Mechanism of the Quorum-Quenching Lactonase (AiiA) from Bacillus thuringiensis. 1. Product-Bound Structures†‡

ABSTRACT: The N-acyl-L-homoserine lactone hydrolases (AHL lactonases) have attracted considerable attention because of their ability to quench AHL-mediated quorum-sensing pathways in Gram-negative bacteria and because of their relation to other enzymes in the metallo--lactamase superfamily. To elucidate the detailed catalytic mechanism of AHL lactonase, mutations are made on residues that presumably contribute to substrate binding and catalysis. Steady-state kinetic studies are carried out on both the wild-type and mutant enzymes using a spectrum of substrates. Two mutations, Y194F and D108N, present significant effects on the overall catalysis. On the basis of a high-resolution structural model of the enzyme-product complex, a hybrid quantum mechanical/molecular mechanical method is used to model the substrate binding orientation and to probe the effect of the Y194F mutation. Combining all experimental and computational results, we propose a detailed mechanism for the ring-opening hydrolysis of AHL substrates as catalyzed by the AHL lactonase from Bacillus thuringiensis. Several features of the mechanism that are also found in related enzymes are discussed and may help to define an evolutionary thread that connects the hydrolytic enzymes of this mechanistically diverse superfamily. Proteins in the metallo--lactamase superfamily span all three domains of life and are quite diverse, encompassin

Characterization of Quorum Sensing and Quorum Quenching Soil Bacteria Isolated from Malaysian Tropical Montane Forest

We report the production and degradation of quorum sensing N-acyl-homoserine lactones by bacteria isolated from Malaysian montane forest soil. Phylogenetic analysis indicated that these isolates clustered closely to the genera of Arthrobacter, Bacillus and Pseudomonas. Quorum quenching activity was detected in six isolates of these three genera by using a series of bioassays and rapid resolution liquid chromatography analysis. Biosensor screening and high resolution liquid chromatography-mass spectrometry analysis revealed the production of N-dodecanoyl-L-homoserine lactone (C12-HSL) by Pseudomonas frederiksbergensis (isolate BT9). In addition to degradation of a wide range of N-acyl-homoserine lactones, Arthrobacter and Pseudomonas spp. also degraded p-coumaroyl-homoserine lactone. To the best of our knowledge, this is the first documentation of Arthrobacter and Pseudomonas spp. capable of degrading p-coumaroyl-homoserine lactone and the production of C12-HSL by P. frederiksbergensis

Spectroscopic and Mechanistic Studies of Heterodimetallic Forms of Metallo-β-lactamase NDM-1

In an effort to characterize the roles of each metal ion in metallo-β-lactamase NDM-1, heterodimetallic analogues (CoCo-, ZnCo-, and CoCd-) of the enzyme were generated and characterized. UV–vis, 1H NMR, EPR, and EXAFS spectroscopies were used to confirm the fidelity of the metal substitutions, including the presence of a homogeneous, heterodimetallic cluster, with a single-atom bridge. This marks the first preparation of a metallo-β-lactamase selectively substituted with a paramagnetic metal ion, Co(II), either in the Zn1 (CoCd-NDM-1) or in the Zn2 site (ZnCo-NDM-1), as well as both (CoCo-NDM-1). We then used these metal-substituted forms of the enzyme to probe the reaction mechanism, using steady-state and stopped-flow kinetics, stopped-flow fluorescence, and rapid-freeze-quench EPR. Both metal sites show significant effects on the kinetic constants, and both paramagnetic variants (CoCd- and ZnCo-NDM-1) showed significant structural changes on reaction with substrate. These changes are discussed in terms of a minimal kinetic mechanism that incorporates all of the data

Measurement of CP observables in B-+/- -> DK +/- and B-+/- -> D pi(+/-) with D -> KS0<mml:msup>K +/-</mml:msup><mml:msup>pi -/+</mml:msup> decays

Measurements of $CP$ observables in $B^\pm \to D K^\pm$ and $B^\pm \to D \pi^\pm$ decays are presented, where $D$ represents a superposition of $D^0$ and $\bar{D}^0$ states. The $D$ meson is reconstructed in the three-body final state $K_{\rm{S}}^0K^\pm \pi^\mp$. The analysis uses samples of $B$ mesons produced in proton-proton collisions, corresponding to an integrated luminosity of 1.0, 2.0, and 6.0 fb$^{-1}$ collected with the LHCb detector at centre-of-mass energies of $\sqrt{s} =$ 7, 8, and 13 TeV, respectively. These measurements are the most precise to date, and provide important input for the determination of the CKM angle $\gamma$

