Zinc ion-dependent β-lactamases (MBLs) catalyze the hydrolysis of almost all β-lactam antibiotics and resist the action of clinically available β-lactamase inhibitors. We report how application of in ...silico fragment-based molecular design employing thiol-mediated metal anchorage leads to potent MBL inhibitors. The new inhibitors manifest potent inhibition of clinically important B1 subfamily MBLs, including the widespread NDM-1, IMP-1, and VIM-2 enzymes; with lower potency, some of them also inhibit clinically relevant Class A and D serine-β-lactamases. The inhibitors show selectivity for bacterial MBL enzymes compared to that for human MBL fold nucleases. Cocrystallization of one inhibitor, which shows potentiation of Meropenem activity against MBL-expressing Enterobacteriaceae, with VIM-2 reveals an unexpected binding mode, involving interactions with residues from conserved active site bordering loops.
Post-translational ribosomal protein hydroxylation is catalyzed by 2-oxoglutarate (2OG) and ferrous iron dependent oxygenases, and occurs in prokaryotes and eukaryotes. OGFOD1 catalyzes trans-3 ...prolyl hydroxylation at Pro62 of the small ribosomal subunit protein uS12 (RPS23) and is conserved from yeasts to humans. We describe crystal structures of the human uS12 prolyl 3-hydroxylase (OGFOD1) and its homolog from Saccharomyces cerevisiae (Tpa1p): OGFOD1 in complex with the broad-spectrum 2OG oxygenase inhibitors; N-oxalylglycine (NOG) and pyridine-2,4-dicarboxylate (2,4-PDCA) to 2.1 and 2.6 Å resolution, respectively; and Tpa1p in complex with NOG, 2,4-PDCA, and 1-chloro-4-hydroxyisoquinoline-3-carbonylglycine (a more selective prolyl hydroxylase inhibitor) to 2.8, 1.9, and 1.9 Å resolution, respectively. Comparison of uS12 hydroxylase structures with those of other prolyl hydroxylases, including the human hypoxia-inducible factor (HIF) prolyl hydroxylases (PHDs), reveals differences between the prolyl 3- and prolyl 4-hydroxylase active sites, which can be exploited for developing selective inhibitors of the different subfamilies.
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•Crystal structures of human OGFOD1 in complex with inhibitors are presented•The OGFOD1 and PHDs likely share a common ancestor•The OGFOD1 active site is comparable with hypoxia-inducible factor prolyl hydroxylase•Differences between OGFOD1 and PHD2 can be exploited for inhibitor design
Horita et al. present structures of the first prolyl 3-hydroxylase from humans, OGFOD1, in complex with small-molecule inhibitors. The results shed light on the catalytic mechanisms and evolution of the OGFOD1 subfamily and the related “oxygen-sensing” hypoxia-inducible factor trans-P4Hs (PHDs).
There are no clinically useful inhibitors of metallo-β-lactamases (MBLs), which are a growing problem because they hydrolyse almost all β-lactam antibacterials. Inhibition by most reported MBL ...inhibitors involves zinc ion chelation. A structure-based virtual screening approach combined with NMR filtering led to the identification of inhibitors of the clinically relevant Verona Integron-encoded MBL (VIM)-2. Crystallographic analyses reveal a new mode of MBL inhibition involving binding adjacent to the active site zinc ions, but which does not involve metal chelation. The results will aid efforts to develop new types of clinically useful inhibitors targeting MBLs/MBL-fold metallo-enzymes involved in antibacterial and anticancer drug resistance.
Variants in the FTO (fat mass and obesity associated) gene are associated with increased body mass index in humans. Here, we show by bioinformatics analysis that FTO shares sequence motifs with ...Fe(II)- and 2-oxoglutarate-dependent oxygenases. We find that recombinant murine Fto catalyzes the Fe(II)- and 2OG-dependent demethylation of 3-methylthymine in single-stranded DNA, with concomitant production of succinate, formaldehyde, and carbon dioxide. Consistent with a potential role in nucleic acid demethylation, Fto localizes to the nucleus in transfected cells. Studies of wild-type mice indicate that Fto messenger RNA (mRNA) is most abundant in the brain, particularly in hypothalamic nuclei governing energy balance, and that Fto mRNA levels in the arcuate nucleus are regulated by feeding and fasting. Studies can now be directed toward determining the physiologically relevant FTO substrate and how nucleic acid methylation status is linked to increased fat mass.
Poly- and perfluoroalkyl substances (PFAS) have become ubiquitous contaminants in the environment. Contamination of the terrestrial ecosystem can occur from the release of aqueous film forming foams ...(AFFF) used in firefighting operations. Following soil contamination with AFFF, studies report root uptake and translocation of PFAS to other plant organs, typically favouring the short chain moiety. This body of experimental work often focuses on edible organs and generally lacks entire PFAS budgets. Here, we calculate short chain (≤6 carbons) and long chain (≥6 or ≥ 7 carbons) PFAS concentrations and respective budgets for terrestrial multimedia mesocosms (plants, soil and lysimeter) of three common agricultural plants (tomato, lettuce and beet) following irrigation with low level PFAS (<1 μg L−1) contaminated river water (short chain: 167 ng L−1; long chain 526 ng L−1). Total net recoveries were strong, ranging between 91% and 118% of added PFAS across all media. While soil was the largest receptor of PFAS in general (∼70% and 115%), there was considerable mobility to various media, including vegetation (∼3% and 20%) and leachate (∼1%). Translocation of short chain PFAS to tomato flowers resulted with biomagnified concentrations (maximus >4000 ng g−1) and accounted for 1.4% of PFAS additions. While smaller tomato fruits had higher concentrations of short chain PFAS, larger fruit had more total PFAS mass. This work provides a detailed description of the fate of short and long chain PFAS when added to relatively uncontaminated terrestrial agricultural systems. We show low-level PFAS concentrations from real-world irrigation sources can affect various receptors across the multimedia landscape. This is most evident in tomato flowers and fruit where biomagnification and high total masses of short chain PFAS occurred which could have implications for pollinators and consumption, respectively.
