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Recombination-promoting nuclease (Rpn) proteins are broadly distributed across bacterial phyla, yet their functions remain unclear. Here we report these proteins are new toxin-antitoxin systems, comprised of genes-within-genes, that combat phage infection. We show the small, highly variable RpnC-terminal domains (RpnS), which are translated separately from the full-length proteins (RpnL), directly block the activities of the toxic full-length proteins. The crystal structure of RpnASrevealed a dimerization interface encompassing a helix that can have four amino acid repeats whose number varies widely among strains of the same species. Consistent with strong selection for the variation, we document plasmid-encoded RpnP2LprotectsEscherichia coliagainst certain phages. We propose many more intragenic-encoded proteins that serve regulatory roles remain to be discovered in all organisms.SignificanceHere we document the function of small genes-within-genes, showing they encode antitoxin proteins that block the functions of the toxic DNA endonuclease proteins encoded by the longerrpngenes. Intriguingly, a sequence present in both long and short protein shows extensive variation in the number of four amino acid repeats. Consistent with a strong selection for the variation, we provide evidence that the Rpn proteins represent a phage defense system.

Oxetanocin A and albucidin are two oxetane natural products. While the biosynthesis of oxetanocin A has been described, less is known about albucidin. In this work, the albucidin biosynthetic gene cluster is identified in Streptomyces. Heterologous expression in a nonproducing strain demonstrates that the genes alsA and alsB are necessary and sufficient for albucidin biosynthesis confirming a previous study (Myronovskyi et al. Microorganisms 2020, 8, 237). A two-step construction of albucidin 4'-phosphate from 2'-deoxyadenosine monophosphate (2'-dAMP) is shown to be catalyzed in vitro by the cobalamin dependent radical S-adenosyl-l-methionine (SAM) enzyme AlsB, which catalyzes a ring contraction, and the radical SAM enzyme AlsA, which catalyzes elimination of a one-carbon fragment. Isotope labelling studies show that AlsB catalysis begins with stereospecific H-atom transfer of the C2'-pro-R hydrogen from 2'-dAMP to 5'-deoxyadenosine, and that the eliminated one-carbon fragment originates from C3' of 2'-dAMP.

Oxetanocin-A is an antitumor, antiviral, and antibacterial nucleoside. It is biosynthesized via the oxidative ring contraction of a purine nucleoside co-opted from primary metabolism. This reaction is catalyzed by a B12-dependent radical S-adenosyl-l-methionine (SAM) enzyme, OxsB, and a phosphohydrolase, OxsA. Previous experiments showed that the product of the OxsB/OxsA-catalyzed reaction is an oxetane aldehyde produced alongside an uncharacterized byproduct. Experiments reported herein reveal that OxsB/OxsA complex formation is crucial for the ring contraction reaction and that reduction of the aldehyde intermediate is catalyzed by a nonspecific dehydrogenase from the general cellular pool. In addition, the byproduct is identified as a 1,3-thiazinane adduct between the aldehyde and l-homocysteine. While homocysteine was never included in the OxsB/OxsA assays, the data suggest that it can be generated from SAM via S-adenosyl-l-homocysteine (SAH). Further study revealed that conversion of SAM to SAH is facilitated by OxsB; however, the subsequent conversion of SAH to homocysteine is due to protein contaminants that co-purify with OxsA. Nevertheless, the observed demethylation of SAM to SAH suggests possible methyltransferase activity of OxsB, and substrate methylation was indeed detected in the OxsB-catalyzed reaction. This work is significant because it not only completes the description of the oxetanocin-A biosynthetic pathway but also suggests that OxsB may be capable of methyltransferase activity.

Significance Dual-function RNAs base pair with target messenger RNAs as small regulatory RNAs and encode small protein regulators. However, only a limited number of these dual-function regulators have been identified. In this study, we show that a well-characterized base-pairing small RNA surprisingly also encodes a 15–amino acid protein. The very small protein binds the cyclic adenosine monophosphate receptor protein transcription factor to block activation of some promoters, raising the question of how many other transcription factors are modulated by unidentified small proteins.

Radical SAM enzymes use S-adenosyl-L-methionine as an oxidant to initiate radical-mediated transformations that would otherwise not be possible with Lewis acid/base chemistry alone. These reactions are either redox neutral or oxidative leading to certain expectations regarding the role of SAM as either a reusable cofactor or the ultimate electron acceptor during each turnover. However, these expectations are frequently not realized resulting in fundamental questions regarding the redox handling and movement of electrons associated with these biological catalysts. Herein we provide a focused perspective on several of these questions and associated hypotheses with an emphasis on recently discovered radical SAM enzymes.