Measurement of the mass difference and relative production rate of the Ωb− and Ξb− baryons

The mass difference between the Ω − b and Ξ − b baryons is measured using proton-proton collision data collected by the LHCb experiment, corresponding to an integrated luminosity of 9     fb − 1 , and is found to be m ( Ω − b ) − m ( Ξ − b ) = 248.54 ± 0.51 ( stat ) ± 0.38 ( syst )     MeV / c 2 . The mass of the Ω − b baryon is measured to be m ( Ω − b ) = 6045.9 ± 0.5 ( stat ) ± 0.6 ( syst )     MeV / c 2 . This is the most precise determination of the Ω − b mass to date. In addition, the production rate of Ω − b baryons relative to that of Ξ − b baryons is measured for the first time in p p collisions, using an LHCb dataset collected at a center-of-mass energy of 13 TeV and corresponding to an integrated luminosity of 6     fb − 1 . Reconstructing beauty baryons in the kinematic region 2 &lt; η &lt; 6 and p T &lt; 20     GeV / c with their decays to a J / ψ meson and a hyperon, the ratio f Ω − b f Ξ − b × B ( Ω − b → J / ψ Ω − ) B ( Ξ − b → J / ψ Ξ − ) = 0.120 ± 0.008 ( stat ) ± 0.008 ( syst ) , is obtained, where f Ω − b and f Ξ − b are the fragmentation fractions of b quarks into Ω − b and Ξ − b baryons, respectively, and B represents the branching fractions of their respective decays

An investigation of a quorum-quenching lactonase from Bacillus thuringiensis

textGram-negative bacteria use N-acyl homoserine lactones (AHLs) to sense population density and regulate gene expression, including virulent phenotypes. The quorum-quenching AHL lactonase from Bacillus thuringiensis cleaves the lactone ring of AHLs, disabling this mode of gene regulation. Despite the potential applications of this enzyme as an antibacterial weapon, little was known about it's lactone ring-opening mechanism. As a member of the metallo-beta-lactamase superfamily, AHL lactonase requires two divalent metal ions for catalysis. NMR experiments confirm that these metal ions are also involved in proper enzyme folding. The chemical mechanism of ring opening was explored using isotope incorporation studies, and hydrolysis was determined to proceed via a nucleophilic attack by a solvent-derived hydroxide at the carbonyl of the lactone ring. A transient, kinetically significant metal-leaving group interaction was detected in steady-state kinetic assays with AHL lactonase containing alternative divalent metal ions hydrolyzing a sulfur-containing substrate. High-resolution crystal structures implicated two residues in substrate binding and hydrolysis, Tyr194 and Asp108. Site-directed mutagenesis of these residues followed by steady-state kinetic studies with wild-type and mutant enzymes hydrolyzing a spectrum of AHL substrates revealed that mutations Y194F and D108N significantly affect catalysis. Combining these results allows the proposal of a detailed hydrolytic mechanism. The binding site for the N-acyl hydrophobic moiety was probed using steady-state kinetics with a variety of naturally occurring and non-natural AHL substrates, and these studies indicate that AHL lactonase will accept a broad range of homoserine lactone containing substrates. Crystal structures with AHL substrates and non-hydrolyzable analogs reveal two distinct binding sites for this N-acyl group. Based on the ability of this enzyme to accommodate a variety of substrates, AHL lactonase was shown to have the ability to quench quorum sensing regulated by a newly discovered class of homoserine lactone signal molecules possessing an N-aryl group using a bioassay. Steady-state kinetic studies confirm that this class of signal molecules are indeed substrates for AHL lactonase.Chemistry and Biochemistr

Human Muscle Fiber Function when Altering Phosphate, Hydrogen Ion, and Regulatory Light Chain Phosphorylation Status at Physiological Temperatures

