Potent and specific antibiotic combination therapy against Clostridioides difficile.

Keratinicyclins and keratinimicins are recently discovered glycopeptide antibiotics. Keratinimicins show broad-spectrum activity against Gram-positive bacteria, while keratinicyclins form a new chemotype by virtue of an unusual oxazolidinone moiety and exhibit specific antibiosis against Clostridioides difficile. Here we report the mechanism of action of keratinicyclin B (KCB). We find that steric constraints preclude KCB from binding peptidoglycan termini. Instead, KCB inhibits C. difficile growth by binding wall teichoic acids (WTAs) and interfering with cell wall remodeling. A computational model, guided by biochemical studies, provides an image of the interaction of KCB with C. difficile WTAs and shows that the same H-bonding framework used by glycopeptide antibiotics to bind peptidoglycan termini is used by KCB for interacting with WTAs. Analysis of KCB in combination with vancomycin (VAN) shows highly synergistic and specific antimicrobial activity, and that nanomolar combinations of the two drugs are sufficient for complete growth inhibition of C. difficile, while leaving common commensal strains unaffected.

Ovoselenol, a Selenium-containing Antioxidant Derived from Convergent Evolution.

Selenium is an essential micronutrient, but its presence in biology has been limited to protein and nucleic acid biopolymers. The recent identification of the first biosynthetic pathway for selenium-containing small molecules suggests that there is a larger family of selenometabolites that remains to be discovered. Using a bioinformatic search strategy that relies on mapping of composite active site motifs, we identify a recently evolved branch of abundant and uncharacterized metalloenzymes that we predict are involved in selenometabolite biosynthesis. Biochemical studies confirm this prediction and show that these enzymes form an unusual C-Se bond onto histidine, thus giving rise to a novel selenometabolite and potent antioxidant that we have termed ovoselenol. Aside from providing insights into the evolution of this enzyme class and the structural basis of C-Se bond formation, our work offers a blueprint for charting the microbial selenometabolome in the future.

Care Transitions for Incarcerated Pregnant People: A Needs Assessment.

Incarcerated pregnant people face significant barriers when seeking health care services in prisons and jails, but little is known about their transitions from state prison health care systems to outside hospitals. This project analyzed current policies and procedures for care transitions for incarcerated people and presents policy recommendations to address issues of concern. We conducted in-depth interviews with stakeholders at a state prison, academic hospital, and private hospital to identify the barriers and facilitators to care transitions. Themes emerging from these interviews were operational, including medical records, communication, and education; and structural, including implicit biases and care of marginalized groups. These findings are likely applicable to similar facilities throughout the United States. A multipronged, interdisciplinary approach is needed to address challenges of care transitions.

Structural Characterization and Ligand-Induced Conformational Changes of SenB, a Se-Glycosyltransferase Involved in Selenoneine Biosynthesis.

Selenium (Se) is an essential micronutrient that is found naturally in proteins, nucleic acids, and natural products. Unlike selenoproteins and selenonucleic acids, little is known about the structures of biosynthetic enzymes that incorporate Se into small molecules. Here, we report the X-ray crystal structure of SenB, the first known Se-glycosyltransferase that was recently found to be involved in the biosynthesis of the Se-containing metabolite selenoneine. SenB catalyzes C-Se bond formation using selenophosphate and an activated uridine diphosphate sugar as a Se and glycosyl donor, respectively, making it the first known selenosugar synthase and one of only four bona fide C-Se bond-forming enzymes discovered to date. Our crystal structure, determined to 2.25 Å resolution, reveals that SenB is a type B glycosyltransferase, displaying the prototypical fold with two globular Rossmann-like domains and a catalytic interdomain cleft. By employing complementary structural biology techniques, we find that SenB undergoes both local and global substrate-induced conformational changes, demonstrating a significant increase in α-helicity and a transition to a more compact conformation. Our results provide the first structure of SenB and set the stage for further biochemical characterization in the future.

Insights into Substrate Recognition by the Unusual Nitrating Enzyme RufO.