Oxetanocin A (OXT-A) is a potent antitumour, antiviral and antibacterial compound. Biosynthesis of OXT-A has been linked to a plasmid-borne Bacillus megaterium gene cluster that contains four genes: oxsA, oxsB, oxrA and oxrB. Here we show that both the oxsA and oxsB genes are required for the production of OXT-A. Biochemical analysis of the encoded proteins, a cobalamin (Cbl)-dependent S-adenosylmethionine (AdoMet) radical enzyme, OxsB, and an HD-domain phosphohydrolase, OxsA, reveals that OXT-A is derived from a 2'-deoxyadenosine phosphate in an OxsB-catalysed ring contraction reaction initiated by hydrogen atom abstraction from C2'. Hence, OxsB represents the first biochemically characterized non-methylating Cbl-dependent AdoMet radical enzyme. X-ray analysis of OxsB reveals the fold of a Cbl-dependent AdoMet radical enzyme, a family of enzymes with an estimated 7,000 members. Overall, this work provides a framework for understanding the interplay of AdoMet and Cbl cofactors and expands the catalytic repertoire of Cbl-dependent AdoMet radical enzymes.

HD domain phosphohydrolase enzymes are characterized by a conserved set of histidine and aspartate residues that coordinate an active site metallocenter. Despite the important roles these enzymes play in nucleotide metabolism and signal transduction, few have been both biochemically and structurally characterized. Here, we present X-ray crystal structures and biochemical characterization of the Bacillus megaterium HD domain phosphohydrolase OxsA, involved in the biosynthesis of the antitumor, antiviral, and antibacterial compound oxetanocin-A. These studies reveal a previously uncharacterized reaction for this family; OxsA catalyzes the conversion of a triphosphorylated compound into a nucleoside, releasing one molecule of inorganic phosphate at a time. Remarkably, this functionality is a result of the OxsA active site, which based on structural and kinetic analyses has been tailored to bind the small, four-membered ring of oxetanocin-A over larger substrates. Furthermore, our OxsA structures show an active site that switches from a dinuclear to a mononuclear metal center as phosphates are eliminated from substrate.

Three 2,3-bis(5-hexylthiophen-2-yl)-6,7-bis(octyloxy)-5,8-di(thiophen-2-yl)-quinoxaline (BTTQ)-based conjugated polymers, namely, PF-BTTQ (P1), PP-BTTQ (P2), and PDCP-BTTQ (P3), were successfully synthesized for efficient polymer solar cells (PSCs) with electron-rich units of fluorene and dialkoxybenzene and electron-deficient unit dicyanobenzene, respectively. All the polymers exhibited good solubility in common organic solvents and good thermal stability. Their deep-lying HOMO energy levels enabled them good stability in the air and the relatively low HOMO energy level assured a higher open circuit potential when used in PSCs. Bulk-heterojunction solar cells were fabricated using these copolymers blended with a fullerene derivative as an acceptor. All of them exhibited promising performance, and the best device performance with power conversion efficiency up to 3.30% was achieved under one sun of AM 1.5 solar simulator illumination (100 mW/cm2). © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012

A new series of intramolecular-charge-transfer (ICT) molecules (compounds 1, 2, and 3) were synthesized by attaching various electron-donating thiophenes groups to a triphenylamine backbone with an aldehyde group as the electron acceptor. Based on the protection reaction between ethanethiol and aldehyde, the corresponding dithioacetals (compounds S1, S2, and S3) were prepared to serve as novel colorimetric and fluorescent chemosensors for Hg(2+) ions. Also, compound S1 was further utilized to construct the chemical-reaction-based conjugated polymer probe (PS1) towards Hg(2+) ions. In the presence of as little as 10 nM Hg(2+), compound PS1 displayed an apparent change in the fluorescent intensity. The sensing processes were revealed to be mediated by ICT, as confirmed by time-dependent DFT calculations. Furthermore, compound S1 was successfully applied to microscopic imaging for the detection of Hg(2+) in HeLa cells with ratiometric fluorescent methods.

A series of conjugated copolymers ( P1 and P2-CN–P4-CN ) were prepared, in which two kinds of side chains were designed: one was the repeating units similar to that of P3HT, with the aim to increase the compatible with PC 61 BM and the hole mobility; another one was the acceptor groups connected with the electron-rich backbone through the conjugated 3,4-ethylenedioxythiophene bridge, in order to broaden the absorption and lower the LUMO level. By controlling the ratios of these two kinds of side chains, the absorption band of the resultant conjugated polymers could be fine-tuned, while their energy levels nearly remained unchanged. As the result, the performance of the corresponding devices increased first, then decreased, indicating that there would be a balance between the different function of these two side chains.