Introduction: The impact of skeletal muscle fatigue, which is the contraction-induced decline in muscle force or power, is associated with elements that are temperature-sensitive, such as myosin regulatory light chain (RLC) phosphorylation, increased phosphate (Pi) and hydrogen (H+) ion accumulation. However, to maintain protein stability, molecular and cellular experiments that illuminate the underlying mechanisms of muscle fatigue are typically examined at temperatures from 15-30°C, which is lower than in vivo (37°C). This study sought to characterize the molecular and cellular effects of fatiguing conditions, including the rarely-studied effect of RLC phosphorylation, at physiological temperatures. Methods: Biopsies of the vastus lateralis muscle of females (n = 8) aged 71.8 + 1.3 years and males (n = 5) aged 69.4 + 1.7 years in ongoing studies (Cultivating Healthy Aging in Older Adults and Understanding Fatigue in Older Adults) were completed and single fibers used for mechanical testing. Maximal calcium-activated cellular force production and molecular-level interactions (myosin-actin cross-bridge kinetics and mechanical myofilament properties) in slow-contracting myosin heavy chain (MHC) I and fast-contracting MHC IIA fibers were tested at physiological temperatures (37°) to examine the effects of elevated Pi, H+, and RLC phosphorylation. To explore the effects of Pi and H+, repeated measures were performed on single fibers under control (pH = 7, Pi = 5 mM), high phosphate (pH = 7, Pi = 30 mM), high hydrogen ion (pH = 6.2, Pi = 5 mM) and fatigue (pH = 6.2, Pi = 30 mM) conditions. To explore the effects of RLC phosphorylation, repeated measures were performed on single fibers under control and fatigue, then control with RLC phosphorylation and fatigue with RLC phosphorylation. Results: At 37°C, specific tension (force normalized to cross-sectional area) was greater than 25°C, apparently due to greater numbers of strongly-bound cross-bridges, which in turn were established by quicker cross-bridge kinetics. With fatigue, specific tension was lower at 37° and 25°C, presumably due to fewer strongly-bound cross-bridges and slowed cross-bridge kinetics observed compared to control conditions in MHC I fibers. In MHC IIA fibers at 25°C, fatigue was accompanied by a reduction in strongly-bound cross-bridges and slower cross-bridge kinetics, but at 37°C due to increases in the work-absorbing properties of single fibers, and faster cross-bridge kinetics compared to control. When examining Pi and pH independently at 37°C, no change in specific tension was found due to increased myofilament stiffness and decreased strongly-bound cross-bridges in both MHC I and IIA fibers. In both cases for MHC I and IIA fibers, oscillatory work and power were depressed with alterations to Pi and pH, but recovered completely for MHC I fibers with fatigue due to alterations in the cross-bridge kinetic ratio, and slightly in MHC IIA due to maintenance of strongly-bound cross-bridges and faster cross-bridge kinetics. With RLC phosphorylation, specific tension was reduced compared to control conditions due to a loss of strongly-bound cross-bridges in both MHC I and IIA fibers, with an additional drop in myofilament stiffness in MHC IIA fibers. However, the relative drop in specific tension from control to fatigue was lower with RLC phosphorylation in both MHC I and IIA fibers. Shifts in the cross-bridge kinetic ratios lead to differing results for oscillatory work and power, such that MHC I fibers had dramatically increased work and power under fatigue with RLC, and in MHC IIA fibers work and power were dramatically increased under control with RLC phosphorylation. Summary: Fiber type specific changes occurred with alterations in temperature in fatiguing conditions indicate the need to conduct experiments at physiological temperatures when attempting to extrapolate to in vivo conditions. While the alterations in specific tension may not occur with changes in phosphate or hydrogen ion concentration, the change in oscillatory work and power production, i.e. force transmission or generation, may be substantial. Additionally, RLC phosphorylation brought about differing effects dependent upon the fiber type examined and fatigue status, thus the relevance of phosphorylation combined with other fatiguing metabolites, should be further questioned and quantified.Doctor of Philosophy (PhD

Calcium formate as a supplement to prevent neural tube defects

Compositions and method for treating a folate-resistant disease in a subject are disclosed. The methods involve administering to the subject an effective amount of a composition containing a formate. For example, the method can be used to reducing the risk of neural tube defects during pregnancy. The method can also be used to treat other conditions normally treatable by folate supplementation.Board of Regents, University of Texas Syste

Human mitochondrial MTHFD2 is a dual redox cofactor-specific methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase

Abstract Background Folate-dependent one-carbon metabolism provides one-carbon units for several biological processes. This pathway is highly compartmentalized in eukaryotes, with the mitochondrial pathway producing formate for use in cytoplasmic processes. The mitochondrial enzyme MTHFD2 has been reported to use NAD+ as a cofactor while the isozyme MTHFD2L utilizes NAD+ or NADP+ at physiologically relevant conditions. Because MTHFD2 is highly expressed in many cancer types, we sought to determine the cofactor preference of this enzyme. Results Kinetic analysis shows that purified human MTHFD2 exhibits dual redox cofactor specificity, utilizing either NADP+ or NAD+ with the more physiologically relevant pentaglutamate folate substrate. Conclusion These results show that the mitochondrial folate pathway isozymes MTHFD2 and MTHFD2L both exhibit dual redox cofactor specificity. Our kinetic analysis clearly supports a role for MTHFD2 in mitochondrial NADPH production, indicating that this enzyme is likely responsible for mitochondrial production of both NADH and NADPH in rapidly proliferating cells