Aspartate/asparagine-β-hydroxylase (AspH) is a human 2-oxoglutarate (2OG) and FeII oxygenase that catalyses C3 hydroxylations of aspartate/asparagine residues of epidermal growth factor-like domains (EGFDs). Unusually, AspH employs two histidine residues to chelate FeII rather than the typical triad of two histidine and one glutamate/aspartate residue. We report kinetic, inhibition, and crystallographic studies concerning human AspH variants in which either of its FeII binding histidine residues are substituted for alanine. Both the H725A and, in particular, the H679A AspH variants retain substantial catalytic activity. Crystal structures clearly reveal metal-ligation by only a single protein histidine ligand. The results have implications for the functional assignment of 2OG oxygenases and for the design of non-protein biomimetic catalysts.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

AspH is an endoplasmic reticulum (ER) membrane-anchored 2-oxoglutarate oxygenase whose C-terminal oxygenase and tetratricopeptide repeat (TPR) domains present in the ER lumen. AspH catalyses hydroxylation of asparaginyl- and aspartyl-residues in epidermal growth factor-like domains (EGFDs). Here we report crystal structures of human AspH, with and without substrate, that reveal substantial conformational changes of the oxygenase and TPR domains during substrate binding. Fe(II)-binding by AspH is unusual, employing only two Fe(II)-binding ligands (His679/His725). Most EGFD structures adopt an established fold with a conserved Cys1–3, 2–4, 5–6 disulfide bonding pattern; an unexpected Cys3–4 disulfide bonding pattern is observed in AspH-EGFD substrate complexes, the catalytic relevance of which is supported by studies involving stable cyclic peptide substrate analogues and by effects of Ca(II) ions on activity. The results have implications for EGFD disulfide pattern processing in the ER and will enable medicinal chemistry efforts targeting human 2OG oxygenases.

AbstractResistance to β‐lactam antibiotics mediated by metallo‐β‐lactamases (MBLs) is a growing problem. We describe the use of protein‐observe 19F‐NMR (PrOF NMR) to study the dynamics of the São Paulo MBL (SPM‐1) from β‐lactam‐resistant Pseudomonas aeruginosa. Cysteinyl variants on the α3 and L3 regions, which flank the di‐ZnII active site, were selectively 19F‐labeled using 3‐bromo‐1,1,1‐trifluoroacetone. The PrOF NMR results reveal roles for the mobile α3 and L3 regions in the binding of both inhibitors and hydrolyzed β‐lactam products to SPM‐1. These results have implications for the mechanisms and inhibition of MBLs by β‐lactams and non‐β‐lactams and illustrate the utility of PrOF NMR for efficiently analyzing metal chelation, identifying new binding modes, and studying protein binding from a mixture of equilibrating isomers.

Isocitrate dehydrogenase 1 mutations drive human gliomagenesis, probably through neomorphic enzyme activity that produces D-2-hydroxyglutarate. To model this disease, we conditionally expressed Idh1R132H in the subventricular zone (SVZ) of the adult mouse brain. The mice developed hydrocephalus and grossly dilated lateral ventricles, with accumulation of 2-hydroxyglutarate and reduced α-ketoglutarate. Stem and transit amplifying/progenitor cell populations were expanded, and proliferation increased. Cells expressing SVZ markers infiltrated surrounding brain regions. SVZ cells also gave rise to proliferative subventricular nodules. DNA methylation was globally increased, while hydroxymethylation was decreased. Mutant SVZ cells overexpressed Wnt, cell-cycle and stem cell genes, and shared an expression signature with human gliomas. Idh1R132H mutation in the major adult neurogenic stem cell niche causes a phenotype resembling gliomagenesis.

A unique structural and biophysical characterization using crystallographic, native ion-mobility mass spectrometry and 19F NMR identifies movement of a loop as being important in SPM-1 catalysis.

Objectives: Metallo-beta-lactamase (MBL)-based resistance is a threat to the use of most beta-lactam antibiotics. Multiple variants of the New Delhi MBL (NDM) have recently been reported. Previous reports indicate that the substitutions affect NDM activity despite being located outside the active site. This study compares the biochemical properties of seven clinically reported NDM variants.Methods: NDM variants were generated by site-directed mutagenesis; recombinant proteins were purified to near homogeneity. Thermal stability and secondary structures of the variants were investigated using differential scanning fluorimetry and circular dichroism; kinetic parameters and MIC values were investigated for representative carbapenem, cephalosporin and penicillin substrates.Results: The substitutions did not affect the overall folds of the NDM variants, within limits of detection; however, differences in thermal stabilities were observed. NDM-8 was the most stable variant with a melting temperature of 72 degrees C compared with 60 degrees C for NDM-1. In contrast to some previous studies, k(cat)/K-M values were similar for carbapenem and penicillin substrates for NDM variants, but differences in kinetics were observed for cephalosporin substrates. Apparent substrate inhibition was observed with nitrocefin for variants containing the M154L substitution. In all cases, cefoxitin and ceftazidime were poorly hydrolysed with k(cat)/K-M values <1 s(-1) mu M-1.Conclusions: These results do not define major differences in the catalytic efficiencies of the studied NDM variants and carbapenem or penicillin substrates. Differences in the kinetics of cephalosporin hydrolysis were observed. The results do reveal that the clinically observed substitutions can make substantial differences in thermodynamic stability, suggesting that this may be a factor in MBL evolution.

