Mechanistic and Structural Studies of Salicylate Biosynthesis in Pseudomonas aeruginosa

Iron is an essential element for most pathogenic bacteria. To survive and establish infections in host tissues, these pathogens must compete with the host organism for iron. One strategy is to excrete iron-chelator siderophores with very high affinity to ferric iron in the low iron environment of the host. The phenolate type siderophore, such as pyochelin in Pseudomonas aeruginosa, uses salicylate derived from chorismate as a precursor. Studies have shown that the salicylate activation by adenylation for incorporation to siderophores is associated with the growth and virulence of some pathogens. The inhibition of salicylate biosynthesis, and hence, siderophore production is considered an attractive target for the development of novel antimicrobial agents. In P. aeruginosa, salicylate is derived from chorismate via isochorismate by two enzymes: isochorismate synthase (PchA) and isochorismate-pyruvate lyase (IPL, PchB). PchB eliminates the enolpyruvyl side chain from isochorismate through a biologically unusual pericyclic reaction mechanism. PchB can also perform an adventitious pericyclic reaction that rearranges chorismate to prephenate possibly due to the homology to the E. coli chorismate mutase (CM). The primary contribution to lower the activation energy for enzymatic pericyclic reactions is controversial and may be arise from electrostatic stabilization of the transition state, or conformational stabilization of the reactive substrate. Structural and mutational studies on a key residue, lysine 42, of the active site loop suggest that rate enhancement of the two pericyclic reactions (IPL and CM) performed by PchB results from both the transition state stabilization and the reactive substrate conformation, but the relative contributions are different for each reaction. A mutation with less active site loop mobility, A43P indicates that the loop dynamics is related to catalysis. The I87T structure reveals a larger disordered region compared to the wild type structure, suggesting that conformational mobility may play a role in catalysis. PchA is an isochorismate synthase (ICS) in P. aeruginosa that removes the C4 hydroxyl group and adds a hydroxyl group to C2-chorismate. PchA is homologous to salicylate synthases from Yersinia spp. and Mycobacterium tuberculosis that convert chorismate to salicylate without requirement of an additional lyase such as PchB in P. aeruginosa. A sequence comparison between PchA with salicylate synthases of known structure suggests that two conserved residues are directly involved in the general acid and base chemistry in PchA: K221 as the general base and E269 as the general acid. Replacement of K221 and E269 with alanine respectively led to catalytically inactive enzymes, suggesting that K221 and E269 are critical for ICS catalysis. Preliminary pH dependence data for PchA supports the general acid and base mechanism of PchA catalysis. Two nonconserved residues A375 and D310 were also examined. Replacement of A375 by threonine, the corresponding residue in salicylate synthase, resulted in only residual ICS activity, indicating that A375 is not associated with the IPL-deficiency in PchA. The D310E mutant leads to two additional activities, IPL and CM. The additional activities in three reactions may due to the preferential orientation of substrates in the active site

Diabetes Alters Diurnal Rhythm of Electroretinogram in db/db Mice

Diabetic retinopathy (DR) is the most common complications of diabetes and a leading cause of blindness in the United States. The retinal neuronal changes precede the vascular dysfunction observed in DR. The electroretinogram (ERG) determines the electrical activity of retinal neural and non-neuronal cells. The retinal ERG amplitude is reduced gradually on the progression of DR to a more severe form. Circadian rhythms play an important role in the physiological function of the body. While ERG is known to exhibit a diurnal rhythm, it is not known whether a progressive increase in the duration of diabetes affects the physiological rhythm of retinal ERG. To study this, we determined the ERG rhythm of db/db mice, an animal model of type 2 diabetes at 2, 4, and 6 months of diabetes under a regular light-dark cycle and constant dark. Our studies demonstrate that the diurnal rhythm of ERG amplitude for retinal a-wave and b-wave was altered in diabetes. The implicit time was increased in db/db mice while the oscillatory potential was reduced. Moreover, there was a progressive decline in an intrinsic rhythm of ERG upon an increase in the duration of diabetes. In conclusion, our studies provide novel insights into the pathogenic mechanism of DR by showing an altered circadian rhythm of the ERG