Nitration reactions are crucial for many industrial syntheses; however, current protocols lack site specificity and employ hazardous chemicals. The noncanonical cytochrome P450 enzymes RufO and TxtE catalyze the only known direct aromatic nitration reactions in nature, making them attractive model systems for the development of analogous biocatalytic and/or biomimetic reactions that proceed under mild conditions. While the associated mechanism has been well-characterized in TxtE, much less is known about RufO. Herein we present the first structure of RufO alongside a series of computational and biochemical studies investigating its unusual reactivity. We demonstrate that free l-tyrosine is not readily accepted as a substrate despite previous reports to the contrary. Instead, we propose that RufO natively modifies l-tyrosine tethered to the peptidyl carrier protein of a nonribosomal peptide synthetase encoded by the same biosynthetic gene cluster and present both docking and molecular dynamics simulations consistent with this hypothesis. Our results expand the scope of direct enzymatic nitration reactions and provide the first evidence for such a modification of a peptide synthetase-bound substrate. Both of these insights may aid in the downstream development of biocatalytic approaches to synthesize rufomycin analogues and related drug candidates.

Robust Chemoenzymatic Synthesis of Keratinimicin Aglycone Analogues Facilitated by the Structure and Selectivity of OxyB.

The emergence of multidrug-resistant pathogens poses a threat to public health and requires new antimicrobial agents. As the archetypal glycopeptide antibiotic (GPA) used against drug-resistant Gram-positive pathogens, vancomycin provides a promising starting point. Peripheral alterations to the vancomycin scaffold have enabled the development of new GPAs. However, modifying the core remains challenging due to the size and complexity of this compound family. The recent successful chemoenzymatic synthesis of vancomycin suggests that such an approach can be broadly applied. Herein, we describe the expansion of chemoenzymatic strategies to encompass type II GPAs bearing all aromatic amino acids through the production of the aglycone analogue of keratinimicin A, a GPA that is 5-fold more potent than vancomycin against Clostridioides difficile. In the course of these studies, we found that the cytochrome P450 enzyme OxyBker boasts both broad substrate tolerance and remarkable selectivity in the formation of the first aryl ether cross-link on the linear peptide precursors. The X-ray crystal structure of OxyBker, determined to 2.8 Å, points to structural features that may contribute to these properties. Our results set the stage for using OxyBker broadly as a biocatalyst toward the chemoenzymatic synthesis of diverse GPA analogues.

Successional dynamics of the cultivated kelp microbiome.

Kelp are important primary producers that are colonized by diverse microbes that can have both positive and negative effects on their hosts. The kelp microbiome could support the burgeoning kelp cultivation sector by improving host growth, stress tolerance, and resistance to disease. Fundamental questions about the cultivated kelp microbiome still need to be addressed before microbiome-based approaches can be developed. A critical knowledge gap is how cultivated kelp microbiomes change as hosts grow, particularly following outplanting to sites that vary in abiotic conditions and microbial source pools. In this study we assessed if microbes that colonize kelp in the nursery stage persist after outplanting. We characterized microbiome succession over time on two species of kelp, Alaria marginata and Saccharina latissima, outplanted to open ocean cultivation sites in multiple geographic locations. We tested for host-species specificity of the microbiome and the effect of different abiotic conditions and microbial source pools on kelp microbiome stability during the cultivation process. We found the microbiome of kelp in the nursery is distinct from that of outplanted kelp. Few bacteria persisted on kelp following outplanting. Instead, we identified significant microbiome differences correlated with host species and microbial source pools at each cultivation site. Microbiome variation related to sampling month also indicates that seasonality in host and/or abiotic factors may influence temporal succession and microbiome turnover in cultivated kelps. This study provides a baseline understanding of microbiome dynamics during kelp cultivation and highlights research needs for applying microbiome manipulation to kelp cultivation.

X-ray Emission Spectroscopy of Single Protein Crystals Yields Insights into Heme Enzyme Intermediates.

