Prochlorococcus Marinus MIT9313 Research Articles

The distribution of divinyl chlorophylls a and b and the presence of ferredoxin-NADP+ reductase in Prochlorococcus marinus MIT9313 thylakoid membranes

The marine unicellular green cyanobacterium Prochlorococcus marinus MIT9313 belongs to the most abundant and photosynthetically productive genus of cyanobacteria in the oceans. This monophyletic genus use divinyl chlorophyll a (Chl a2) and b (Chl b2) to build the photosystems and the membrane-intrinsic Pcb-type antennae. We used the mild detergent n-dodecyl β D-maltopyranoside to solubilize the thylakoid membranes. Gel electrophoresis and sucrose gradient ultracentrifugation was then used to separate the complexes of the photosynthetic apparatus. The proteins and the pigments were identified by mass spectrometry. Protein complexes were characterized biochemically, and the distribution of Chl a2 and Chl b2 was determined. The photosynthetic apparatus was shown as supercomplexes formed by Photosystem II dimers with up to eight PcbB proteins; Photosystem I was present as trimers. A heterogeneous distribution of pigments was shown using sucrose gradient-enriched fractions with ratios of [Chl b2]/[Chl a2] of 2.16 ± 0.13, 1.86 ± 0.08, and 2.61 ± 0.07, for Photosystem I, Photosystem II, and PcbB, respectively. These ratios of Chl b/a are without precedent in organisms with oxygenic photosynthesis. Diaphorase activity was measured in the fractions of the sucrose gradient. Gel electrophoresis, immunodetection, and mass spectrometry were used to conclude that the commonly soluble protein ferredoxin-NADP+ reductase (FNR) is a membrane-anchored protein (probably associated to cytochrome b6f complex) in the low-light adapted Prochlorococcus marinus MIT9313.

Comparison of orthologous cyanobacterial aldehyde deformylating oxygenases in the production of volatile C3-C7 alkanes in engineered E. coli

Aldehyde deformylating oxygenase (ADO) is a unique enzyme found exclusively in photosynthetic cyanobacteria, which natively converts acyl aldehyde precursors into hydrocarbon products embedded in cellular lipid bilayers. This capacity has opened doors for potential biotechnological applications aiming at biological production of diesel-range alkanes and alkenes, which are compatible with the nonrenewable petroleum-derived end-products in current use. The development of production platforms, however, has been limited by the relative inefficiency of ADO enzyme, promoting research towards finding new strategies and information to be used for rational design of enhanced pathways for hydrocarbon over-expression. In this work we present an optimized approach to study different ADO orthologs derived from different cyanobacterial species in an in vivo set-up in Escherichia coli. The system enabled comparison of alternative ADOs for the production efficiency of short-chain volatile C3-C7 alkanes, propane, pentane and heptane, and provided insight on the differences in substrate preference, catalytic efficiency and limitations associated with the enzymes. The work concentrated on five ADO orthologs which represent the most extensively studied cyanobacterial species in the field, and revealed distinct differences between the enzymes. In most cases the ADO from Nostoc punctiforme PCC 73102 performed the best in respect to yields and initial rates for the production of the volatile hydrocarbons. At the other extreme, the system harboring the ADO form Synechococcus sp. RS9917 produced very low amounts of the short-chain alkanes, primarily due to poor accumulation of the enzyme in E. coli. The ADOs from Synechocystis sp. PCC 6803 and Prochlorococcus marinus MIT9313, and the corresponding variant A134F displayed less divergence, although variation between chain-length preferences could be observed. The results confirmed the general trend of ADOs having decreasing catalytic efficiency towards precursors of decreasing chain-length, while expanding the knowledge on the species-specific traits, which may aid future pathway design and structure-based engineering of ADO for more efficient hydrocarbon production systems.

Correction to Oxygen-Independent Alkane Formation by Non-Heme Iron-Dependent Cyanobacterial Aldehyde Decarbonylase: Investigation of Kinetics and Requirement for an External Electron Donor

