BilR is a gut microbial enzyme that reduces bilirubin to urobilinogen.

Metabolism of haem by-products such as bilirubin by humans and their gut microbiota is essential to human health, as excess serum bilirubin can cause jaundice and even neurological damage. The bacterial enzymes that reduce bilirubin to urobilinogen, a key step in this pathway, have remained unidentified. Here we used biochemical analyses and comparative genomics to identify BilR as a gut-microbiota-derived bilirubin reductase that reduces bilirubin to urobilinogen. We delineated the BilR sequences from similar reductases through the identification of key residues critical for bilirubin reduction and found that BilR is predominantly encoded by Firmicutes species. Analysis of human gut metagenomes revealed that BilR is nearly ubiquitous in healthy adults, but prevalence is decreased in neonates and individuals with inflammatory bowel disease. This discovery sheds light on the role of the gut microbiome in bilirubin metabolism and highlights the significance of the gut-liver axis in maintaining bilirubin homeostasis.

Toxic anti-phage defense proteins inhibited by intragenic antitoxin proteins.

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 Rpn C -terminal domains (Rpn S ), which are translated separately from the full-length proteins (Rpn L ), directly block the activities of the toxic full-length proteins. The crystal structure of RpnA S revealed 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 RpnP2 L protects Escherichia coli against certain phages. We propose many more intragenic-encoded proteins that serve regulatory roles remain to be discovered in all organisms. Significance: Here 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 longer rpn genes. 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.

Toxic antiphage defense proteins inhibited by intragenic antitoxin proteins.

Recombination-promoting nuclease (Rpn) proteins are broadly distributed across bacterial phyla, yet their functions remain unclear. Here, we report that these proteins are toxin-antitoxin systems, comprised of genes-within-genes, that combat phage infection. We show the small, highly variable Rpn C-terminal domains (RpnS), which are translated separately from the full-length proteins (RpnL), directly block the activities of the toxic RpnL. The crystal structure of RpnAS revealed a dimerization interface encompassing α 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 that plasmid-encoded RpnP2L protects Escherichia coli against certain phages. We propose that many more intragenic-encoded proteins that serve regulatory roles remain to be discovered in all organisms.

Changing Fates of the Substrate Radicals Generated in the Active Sites of the B12-Dependent Radical SAM Enzymes OxsB and AlsB.

OxsB is a B12-dependent radical SAM enzyme that catalyzes the oxidative ring contraction of 2'-deoxyadenosine 5'-phosphate to the dehydrogenated, oxetane containing precursor of oxetanocin A phosphate. AlsB is a homologue of OxsB that participates in a similar reaction during the biosynthesis of albucidin. Herein, OxsB and AlsB are shown to also catalyze radical mediated, stereoselective C2'-methylation of 2'-deoxyadenosine monophosphate. This reaction proceeds with inversion of configuration such that the resulting product also possesses a C2' hydrogen atom available for abstraction. However, in contrast to methylation, subsequent rounds of catalysis result in C-C dehydrogenation of the newly added methyl group to yield a 2'-methylidene followed by radical addition of a 5'-deoxyadenosyl moiety to produce a heterodimer. These observations expand the scope of reactions catalyzed by B12-dependent radical SAM enzymes and emphasize the susceptibility of radical intermediates to bifurcation along different reaction pathways even within the highly organized active site of an enzyme.

Two Radical SAM Enzymes Are Necessary and Sufficient for the In Vitro Production of the Oxetane Nucleoside Antiviral Agent Albucidin.

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.

Dual-function Spot 42 RNA encodes a 15-amino acid protein that regulates the CRP transcription factor.

SignificanceDual-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.

Biosynthesis of Oxetanocin-A Includes a B12-Dependent Radical SAM Enzyme That Can Catalyze both Oxidative Ring Contraction and the Demethylation of SAM.

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.

Following the electrons: peculiarities in the catalytic cycles of radical SAM enzymes.

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.

A B12-dependent radical SAM enzyme involved in oxetanocin A biosynthesis.

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.

An HD domain phosphohydrolase active site tailored for oxetanocin-A biosynthesis.

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.

In vitro characterization of LmbK and LmbO: identification of GDP-D-erythro-α-D-gluco-octose as a key intermediate in lincomycin A biosynthesis.

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.

A new low-bandgap polymer containing benzene-fused quinoxaline: significantly enhanced performance caused by one additional benzene ring.

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.

Fluorescent and colorimetric probes for mercury(II): tunable structures of electron donor and π-conjugated bridge.

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.

N-arylpyrrole-based chromophores of donor-π-donor type displaying high two-photon absorption.

A series of new N-arylpyrrole-based chromophores with the donor-π-donor structure have been designed and synthesized via Wittig-Horner-Emmons olefination and Suzuki coupling reactions. The electronic and optical properties of the designed chromophores could be well tuned by modifying the conjugation bridges and changing the groups linked to the pyrrole moieties through the C-N single bond. All the chromophores exhibited two-photon absorption activity in the range of 730-900 nm with a large two-photon absorption cross section (∼1000-1700 GM).