Three novel conjugated polymers with N-arylpyrrole as the conjugated bridge were designed and synthesized, which emitted strong one- or two-photon excitation fluorescence in dilute tetrahydrofuran (THF) solution with high quantum yields. The maximal two-photon absorption (TPA) cross-sections of the polymers, measured by the two-photon-induced fluorescence method using femtosecond laser pulses in THF, were 752, 1114, and 1869 GM, respectively, indicating that the insertion of electron-donating or electron-withdrawing moieties into the polymer backbone could benefit to the increase of the TPA cross-section. Their large TPA cross-sections, coupled with the relatively high emission quantum yields, made these conjugated polymers attractive for practical applications, especially two-photon excited fluorescence. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011

A series of V-type chromophores with electron-rich heterocyclevinylene bridges have been synthesized via Wittig-Horner-Emmons and Vilsmeier reactions. All the target chromophores showed strong one-photon and two-photon excited emission, and their electron properties could be tuned by using different heterocycles such as furan, thiophene and pyrrole moieties in I–III. The maximum two-photon absorption (TPA) cross sections occurred at 760 nm and were measured to be in the range of 400–800 GM.

A new pyrrole-based conjugated oligomer (P1) was obtained with phenyl-linked triphenylamine moieties as an isolation group. Little aggregations were observed whether oligomer P1 was absorbed on titanium dioxide (TiO2) surface or in solid state. Since the pyrrole-based moieties in donor-π-acceptor type was the core component of oligomer P1 for light absorption, the corresponding dye-sensitized solar cell (DSSC) demonstrated the efficiency of light-to-electrical conversion by 0.48%. Higher conversion efficiency could be achieved by tuning the size of the isolation groups and the structure of the donor-π-acceptor type dyes.

Lincomycin A is a clinically useful antibiotic isolated from Streptomyces lincolnensis. It contains an unusual methylmercapto-substituted octose, methylthiolincosamide (MTL). While it has been demonstrated that the C8 backbone of MTL moiety is derived from D-fructose 6-phosphate and D-ribose 5-phosphate via a transaldol reaction catalyzed by LmbR, the subsequent enzymatic transformations leading to the MTL moiety remain elusive. Here, we report the identification of GDP-D-erythro-α-D-gluco-octose (GDP-D-α-D-octose) as a key intermediate in the MTL biosynthetic pathway. Our data show that the octose 1,8-bisphosphate intermediate is first converted to octose 1-phosphate by a phosphatase, LmbK. The subsequent conversion of the octose 1-phosphate to GDP-D-α-D-octose is catalyzed by the octose 1-phosphate guanylyltransferase, LmbO. These results provide significant insight into the lincomycin biosynthetic pathway, because the activated octose likely serves as the acceptor for the installation of the C1 sulfur appendage of MTL.

Two new alkoxy-substituted quinoxaline (Qx)-based copolymers, PBDTQx and PBDTPz, are designed and synthesized. The only difference between these two polymers is that two methyl groups of the Qx are replaced by one additional fused benzene ring. The UV-Vis absorptions, thermal stability, energy levels, field-effect carrier mobility, and photovoltaic characteristics of the two copolymers are systematically evaluated to understand the relationships between the polymer structure at the molecular level and the photovoltaic performances. Photovoltaic cells based on the PBDTPz with a structure of ITO/PEDOT:PSS/Polymer:PC(71) BM/PEO/Ca/Al exhibit a promising efficiency of 4.40%, while that of PBDTQx is relatively much poorer.

Two new low bandgap copolymers, PBDT-DBPz and PBDT-DTDBPz, were prepared, in which the only difference was the additional added thiophene bridge between the BDT donor unit and DBPz acceptor unit for PBDT-DTDBPz. In addition to the improvements of UV-vis absorption and hole mobility, the PSC based on PBDT-DTDBPz:PC71BM demonstrated a power conversion efficiency of 4.75%, much higher than that of the device based on PBDT-DBPz:PC71BM (0.46%), under the same experimental conditions.

Two new alternating copolymers. PCT-10,13-BPz (P1) and PCT-2,7-BPz (P2), which were constructed from the same monomers and only different in the connecting points, were synthesized via Suzuki coupling reaction. The UV-vis absorptions, thermal stability, energy levels, field-effect carrier mobility and photovoltaic characteristics of the two copolymers were systematically evaluated to understand the relationships between the polymer structure at the molecular level and the photovoltaic performances. Photovoltaic cells based on the two copolymers with a structure of ITO/PEDOT : PSS/Polymer : PC71BM/PFN/Al exhibited PCEs of 4.31% and 0.64%, respectively, under one sun of AM 1.5 solar simulator illumination (100 mW cm−2).