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Carotenoid (Car) pigments perform central roles in photosynthesis-related light harvesting (LH), photoprotection, and assembly of functional pigment-protein complexes. However, the relationships between Car depletion in the LH, assembly of the prokaryotic reaction center (RC)-LH complex, and quinone exchange are not fully understood. Here, we analyzed native RC-LH (nRC-LH) and Car-depleted RC-LH (dRC-LH) complexes in Roseiflexus castenholzii, a chlorosome-less filamentous anoxygenic phototroph that forms the deepest branch of photosynthetic bacteria. Newly identified exterior Cars functioned with the bacteriochlorophyll B800 to block the proposed quinone channel between LHαβ subunits in the nRC-LH, forming a sealed LH ring that was disrupted by transmembrane helices from cytochrome c and subunit X to allow quinone shuttling. dRC-LH lacked subunit X, leading to an exposed LH ring with a larger opening, which together accelerated the quinone exchange rate. We also assigned amino acid sequences of subunit X and two hypothetical proteins Y and Z that functioned in forming the quinone channel and stabilizing the RC-LH interactions. This study reveals the structural basis by which Cars assembly regulates the architecture and quinone exchange of bacterial RC-LH complexes. These findings mark an important step forward in understanding the evolution and diversity of prokaryotic photosynthetic apparatus.

ABSTRACT Malonyl-CoA reductase (MCR) is a NADPH-dependent bi-functional enzyme that performs alcohol dehydrogenase and aldehyde dehydrogenase (CoA-acylating) activities in the N- and C-terminal fragments, respectively. It catalyzes the two-step reduction of malonyl-CoA to 3-hydroxypropionate (3-HP), a key reaction in the autotrophic CO2 fixation cycles of Chloroflexaceae green non-sulfur bacteria and the archaea Crenarchaeota. However, the structural basis underlying substrate selection, coordination, and the subsequent catalytic reactions of full-length MCR is largely unknown. For the first time, we here determined the structure of full-length MCR from the photosynthetic green non-sulfur bacterium Roseiflexus castenholzii (RfxMCR) at 3.35 Å resolution. Furthermore, we determined the crystal structures of the N- and C-terminal fragments bound with reaction intermediates NADP+ and malonate semialdehyde (MSA) at 2.0 Å and 2.3 Å, respectively, and elucidated the catalytic mechanisms using a combination of molecular dynamics simulations and enzymatic analyses. Full-length RfxMCR was a homodimer of two cross-interlocked subunits, each containing four tandemly arranged short-chain dehydrogenase/reductase (SDR) domains. Only the catalytic domains SDR1 and SDR3 incorporated additional secondary structures that changed with NADP+–MSA binding. The substrate, malonyl-CoA, was immobilized in the substrate-binding pocket of SDR3 through coordination with Arg1164 and Arg799 of SDR4 and the extra domain, respectively. Malonyl-CoA was successively reduced through protonation by the Tyr743–Arg746 pair in SDR3 and the catalytic triad (Thr165–Tyr178–Lys182) in SDR1 after nucleophilic attack from NADPH hydrides. IMPORTANCE The bi-functional MCR catalyzes NADPH-dependent reduction of malonyl-CoA to 3-HP, an important metabolic intermediate and platform chemical, from biomass. The individual MCR-N and MCR-C fragments, which contain the alcohol dehydrogenase and aldehyde dehydrogenase (CoA-acylating) activities, respectively, have previously been structurally investigated and reconstructed into a malonyl-CoA pathway for the biosynthetic production of 3-HP. However, no structural information for full-length MCR has been available to illustrate the catalytic mechanism of this enzyme, which greatly limits our capacity to increase the 3-HP yield of recombinant strains. Here, we report the cryo-electron microscopy structure of full-length MCR for the first time and elucidate the mechanisms underlying substrate selection, coordination, and catalysis in the bi-functional MCR. These findings provide a structural and mechanistic basis for enzyme engineering and biosynthetic applications of the 3-HP carbon fixation pathways.

