri b oxygenicPgis reduced by ≈50%. Effective elimination of near IR limitsPgby photosynthetic bacteria (<10% of the surface value). The spectrum of Proxima Centauri b is unfavourable for anoxygenic aquatic photosynthesis. Nevertheless, a substantial aerobic or anaerobic ecology is possible on Proxima Centauri b. Protocols to recognize the biogenic signature of anoxygenic photosynthesis are needed.

Asymmetry in serial femtosecond crystallography data

Serial crystallography is an increasingly important approach to protein crystallography that exploits both X-ray free-electron laser (XFEL) and synchrotron radiation. Serial crystallography recovers complete X-ray diffraction data by processing and merging diffraction images from thousands of randomly oriented non-uniform microcrystals, of which all observations are partial Bragg reflections. Random fluctuations in the XFEL pulse energy spectrum, variations in the size and shape of microcrystals, integrating over millions of weak partial observations and instabilities in the XFEL beam position lead to new types of experimental errors. The quality of Bragg intensity estimates deriving from serial crystallography is therefore contingent upon assumptions made while modeling these data. Here it is observed that serial femtosecond crystallography (SFX) Bragg reflections do not follow a unimodal Gaussian distribution and it is recommended that an idealized assumption of single Gaussian peak profiles be relaxed to incorporate apparent asymmetries when processing SFX data. The phenomenon is illustrated by re-analyzing data collected from microcrystals of theBlastochloris viridisphotosynthetic reaction center and comparing these intensity observations with conventional synchrotron data. The results show that skewness in the SFX observations captures the essence of the Wilson plot and an empirical treatment is suggested that can help to separate the diffraction Bragg intensity from the background.

Revised Genome Sequence of the Purple Photosynthetic Bacterium Blastochloris viridis

Blastochloris viridis is a unique anaerobic, phototrophic purple bacterium that produces bacteriochlorophyll b . Here we report an improved genome sequence of Blastochloris viridis DSM133, which is instrumental to the studies of photosynthesis, metabolic versatility, and genetic engineering of this microorganism.

Complete Genome Sequence of the Bacteriochlorophyll b -Producing Photosynthetic Bacterium Blastochloris viridis

We report the complete genome sequence of the purple photosynthetic bacterium Blastochloris viridis belonging to α- Proteobacteria . This is the first completed genome sequence of a phototroph producing bacteriochlorophyll b . The genome information will be useful for further analysis of the photosynthetic energy conversion system and bacteriochlorophyll pigment biosynthesis.

New insights into the structure of the reaction centre from Blastochloris viridis: evolution in the laboratory

Newly determined crystal structures of the photosynthetic RC (reaction centre) from two substrains of the non-sulfur purple bacterium Blastochloris viridis strain DSM 133, together with analysis of their gene sequences, has revealed intraspecies evolutionary changes over a period of 14 years. Over 100 point mutations were identified between these two substrains in the four genes encoding the protein subunits of the RC, of which approximately one-fifth resulted in a total of 16 amino acid changes. The most interesting difference was in the M subunit where the change from a leucine residue to glycine in the carotenoid-binding pocket allowed NS5 (1,2-dihydroneurosporene) to adopt a more sterically favoured conformation, similar to the carotenoid conformation found in other related RCs. The results of the present study, together with a high rate of mutations in laboratory bacterial cultures described recently, suggest that bacteria evolve faster than has been generally recognized. The possibility that amino acid changes occur within protein sequences, without exhibiting any immediately observable phenotype, should be taken into account in studies that involve long-term continuous growth of pure bacterial cultures. The Blc. viridis RC is often studied with sophisticated biophysical techniques and changes such as those described here may well affect their outcome. In other words, there is a danger that laboratory-to-laboratory variation could well be due to different groups not realising that they are actually working with slightly different proteins. A way around this problem is suggested.