AbstractThe New Delhi metallo‐β‐lactamase (NDM‐1) is involved in the emerging antibiotic resistance problem. Development of metallo‐β‐lactamases (MBLs) inhibitors has proven challenging, due to their conformational flexibility. Here we report site‐selective labeling of NDM‐1 with 1,1,1‐trifluoro‐3‐bromo acetone (BFA), and its use to study binding events and conformational changes upon ligand–metal binding using 19F NMR spectroscopy. The results demonstrate different modes of binding of known NDM‐1 inhibitors, including L‐ and D‐captopril by monitoring the changing chemical environment of the active‐site loop of NDM‐1. The method described will be applicable to other MBLs and more generally to monitoring ligand‐induced conformational changes.

Antibiotikaresistenz ist ein weltweites Gesundheitsproblem. Metallo-β-lactamasen katalysieren die Hydrolyse von nahezu allen β-Lactam-Antibiotika. T. D. W. Claridge, C. J. Schofield et al. zeigen in ihrer Zuschrift auf S. 3193 ff., wie 19F-NMR-Spektroskopie genutzt werden kann, um die Bindungsweise von Inhibitoren der New-Delhi-Metallo-β-lactamase I (NDM-1) zu untersuchen: Man markiert eine Schleife im aktiven Zentrum mit 19F und kann dann Änderungen der lokalen chemischen Umgebung, die durch die Bindung des Inhibitors verursacht werden, NMR-spektroskopisch verfolgen. Antibiotikaresistenz ist ein weltweites Gesundheitsproblem. Metallo-β-lactamasen katalysieren die Hydrolyse von nahezu allen β-Lactam-Antibiotika. T. D. W. Claridge, C. J. Schofield et al. zeigen in ihrer Zuschrift auf S. 3193 ff., wie 19F-NMR-Spektroskopie genutzt werden kann, um die Bindungsweise von Inhibitoren der New-Delhi-Metallo-β-lactamase I (NDM-1) zu untersuchen: Man markiert eine Schleife im aktiven Zentrum mit 19F und kann dann Änderungen der lokalen chemischen Umgebung, die durch die Bindung des Inhibitors verursacht werden, NMR-spektroskopisch verfolgen. Antiaromatische Batterien Eine wiederaufladbare Batterie mit einem antiaromatischen Norcorrol-Nickel(II)-Komplex als aktives Kathodenmaterial und einer Anode aus Lithiummetall wird in der Zuschrift von H. Yoshikawa, K. Awaga, H. Shinokubo und Mitarbeitern auf S. 3160 ff. vorgestellt.1 Metallofullerene In ihrer Zuschrift auf S. 3166 ff. beschreiben K. Itami, H. Shinohara et al. eine neue Strategie zur nichtchromatographischen Extraktion von Metallofullerenen aus Lösungen von Lichtbogenruß.1 C-H-Aktivierung In ihrer Zuschrift auf S. 3184 ff. entwickeln Z. Li et al. ein Enzym zur regio- und enantioselektiven subterminalen Hydroxylierung von Alkanen durch die gerichtete Evolution terminal-selektiver P450pyr-Hydroxylase.1

Increasing antibiotic resistance is a global health concern. Metallo-β-lactamases catalyze the hydrolysis of almost all β-lactam antibiotics. T. D. W. Claridge, C. J. Schofield et al. show in their Communication on page 3129 ff. how 19F NMR spectroscopy can be used to investigate the binding mode of inhibitors to the New Delhi Metallo-β-lactamase I (NDM-1). By labeling an active-site loop with 19F, changes in the local chemical environment caused by inhibitor binding can be readily monitored by NMR spectroscopy. Increasing antibiotic resistance is a global health concern. Metallo-β-lactamases catalyze the hydrolysis of almost all β-lactam antibiotics. T. D. W. Claridge, C. J. Schofield et al. show in their Communication on page 3129 ff. how 19F NMR spectroscopy can be used to investigate the binding mode of inhibitors to the New Delhi Metallo-β-lactamase I (NDM-1). By labeling an active-site loop with 19F, changes in the local chemical environment caused by inhibitor binding can be readily monitored by NMR spectroscopy. Antiaromatic Batteries A rechargeable battery with an antiaromatic norcorrole NiII complex as a cathode-active material and a Li metal anode is described in the Communication by H. Yoshikawa, K. Awaga, H. Shinokubo and co-workers on page 3096 ff. 1 Metallofullerenes K. Itami, H. Shinohara et al. describe in their Communication on page 3102 ff., a new strategy for the nonchromatographic extraction of metallofullerenes from solutions of arc-processed raw soot.1 CH Activation In their Communication on page 3120 ff., Z. Li et al. describe the development of an enzyme by the directed evolution of terminal-selective P450pyr hydroxylase for the regio- and enantioselective subterminal hydroxylation of an alkane.1