論曹文軒兒童文學作品裏「身體受虐」對兒童本位觀的體現

曹文軒，1954年出生在江蘇省鹽城市，2016年獲得安徒生獎，該獎素有諾貝爾兒童文學獎的美譽。其創作的兒童文學作品有《草房子》、《細米》、《青銅葵花》等，作品善於觀察兒童的生活，用詩意的語言和悲憫情懷展現兒童生活悲傷和苦難的一面。大多學者研究曹文軒作品的語言技巧和主旨思想，或人物形象的塑造，缺乏從創作理念的角度切入論述，亦欠缺人物的身體書寫分析。 本文以曹文軒作品裏的兒童身體受虐為研究對象，以兒童本位觀的創作理念對照分析。第一章為緒論，分別説明本文之寫作緣起、兒童文學領域對兒童本位觀和身體敘事的研究，及本文採用的研究方法；第二章以曹文軒作品中疾病、殘疾和死亡三種身體受虐形式，探討它們如何與兒童本位觀的創作視角、兒童價值和個體意義三個層面相呼應；第三章承接上章從曹文軒作品內部對於身體書寫和兒童本位觀的分析，擴大至各個時期兒童文學對於兒童本位觀的接受——先從五四時期的兒童的發現開始，後闡釋各個時期對於兒童視角和成人視角的選擇。第四章為結論，整合全文中心要點

Entropic and enthalpic components of catalysis in the mutase and lyase activities of Pseudomonas aeruginosa PchB

This document is the Accepted Manuscript version of a Published Work that appeared in final form in the Journal of the American Chemical Society, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://doi.org/10.1021/ja202091a.The isochorismate-pyruvate lyase from Pseudomonas aeruginosa (PchB) catalyzes two pericyclic reactions, demonstrating the eponymous activity and also chorismate mutase activity. The thermodynamic parameters for these enzyme-catalyzed activities, as well as the uncatalyzed isochorismate decomposition, are reported from temperature dependence of kcat and kuncat data. The entropic effects do not contribute to enzyme catalysis as expected from previously reported chorismate mutase data. Indeed, an entropic penalty for the enzyme-catalyzed mutase reaction (ΔS‡ = -12.1 ± 0.6 cal/molK) is comparable to that of the previously reported uncatalyzed reaction, whereas that of the enzyme-catalyzed lyase reaction (ΔS‡ = -24.3 ± 0.6 cal/molK) is larger than that of the uncatalyzed lyase reaction (-15.77 ± 0.02 cal/molK) documented here. With the assumption that chemistry is rate-limiting, we propose that a reactive substrate conformation is formed upon loop closure of the active site and that ordering of the loop contributes to the entropic penalty for converting the enzyme substrate complex to the transition state

Type 2 diabetes disturbs Kir4.1 rhythm in retinal Müller cells

poster abstractThe Müller cells function as a principal glia of the retina and maintain water homeostasis and K+ concentration via the specialized inwardly rectifying K+ (Kir) channels. About six to seven Kir channels have been found, among which Kir4.1 is expressed abundantly in Müller cells. Diabetes leads to a decrease in Kir4.1 expression and of potassium currents. For this study, we hypothesized that a diurnal change in Müller cell metabolism plays an important role in regulating the Kir4.1 expression. We tested our hypothesis using an animal model of type 2 diabetes (T2D;db/db mice) and in an in vitro study on the rat Müller (rMC-1) cells. The electroretinogram (ERG) assessment was performed on db/db mice to evaluate the Müller cell dysfunction. The rhythm of protein expression of Kir4.1 was examined in rMC-1 cells by western blot. The ‘b’ wave of an ERG, a characteristic of K+ ion distribution across the retina exhibited a diurnal rhythm in a mouse retina. The oscillatory pattern of ERG response was profoundly dampened in db/db mice. The clock synchronized rMC-1 cells in vitro exhibited a consistent oscillatory pattern for clock genes. The Kir4.1 protein in rMC-1 cells showed a regular pattern of the peak and troughs, consistent with the functional cock. Our studies suggest that Kir4.1 channels possess a diurnal rhythm and with T2D this rhythm is dampened