Enzyme reactivity is often enhanced by changes in oxidation state, spin state, and metal-ligand covalency of associated metallocofactors. The development of spectroscopic methods for studying these processes coincidentally with structural rearrangements is essential for elucidating metalloenzyme mechanisms. Herein, we demonstrate the feasibility of collecting X-ray emission spectra of metalloenzyme crystals at a third-generation synchrotron source. In particular, we report the development of a von Hamos spectrometer for the collection of Fe Kß emission optimized for analysis of dilute biological samples. We further showcase its application in crystals of the immunosuppressive heme-dependent enzyme indoleamine 2,3-dioxygenase. Spectra from protein crystals in different states were compared with relevant reference compounds. Complementary density functional calculations assessing covalency support our spectroscopic analysis and identify active site conformations that correlate to high- and low-spin states. These experiments validate the suitability of an X-ray emission approach for determining spin states of previously uncharacterized metalloenzyme reaction intermediates.

Inhibition of Th1 activation and differentiation by dietary guar gum ameliorates experimental autoimmune encephalomyelitis.

Dietary fibers are potent modulators of immune responses that can restrain inflammation in multiple disease contexts. However, dietary fibers encompass a biochemically diverse family of carbohydrates, and it remains unknown how individual fiber sources influence immunity. In a direct comparison of four different high-fiber diets, we demonstrate a potent ability of guar gum to delay disease and neuroinflammation in experimental autoimmune encephalomyelitis, a T cell-mediated mouse model of multiple sclerosis. Guar gum-specific alterations to the microbiota are limited, and disease protection appears to be independent of fiber-induced increases in short-chain fatty acid levels or regulatory CD4+ T cells. Instead, CD4+ T cells of guar gum-supplemented mice are less encephalitogenic due to reduced activation, proliferation, Th1 differentiation, and altered migratory potential. These findings reveal specificity in the host response to fiber sources and define a pathway of fiber-induced immunomodulation that protects against pathologic neuroinflammation.

Structural insights into inhibition of the drug target dihydroorotate dehydrogenase by bacterial hydroxyalkylquinolines.

Hydroxyalkylquinolines (HAQs) are ubiquitious natural products but their interactions with associated protein targets remain elusive. We report X-ray crystal structures of two HAQs in complex with dihydroorotate dehydrogenase (DHODH). Our results reveal the structural basis of DHODH inhibition by HAQs and open the door to downstream structure-activity relationship studies.

Structural basis for an unprecedented enzymatic alkylation in cylindrocyclophane biosynthesis.

The cyanobacterial enzyme CylK assembles the cylindrocyclophane natural products by performing two unusual alkylation reactions, forming new carbon-carbon bonds between aromatic rings and secondary alkyl halide substrates. This transformation is unprecedented in biology, and the structure and mechanism of CylK are unknown. Here, we report X-ray crystal structures of CylK, revealing a distinctive fusion of a Ca2+-binding domain and a ß-propeller fold. We use a mutagenic screening approach to locate CylK's active site at its domain interface, identifying two residues, Arg105 and Tyr473, that are required for catalysis. Anomalous diffraction datasets collected with bound bromide ions, a product analog, suggest that these residues interact with the alkyl halide electrophile. Additional mutagenesis and molecular dynamics simulations implicate Asp440 in activating the nucleophilic aromatic ring. Bioinformatic analysis of CylK homologs from other cyanobacteria establishes that they conserve these key catalytic amino acids, but they are likely associated with divergent reactivity and altered secondary metabolism. By gaining a molecular understanding of this unusual biosynthetic transformation, this work fills a gap in our understanding of how alkyl halides are activated and used by enzymes as biosynthetic intermediates, informing enzyme engineering, catalyst design, and natural product discovery.

Computational Approaches: An Underutilized Tool in the Quest to Elucidate Radical SAM Dynamics.

Enzymes are biological catalysts whose dynamics enable their reactivity. Visualizing conformational changes, in particular, is technically challenging, and little is known about these crucial atomic motions. This is especially problematic for understanding the functional diversity associated with the radical S-adenosyl-L-methionine (SAM) superfamily whose members share a common radical mechanism but ultimately catalyze a broad range of challenging reactions. Computational chemistry approaches provide a readily accessible alternative to exploring the time-resolved behavior of these enzymes that is not limited by experimental logistics. Here, we review the application of molecular docking, molecular dynamics, and density functional theory, as well as hybrid quantum mechanics/molecular mechanics methods to the study of these enzymes, with a focus on understanding the mechanistic dynamics associated with turnover.