Long chain alkanes are produced by a wide variety of organisms including plants (1–2), insects (3–4) and birds (5), in which they function as water-proofing agents, and by green algae where they serve as a store of cellular energy (6). These alkanes, which are typified by an odd number of carbons, are derived from fatty acid biosynthesis through the elongation of the “standard” C16 and C18 fatty acids by specialized fatty acid synthases to “very long chain” fatty acyl-CoA esters of 20 – 34 carbons (2). These acyl-CoA esters are then reduced to the corresponding aldehydes by fatty acyl-CoA reductase (7). In the final, and chemically most unusual step, cleavage of the bond between the aldehyde carbon and the α-carbon produces an odd-chain alkane; this reaction is catalyzed by aldehyde decarbonylase1 (AD) (8–9). The potential utility of this enzyme has not been lost on those seeking to develop new routes to biofuels (10). It appears that there are three distinct AD enzymes that catalyze different chemical reactions to effect removal of the aldehyde carbon. In higher plants and green algae the enzyme is a metal dependent, integral membrane protein that converts the aldehyde carbon to carbon monoxide (9, 11). In insects AD appears to be a cytochrome p450 enzyme that oxidizes the aldehyde carbon to carbon dioxide (4). In cyanobacteria AD (cAD) was recently found to be a small, soluble protein whose structure, shown in Figure 1, is closely related to the non-heme di-iron oxygenases (10). We previously demonstrated that, in the reaction catalyzed by cAD from the cyanobacterium Prochlorococcus marinus MIT9313, the aldehyde carbon is converted to formate (Figure 1) (12). Labeling studies established that the proton in the alkane derives from water and that the aldehyde proton is retained in formate (12), so that the alkane is formally derived by hydrolysis of the aldehyde, albeit in a very unusual reaction. Similar results have been independently demonstrated by Warui et al. (13) for cAD from a related cyanobacterium, Nostoc punctiformes. Figure 1 A: x-ray structure of cAD from P. marinus MIT9313, illustrating the position of the di-iron center and fatty acid bound at the active site. B: detail of the di-iron center and metal coordinating ligands. C: reaction catalyzed by cAD. The color coding ... Our initial characterization of cAD (12) established that it is an iron-dependent enzyme and that an auxiliary reducing system is required for activity. In this respect, the enzyme is similar to other di-iron oxygenases such as methane mono-oxygenase (14–15). However, we determined that oxygen is not a co-substrate in the reaction; the enzyme is in fact more active under anaerobic conditions. Furthermore, the reducing system appears to be absolutely required for alkane formation even under rigorously anaerobic conditions. EPR experiments provided evidence that free radical species are generated in the mechanism. Based on these results we proposed a tentative mechanism, shown in Figure 2, for the enzyme in which the reducing system plays a catalytic role, possibly serving to generate free radicals to facilitate the chemically difficult cleavage of the aldehyde carbon. Figure 2 Mechanistic proposal for the oxygen-independent formation of alkanes by cAD. In this mechanism the external reducing system functions catalytically to generate a reactive ketyl radical anion and facilitate carbon-carbon bond cleavage. For a detailed discussion ... Our findings stand in contrast to a labeling study by Li et al. (16) using cAD from N. punctiformes, which found that cAD activity was dependent upon the presence of oxygen and demonstrated that oxygen was incorporated into formate. To accommodate this finding the authors proposed a “cryptic oxidation” mechanism in which activated oxygen species react with the aldehyde to facilitate C-C bond cleavage. However, the rate of the oxidative reaction was orders of magnitude slower than the rate we measured for the anaerobic reaction. To address the apparent discrepancies noted above, we have directly compared the activities of cAD from representatives of 4 of the major classes of cyanobacteria: Prochlorococcus marinus MIT9313, Nostoc punctiformes PCC73102, Synechococcus sp. RS9917 and Synechocystis sp. PCC6803. These enzymes each exhibit activity under anaerobic conditions. Focusing on the P. marinus enzyme, for which a structure is available, we have undertaken a detailed investigation of the kinetics of the enzyme and the unusual role of the reducing system in the reaction.

Kinetic isotope effect and biochemical characterization of form IA RubisCO from the marine cyanobacterium Prochlorococcus marinus MIT9313

In an effort to better understand the factors influencing carbon fixation by Prochlorococcus, and to elucidate the effects of these cyanobacteria on the ocean carbon cycle, the kinetic parameters and isotopic discrimination of form IA ribulose 1,5‐bisphosphate carboxylase/oxygenase (RubisCO) from Prochlorococcus marinus MIT 9313 were determined. The RubisCO genes (cbbL and cbbS) were cloned and expressed in Escherichia coli. Enzyme was purified via ammonium sulfate precipitation, and the optimum pH and temperature, as well as Michaelis‐Menton constants, were determined radiometrically. The degree to which this RubisCO discriminates against 13CO2 during fixation was determined by the high‐precision substrate depletion method. Purified enzyme had a pH optimum of 7.5, was most active between 20°C and 30°C, had a moderate Vmax (0.41 µmol min−1 mg protein−1), and had the highest ΚCO2 value (0.75 mmol L−1) for a form I RubisCO characterized to date. The  value,  = 1,000[(Κ12/Κ13) ‐ 1], for the enzyme was determined to be 24.0‰ (95% C.I. = 22.2‐25.6‰), within the range observed for other form I RubisCOs. This  value is a critical baseline for interpreting the δ13C values of marine environmental samples, particularly those collected from the open ocean, where P. marinus is responsible for a substantial fraction of carbon fixation.

Phylogenetic analysis of perfect repeats in the genomes of unicellular cyanobacteria

We have fulfilled in silico research of number, structure, distribution and location of direct and inverted repeated sequences in eight complete genomes of unicellular cyanobacteria. Analysis of whole genome repeats has shown utility of this approach for purposes of molecular phylogeny and ecological genomics. Comparative analysis of nonrandom repeats patterns has allowed: 1) to confirm the close genetic relationship of two Prochlorococcus marinus strains (MED4 and SS120) that have reduced genomes and inhabit the econiches with different light intensities; 2) to suggest the close phylogenetic relationship of genomes Prochlorococcus marinus MIT9313 and Synechococcus WH8102 that significantly differ by sets of lightharvesting photosystem; 3) to reveal specific differences in genome organization between marine and freshwater cyanobacteria.