Redesign of MST enzymes to target lyase activity instead promotes mutase and dehydratase activities

The isochorismate and salicylate synthases are members of the MST family of enzymes. The isochorismate synthases establish an equilibrium for the conversion chorismate to isochorismate and the reverse reaction. The salicylate synthases convert chorismate to salicylate with an isochorismate intermediate; therefore, the salicylate synthases perform isochorismate synthase and isochorismate-pyruvate lyase activities sequentially. While the active site residues are highly conserved, there are two sites that show trends for lyase-activity and lyase-deficiency. Using steady state kinetics and HPLC progress curves, we tested the “interchange” hypothesis that interconversion of the amino acids at these sites would promote lyase activity in the isochorismate synthases and remove lyase activity from the salicylate synthases. An alternative, “permute” hypothesis, that chorismate-utilizing enzymes are designed to permute the substrate into a variety of products and tampering with the active site may lead to identification of adventitious activities, is tested by more sensitive NMR time course experiments. The latter hypothesis held true. The variant enzymes predominantly catalyzed chorismate mutase-prephenate dehydratase activities, sequentially generating prephenate and phenylpyruvate, augmenting previously debated (mutase) or undocumented (dehydratase) adventitious activities

pH Dependence of Catalysis by Pseudomonas aeruginosa Isochorismate-Pyruvate Lyase: Implications for Transition State Stabilization and the Role of Lysine 42

This publication was made possible by funds from the American Lung Association of Kansas and from the Kansas Masonic Cancer Research Institute, by the Graduate Training Program in Dynamic Aspects of Chemical Biology NIH grant T32 GM08545 (J.O.) from the National Institute of General Medical Sciences, by NIH grant P20 RR016475 from the INBRE Program of the National Center for Research Resources, and by NIH grants R01 AI77725 and K02 AI093675 from the National Institute for Allergy and Infectious Disease.An isochorismate-pyruvate lyase with adventitious chorismate mutase activity from Pseudomonas aerugionsa (PchB) achieves catalysis of both pericyclic reactions in part by the stabilization of reactive conformations and in part by electrostatic transition-state stabilization. When the active site loop Lys42 is mutated to histidine, the enzyme develops a pH dependence corresponding to a loss of catalytic power upon deprotonation of the histidine. Structural data indicate that the change is not due to changes in active site architecture, but due to the difference in charge at this key site. With loss of the positive charge on the K42H sidechain at high pH, the enzyme retains lyase activity at approximately 100-fold lowered catalytic efficiency, but loses detectable mutase activity. We propose that both substrate organization and electrostatic transition state stabilization contribute to catalysis. However, the dominant reaction path for catalysis is dependent on reaction conditions, which influence the electrostatic properties of the enzyme active site amino acid sidechains

Lysine221 is the general base residue of the isochorismate synthase from Pseudomonas aeruginosa (PchA) in a reaction that is diffusion limited

The isochorismate synthase from Pseudomonas aeruginosa (PchA) catalyzes the conversion of chorismate to isochorismate, which is subsequently converted by a second enzyme (PchB) to salicylate for incorporation into the salicylate-capped siderophore pyochelin. PchA is a member of the MST family of enzymes, which includes the structurally homologous isochorismate synthases from E. coli (EntC and MenF) and salicylate synthases from Yersinia enterocolitica (Irp9) and Mycobacterium tuberculosis (MbtI). The latter enzymes generate isochorismate as an intermediate before generating salicylate and pyruvate. General acid – general base catalysis has been proposed for isochorismate synthesis in all five enzymes, but the residues required for the isomerization are a matter of debate, with both lysine221 and glutamate313 proposed as the general base (PchA numbering). This work includes a classical characterization of PchA with steady state kinetic analysis, solvent kinetic isotope effect analysis and by measuring the effect of viscosogens on catalysis. The results suggest that isochorismate production from chorismate by the MST enzymes is the result of general acid – general base catalysis with a lysine as the base and a glutamic acid as the acid, in reverse protonation states. Chemistry is determined to not be rate limiting, favoring the hypothesis of a conformational or binding step as the slow step