The microbiota of intertidal macroalgae Fucus distichus is site-specific and resistant to change following transplant.

It is unclear how host-associated microbial communities will be affected by future environmental change. Characterizing how microbiota differ across sites with varying environmental conditions and assessing the stability of the microbiota in response to abiotic variation are critical steps towards predicting outcomes of environmental change. Intertidal organisms are valuable study systems because they experience extreme variation in environmental conditions on tractable timescales such as tide cycles and across small spatial gradients in the intertidal zone. Here we show a widespread intertidal macroalgae, Fucus distichus, hosts site-specific microbiota over small (meters to kilometres) spatial scales. We demonstrate stability of site-specific microbial associations by manipulating the host environment and microbial species pool with common garden and reciprocal transplant experiments. We hypothesized that F. distichus microbiota would readily shift to reflect the contemporary environment due to selective filtering by abiotic conditions and/or colonization by microbes from the new environment or nearby hosts. Instead, F. distichus microbiota was stable for days after transplantation in both the laboratory and field. Our findings expand the current understanding of microbiota dynamics on an intertidal foundation species. These results may also point to adaptations for withstanding short-term environmental variation, in hosts and/or microbes, facilitating stable host-microbial associations.

Kelp-associated Microbiota are Structured by Host Anatomy1.

Seaweed-associated microbiota are essential for the health and resilience of nearshore ecosystems, marine biogeochemical cycling, and host health. Yet much remains unknown about the ecology of seaweed-microbe symbioses. In this study, we quantified fine-scale patterns of microbial community structure across distinct anatomical regions of the kelp Laminaria setchellii. These anatomical regions represent a gradient of tissue ages: perennial holdfasts can be several years old, whereas stipe epicortex and blades are younger annual structures. Within blades, new growth occurs at the base, while the blade tips may be several months old and undergoing senescence. We hypothesized that microbial communities will differ across anatomical regions (holdfast, stipe, blade base, and blade tip), such that younger tissues will harbor fewer microbes that are more consistent across replicate individuals. Our data support this hypothesis, with the composition of bacterial (16S rRNA gene) and microeukaryote (18S rRNA gene) communities showing significant differences across the four anatomical regions, with the surfaces of older tissues (holdfast and blade tips) harboring significantly greater microbial richness compared to the younger tissues of the meristematic region. Additional samples collected from the surfaces of new L. setchellii recruits (<1y old) also showed differences in microbial community structure across anatomical regions, which demonstrates that these microbial differences are established early. We also observed this pattern in two additional algal species, suggesting that microbial community structure across host anatomy may be a common feature of the seaweed microbiome.

An Iron(IV)-Oxo Intermediate Initiating l-Arginine Oxidation but Not Ethylene Production by the 2-Oxoglutarate-Dependent Oxygenase, Ethylene-Forming Enzyme.

Ethylene-forming enzyme (EFE) is an ambifunctional iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase. In its major (EF) reaction, it converts carbons 1, 2, and 5 of 2OG to CO2 and carbons 3 and 4 to ethylene, a four-electron oxidation drastically different from the simpler decarboxylation of 2OG to succinate mediated by all other Fe/2OG enzymes. EFE also catalyzes a minor reaction, in which the normal decarboxylation is coupled to oxidation of l-arginine (a required activator for the EF pathway), resulting in its conversion to l-glutamate semialdehyde and guanidine. Here we show that, consistent with precedent, the l-Arg-oxidation (RO) pathway proceeds via an iron(IV)-oxo (ferryl) intermediate. Use of 5,5-[2H2]-l-Arg slows decay of the ferryl complex by >16-fold, implying that RO is initiated by hydrogen-atom transfer (HAT) from C5. That this large substrate deuterium kinetic isotope effect has no impact on the EF:RO partition ratio implies that the same ferryl intermediate cannot be on the EF pathway; the pathways must diverge earlier. Consistent with this conclusion, the variant enzyme bearing the Asp191Glu ligand substitution accumulates ∼4 times as much of the ferryl complex as the wild-type enzyme and exhibits a ∼40-fold diminished EF:RO partition ratio. The selective detriment of this nearly conservative substitution to the EF pathway implies that it has unusually stringent stereoelectronic requirements. An active-site, like-charge guanidinium pair, which involves the l-Arg substrate/activator and is unique to EFE among four crystallographically characterized l-Arg-modifying Fe/2OG oxygenases, may serve to selectively stabilize the transition state leading to the unique EF branch.