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•Only short chain PFAS accumulated in tomato fruit.•>99% short chain translocated from roots to shoots.•PFAS budgets in the soil-plant system can be calculated for low level contamination.•Tomato flowers concentrate shot chain PFAS.
Low level PFAS contamination in river water from historical firefighting activities contaminates soil and plants. Flower concentrations in tomato plants are exceptionally high.
The stability and activity of hypoxia-inducible factor (HIF) are regulated by the post-translational hydroxylation of specific prolyl and asparaginyl residues. We show that the HIF asparaginyl ...hydroxylase, factor inhibiting HIF (FIH), also catalyzes hydroxylation of highly conserved asparaginyl residues within ankyrin repeat (AR) domains (ARDs) of endogenous Notch receptors. AR hydroxylation decreases the extent of ARD binding to FIH while not affecting signaling through the canonical Notch pathway. ARD proteins were found to efficiently compete with HIF for FIH-dependent hydroxylation. Crystallographic analyses of the hydroxylated Notch ARD (2.35Å) and of Notch peptides bound to FIH (2.4–2.6Å) reveal the stereochemistry of hydroxylation on the AR and imply that significant conformational changes are required in the ARD fold in order to enable hydroxylation at the FIH active site. We propose that ARD proteins function as natural inhibitors of FIH and that the hydroxylation status of these proteins provides another oxygen-dependent interface that modulates HIF signaling.
Homeostatic mucosal immune responses are fine-tuned by naturally evolved interactions with native microbes, and integrating these relationships into experimental models can provide new insights into ...human diseases. Here, we leverage a murine-adapted airway microbe, Bordetella pseudohinzii (Bph), to investigate how chronic colonization impacts mucosal immunity and the development of allergic airway inflammation (AAI). Colonization with Bph induces the differentiation of interleukin-17A (IL-17A)-secreting T-helper cells that aid in controlling bacterial abundance. Bph colonization protects from AAI and is associated with increased production of secretory leukocyte protease inhibitor (SLPI), an antimicrobial peptide with anti-inflammatory properties. These findings are additionally supported by clinical data showing that higher levels of upper respiratory SLPI correlate both with greater asthma control and the presence of Haemophilus, a bacterial genus associated with AAI. We propose that SLPI could be used as a biomarker of beneficial host-commensal relationships in the airway.
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•Bordetella pseudohinzii (Bph) colonizes the mouse airway and induces a Th17 response•Bph-colonized mice show reduced allergic airway inflammation (AAI) in an asthma model•The antimicrobial peptide SLPI is linked to diminished AAI and is regulated by IL-17A•In humans, upper airway microbiota composition was correlated with SLPI abundance
Asthma is known to be modified by airway microbes. Jaeger et al. use a murine-adapted bacterium to show that airway colonization evokes a Th17 response associated with increased SLPI, an antimicrobial peptide, and protection from lung inflammation. In people, SLPI was correlated with airway microbiota composition.
Ethylene is important in industry and biological signaling. In plants, ethylene is produced by oxidation of 1-aminocyclopropane-1-carboxylic acid, as catalyzed by 1-aminocyclopropane-1-carboxylic ...acid oxidase. Bacteria catalyze ethylene production, but via the fourelectron oxidation of 2-oxoglutarate to give ethylene in an arginine-dependent reaction. Crystallographic and biochemical studies on the Pseudomonas syringae ethylene-forming enzyme reveal a branched mechanism. In one branch, an apparently typical 2-oxoglutarate oxygenase reaction to give succinate, carbon dioxide, and sometimes pyrroline-5-carboxylate occurs. Alternatively, Grob-type oxidative fragmentation of a 2-oxoglutarate–derived intermediate occurs to give ethylene and carbon dioxide. Crystallographic and quantum chemical studies reveal that fragmentation to give ethylene is promoted by binding of L-arginine in a nonoxidized conformation and of 2-oxoglutarate in an unprecedented high-energy conformation that favors ethylene, relative to succinate formation.
Human prolyl‐hydroxylases (PHDs) are hypoxia‐sensing 2‐oxoglutarate (2OG) oxygenases, catalysis by which suppresses the transcription of hypoxia‐inducible factor target genes. PHD inhibition enables ...the treatment of anaemia/ischaemia‐related disease. The PHD inhibitor Molidustat is approved for the treatment of renal anaemia; it differs from other approved/late‐stage PHD inhibitors in lacking a glycinamide side chain. The first reported crystal structures of Molidustat and IOX4 (a brain‐penetrating derivative) complexed with PHD2 reveal how their contiguous triazole, pyrazolone and pyrimidine/pyridine rings bind at the active site. The inhibitors bind to the active‐site metal in a bidentate manner through their pyrazolone and pyrimidine nitrogens, with the triazole π‐π‐stacking with Tyr303 in the 2OG binding pocket. Comparison of the new structures with other PHD inhibitor complexes reveals differences in the conformations of Tyr303, Tyr310, and a mobile loop linking β2–β3, which are involved in dynamic substrate binding/product release.
Ring binder: Co‐crystal structures of human PHD2 with Molidustat and a related inhibitor provide insight into their mode of inhibition. The pyrazolone‐pyrimidine rings of Molidustat chelate the active‐site metal ion, and its triazole ring makes a π‐π‐stacking interaction with Tyr303. The results reveal altered conformations of PHD2 residues, including Tyr303 and Tyr310 on binding of Fe‐chelating PHD inhibitors.
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