The Diurnal Rhythm of Insulin Receptor Substrate-1 (IRS-1) and Kir4.1 in Diabetes: Implications for a Clock Gene Bmal1

Purpose: Diabetes leads to the downregulation of the retinal Kir4.1 channels and Müller cell dysfunction. The insulin receptor substrate-1 (IRS-1) is a critical regulator of insulin signaling in Müller cells. Circadian rhythms play an integral role in normal physiology; however, diabetes leads to a circadian dysrhythmia. We hypothesize that diabetes will result in a circadian dysrhythmia of IRS-1 and Kir4.1 and disturbed clock gene function will have a critical role in regulating Kir4.1 channels. Methods: We assessed a diurnal rhythm of retinal IRS-1 and Kir4.1 in db/db mice. The Kir4.1 function was evaluated using a whole-cell recording of Müller cells. The rat Müller cells (rMC-1) were used to undertake in vitro studies using a siRNA. Results: The IRS-1 exhibited a diurnal rhythm in control mice; however, with diabetes, this natural rhythm was lost. The Kir4.1 levels peaked and troughed at times similar to the IRS-1 rhythm. The IRS-1 silencing in the rMC-1 led to a decrease in Kir4.1 and BMAL1. The insulin treatment of retinal explants upregulated Kir4.1 possibly via upregulation of BMAL1 and phosphorylation of IRS-1 and Akt-1. Conclusions: Our studies highlight that IRS-1, by regulating BMAL1, is an important regulator of Kir4.1 in Müller cells and the dysfunctional signaling mediated by IRS-1 may be detrimental to Kir4.1

Metformin Corrects Abnormal Circadian Rhythm and Kir4.1 Channels in Diabetes

Purpose Diabetic retinopathy (DR) is a leading cause of visual impairment. Müller cells in DR are dysfunctional due to downregulation of the inwardly rectifying potassium channel Kir4.1. Metformin, a commonly used oral antidiabetic drug, is known to elicit its action through 5′ adenosine monophosphate-activated protein kinase (AMPK), a cellular metabolic regulator; however, its effect on Kir4.1 channels is unknown. For this study, we hypothesized that metformin treatment would correct circadian rhythm disruption and Kir4.1 channel dysfunction in db/db mice. Methods Metformin was given orally to db/db mice. Wheel-running activity, retinal levels of Kir4.1, and AMPK phosphorylation were determined at study termination. In parallel, rat retinal Müller cell line (rMC-1) cells were treated using metformin and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to assess the effect of AMPK activation on the Kir4.1 channel. Results The wheel-running activity of the db/db mice was improved following the metformin treatment. The Kir4.1 level in Müller cells was corrected after metformin treatment. Metformin treatment led to an upregulation of clock regulatory genes such as melanopsin (Opn4) and aralkylamine N-acetyltransferase (Aanat). In rMC-1 cells, AMPK activation via AICAR and metformin resulted in increased Kir4.1 and intermediate core clock component Bmal-1 protein expression. The silencing of Prkaa1 (gene for AMPKα1) led to decreased Kir4.1 and Bmal-1 protein expression. Conclusions Our findings demonstrate that metformin corrects abnormal circadian rhythm and Kir4.1 channels in db/db mouse a model of type 2 diabetes. Metformin could represent a critical pharmacological agent for preventing Müller cell dysfunction observed in human DR