Steric Enforcement of cis-Epoxide Formation in the Radical C-O-Coupling Reaction by Which (S)-2-Hydroxypropylphosphonate Epoxidase (HppE) Produces Fosfomycin.

(S)-2-Hydroxypropylphosphonate [(S)-2-HPP, 1] epoxidase (HppE) reduces H2O2 at its nonheme-iron cofactor to install the oxirane "warhead" of the antibiotic fosfomycin. The net replacement of the C1 pro-R hydrogen of 1 by its C2 oxygen, with inversion of configuration at C1, yields the cis-epoxide of the drug [(1R,2S)-epoxypropylphosphonic acid (cis-Fos, 2)]. Here we show that HppE achieves ∼95% selectivity for C1 inversion and cis-epoxide formation via steric guidance of a radical-coupling mechanism. Published structures of the HppE·FeII·1 and HppE·ZnII·2 complexes reveal distinct pockets for C3 of the substrate and product and identify four hydrophobic residues-Leu120, Leu144, Phe182, and Leu193-close to C3 in one of the complexes. Replacement of Leu193 in the substrate C3 pocket with the bulkier Phe enhances stereoselectivity (cis:trans ∼99:1), whereas the Leu120Phe substitution in the product C3 pocket diminishes it (∼82:18). Retention of C1 configuration and trans-epoxide formation become predominant with the bulk-reducing Phe182Ala substitution in the substrate C3 pocket (∼13:87), trifluorination of C3 (∼23:77), or both (∼1:99). The effect of C3 trifluorination is counteracted by the more constrained substrate C3 pockets in the Leu193Phe (∼56:44) and Leu144Phe/Leu193Phe (∼90:10) variants. The ability of HppE to epoxidize substrate analogues bearing halogens at C3, C1, or both is inconsistent with a published hypothesis of polar cyclization via a C1 carbocation. Rather, specific enzyme-substrate contacts drive inversion of the C1 radical-as proposed in a recent computational study-to direct formation of the more potently antibacterial cis-epoxide by radicaloid C-O coupling.

Structure of a Ferryl Mimic in the Archetypal Iron(II)- and 2-(Oxo)-glutarate-Dependent Dioxygenase, TauD.

Iron(II)- and 2-(oxo)-glutarate-dependent (Fe/2OG) oxygenases catalyze a diverse array of oxidation reactions via a common iron(IV)-oxo (ferryl) intermediate. Although the intermediate has been characterized spectroscopically, its short lifetime has precluded crystallograhic characterization. In solution, the ferryl was first observed directly in the archetypal Fe/2OG hydroxylase, taurine:2OG dioxygenase (TauD). Here, we substitute the iron cofactor of TauD with the stable vanadium(IV)-oxo (vanadyl) ion to obtain crystal structures mimicking the key ferryl complex. Intriguingly, whereas the structure of the TauD·(VIV-oxo)·succinate·taurine complex exhibits the expected orientation of the V≡O bond-trans to the His255 ligand and toward the C-H bond to be cleaved, in what has been termed the in-line configuration-the TauD·(VIV-oxo) binary complex is best modeled with its oxo ligand trans to Asp101. This off-line-like configuration is similar to one recently posited as a means to avoid hydroxylation in Fe/2OG enzymes that direct other outcomes, though neither has been visualized in an Fe/2OG structure to date. Whereas an off-line (trans to the proximal His) or off-line-like (trans to the carboxylate ligand) ferryl is unlikely to be important in the hydroxylation reaction of TauD, the observation that the ferryl may deviate from an in-line orientation in the absence of the primary substrate may explain the enzyme's mysterious self-hydroxylation behavior, should the oxo ligand lie trans to His99. This finding reinforces the potential for analogous functional off-line oxo configurations in halogenases, desaturases, and/or cyclases.

Potent Activation of Indoleamine 2,3-Dioxygenase by Polysulfides.