Roseiflexus castenholzii is an ancient green non-sulfur bacteria that absorbs the solar energy through bacteriochlorophylls (BChls) bound in the only light harvesting (LH) complex, and transfers to the reaction center (RC), wherein primary charge separation occurs and transforms the energy into electrochemical potentials. In contrast to purple bacteria, R. castenholzii RC-LH (rcRC-LH) does not contain an H subunit. Instead, a tightly bound tetraheme cytochrome c subunit is exposed on the P-side of the RC, which contains three BChls, three bacteriopheophytins (BPheos), two menaquinones, and one iron for electron transfer. These novel structural features of the rcRC-LH are advantageous for enhancing the electron transfer efficiency and subsequent photo-oxidation of the c-type hemes. However, the photochemical properties of rcRC-LH and its applications in developing the photo-bioelectrochemical cells (PBECs) have not been characterized. Here, we prepared a PBEC using overlapped fluorine-doped tin oxide (FTO) glass and Pt-coated glass as electrodes, and rcRC-LH mixed with varying mediators as the electrolyte. Absence of the H subunit allows rcRC-LH to be selectively adhered onto the hydrophilic surface of the front electrode with its Q-side. Upon illumination, the photogenerated electrons directly enter the front electrode and transfer to the counter electrode, wherein the accepted electrons pass through the exposed c-type hemes to reduce the excited P+, generating a steady-state current of up to 320 nA/cm2 when using 1-Methoxy-5-methylphenazinium methyl sulfate (PMS) as mediator. This study demonstrated the novel photoelectric properties of rcRC-LH and its advantages in preparing effective PBECs, showcasing a potential of this complex in developing new type PBECs.

Generally, various kinds of cracks are the main type of distresses during the service period of asphalt pavements. To save maintenance costs and improve the crack resistance of asphalt pavements effectively, this paper presents a unique design method for the material composition of small particle-size (SPS) asphalt mixture for controlling cracks in asphalt pavement. First, Stone Mastic Asphalt (SMA)-II was designed as a basic gradation according to the A.N. Talbot curve and SMA-II-1, SMA-II-2 and SMA-II-3 were designed according to the Superpave mix design method, the Bailey method and the Particle interference theory, respectively. Second, based on Marshall test results, the optimal fiber content and optimal asphalt content of three gradations were determined. Then, the influence of the passing rate of 1.18-mm sieve and 0.075-mm sieve on the air voids of SMA-II and the influence of the filler-asphalt ratio on the performance of SMA-II were investigated, and an appropriate range of filler-asphalt ratio was obtained. Finally, a high-temperature performance test, a water stability test, and a skid resistance test demonstrate that the overall performance of SMA-II can satisfy the specifications. A low-temperature bend test and analysis of bending strain energy density show that SMA-II with a crumb rubber modifier and a polymer fiber has better crack resistance performance than SMA-I (SBS-modified mixture). Life-cycle cost analysis shows the economic advantage of SPS asphalt thin overlays over traditional AC-13 thin overlays.

Harmless treatment and resource utilization of wastes like flyash have always been a social difficulty and a research hot spot in the field of pavement. In this paper, samples of fly ash generated from a coal-fired power plant in Yan'an City, Shaanxi Province were collected; and a desulfurized-high-calciumfly- ash (DHCFA) was selected as the representative to be used for the alternative of traditional limestone fine powder (LFP) in asphalt mixture. Specifically, the chemical and physical characteristics of DHCFA and LFP were studied and compared firstly. Then the performance of asphalt mortar and asphalt mixture containing different amounts of DHCFA were further investigated. XRF and XRD results show that DHCFA and LFP both have significant alkalinity, and DHCFA has relatively richer chemical composition and phase distribution. TG-DSC results show DHCFA and LFP both satisfy the construction requirements on thermal stability. Asphalt mortar results showed DHCFA had the similar effect as LFP (control sample) on the whole. Asphalt mixture results revealed that the characteristics of DHCFA such as density, composition and compaction influence the performance of asphalt mixture. DHCFA could improve the low temperature performance and water stability of asphalt mixture, while reducing the high temperature performance and strength. Above results indicates that DHCFA could be used for mineral powder in asphalt mixture, however, its dosage should be carefully verified since it has adverse impact on the high temperature performance and strength of asphalt mixture. [ABSTRACT FROM AUTHOR]