Indoleamine 2,3-dioxygenase (IDO1) is a heme enzyme that catalyzes the oxygenation of the indole ring of tryptophan to afford N-formylkynurenine. This activity significantly suppresses the immune response, mediating inflammation and autoimmune reactions. These consequential effects are regulated through redox changes in the heme cofactor of IDO1, which autoxidizes to the inactive ferric state during turnover. This change in redox status increases the lability of the heme cofactor leading to further suppression of activity. The cell can thus regulate IDO1 activity through the supply of heme and reducing agents. We show here that polysulfides bind to inactive ferric IDO1 and reduce it to the oxygen-binding ferrous state, thus activating IDO1 to maximal turnover even at low, physiologically significant concentrations. The on-rate for hydrogen disulfide binding to ferric IDO1 was found to be >106 M-1 s-1 at pH 7 using stopped-flow spectrometry. Fe K-edge XANES and EPR spectroscopy indicated initial formation of a low-spin ferric sulfur-bound species followed by reduction to the ferrous state. The µM affinity of polysulfides for IDO1 implicates these polysulfides as important signaling factors in immune regulation through the kynurenine pathway. Tryptophan significantly enhanced the relatively lower-affinity binding of hydrogen sulfide to IDO1, inspiring the use of the small molecule 3-mercaptoindole (3MI), which selectively binds to and activates ferric IDO1. 3MI sustains turnover by catalytically transferring reducing equivalents from glutathione to IDO1, representing a novel strategy of upregulating innate immunosuppression for treatment of autoimmune disorders. Reactive sulfur species are thus likely unrecognized immune-mediators with potential as therapeutic agents through these interactions with IDO1.

Structures of Class Id Ribonucleotide Reductase Catalytic Subunits Reveal a Minimal Architecture for Deoxynucleotide Biosynthesis.

Class I ribonucleotide reductases (RNRs) share a common mechanism of nucleotide reduction in a catalytic α subunit. All RNRs initiate catalysis with a thiyl radical, generated in class I enzymes by a metallocofactor in a separate ß subunit. Class Id RNRs use a simple mechanism of cofactor activation involving oxidation of a MnII2 cluster by free superoxide to yield a metal-based MnIIIMnIV oxidant. This simple cofactor assembly pathway suggests that class Id RNRs may be representative of the evolutionary precursors to more complex class Ia-c enzymes. X-ray crystal structures of two class Id α proteins from Flavobacterium johnsoniae ( Fj) and Actinobacillus ureae ( Au) reveal that this subunit is distinctly small. The enzyme completely lacks common N-terminal ATP-cone allosteric motifs that regulate overall activity, a process that normally occurs by dATP-induced formation of inhibitory quaternary structures to prevent productive ß subunit association. Class Id RNR activity is insensitive to dATP in the Fj and Au enzymes evaluated here, as expected. However, the class Id α protein from Fj adopts higher-order structures, detected crystallographically and in solution. The Au enzyme does not exhibit these quaternary forms. Our study reveals structural similarity between bacterial class Id and eukaryotic class Ia α subunits in conservation of an internal auxiliary domain. Our findings with the Fj enzyme illustrate that nucleotide-independent higher-order quaternary structures can form in simple RNRs with truncated or missing allosteric motifs.

A genetics-free method for high-throughput discovery of cryptic microbial metabolites.

Bacteria contain an immense untapped trove of novel secondary metabolites in the form of 'silent' biosynthetic gene clusters (BGCs). These can be identified bioinformatically but are not expressed under normal laboratory growth conditions. Methods to access their products would dramatically expand the pool of bioactive compounds. We report a universal high-throughput method for activating silent BGCs in diverse microorganisms. Our approach relies on elicitor screening to induce the secondary metabolome of a given strain and imaging mass spectrometry to visualize the resulting metabolomes in response to ~500 conditions. Because it does not require challenging genetic, cloning, or culturing procedures, this method can be used with both sequenced and unsequenced bacteria. We demonstrate the power of the approach by applying it to diverse bacteria and report the discovery of nine cryptic metabolites with potentially therapeutic bioactivities, including a new glycopeptide chemotype with potent inhibitory activity against a pathogenic virus.