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UNIVERSITY OF CALIFORNIA RIVERSIDE Engineering Human Paraoxonase 2 for Quorum Quenching A Dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Biochemistry and Molecular Biology by Xin Li September 2017 Dissertation Committee: Dr. Xin Ge, Chairperson Dr. Ashok Mulchandani Dr. Manuela Martins-Green Copyright by Xin Li 2017 The Dissertation of Xin Li is approved: Committee Chairperson University of California, Riverside Acknowledgement After an intensive period, today is the day: writing this note of thanks is the finishing touch on my dissertation. It has been five years of intense learning for me, not only in the scientific arena, but also on a personal level. Writing this dissertation has had a big impact on me. I would like to reflect on the people who have supported and helped me so much throughout this time. I would express my deepest gratitude to my parents for their continuous love and non-stop support. They are my dearest friends, great listeners, talented cook, and strong allies. At the most depressed time, with their endless love and giving, I did not give up myself. They always wish their baby girl happy, respect her decisions even though sometimes they are ridiculous, and most importantly, they have faith in her. I cannot thank you enough. My sincere thanks to my advisor Dr. Xin Ge. Thank you for giving me the opportunity to be one of your graduate students. During the past four years working with you, I have learnt quite a few from you, such as scientific thinking, efficient planning, abilities to solve research problems. I would like to thank you for your patience. This training makes me become stronger, smarter, and wiser (at least in my opinion, haha!). I also want to give my sincere thanks to Dr. Ashok Mulchandani and Dr. Manuela Martins-Green. You gave me enlightening suggestions for my projects when I had difficulty solving the puzzles. Also, your support and understanding iv game me a lot of strength when I had concerns about my abilities. I am very grateful for your unselfish assistance. I would like to thank Dr. Richard Debus, Dr. Dr. Meng Chen, lab safety manager Joshua Halford, TA coworkers Kyu Lee, Carlos Rodriguez, Matthew Young. During many of these busy quarters, you all like great friends to me. Your wisdom, your stories and your experience were warm, funny, and impressive. You were all brightened and smoothed my days. I would like to thank my lab members, Long Chen, Tyler Lopez, Dr. Chuan Chen, Kibaek Lee, Matthew Chow, Evan Kruchowy; and my undergraduates, Christopher Wang, Guan-Ju Lai, Ran Ye, Jaspreet Wadhwa, Anson Tsang. Thank you all for your daily support, suggestions and discussions, all the scientific sparkling during this time. I am lucky to have you all as my colleagues and closest friends. I want to take this opportunity to thank my friends, Fei Bu, Yang Yu, Xuye Lang, Chuanxiao Chen, Chen Geng, Qi Ai, Chunyu Chen, and my roommates Jie Shi, Chenwei Tian, Honghong Wu who have helped me and had great life experience with me. Last but not the least, I would like to express my heart-felt gratitude to Jan Schlemmer. Your love and support, your humor and kindness, made my life colorful. I cannot walk this far without you. v ABSTRACT OF THE DISSERTATION Engineering Human Paraoxonase 2 for Quorum Quenching by Xin Li Doctor of Philosophy, Graduate Program in Biochemistry and Molecular Biology University of California, Riverside, September 2017 Dr. Xin Ge, Chairperson The rapid emergence of antibiotic-resistant bacteria results in a global health crisis. Among approaches to fighting against antibiotic resistance, this study focuses on quorum sensing (QS) quenching. The QS system regulates the expression of non-essential functions – virulence factors – by sensing their own population density. QS inhibitors interfere with QS communication, thus disrupting biofilm formation and inhibiting the expression of virulence factors. Different from antibiotics, manipulating QS system may halt the development of resistance. Another approach to disrupting QS is the use of quorum quenching (QQ) enzymes to abolish the biological activity of autoinducers (AIs). The first report of degradation AIs was lactonase aiiA isolated from Bacillus sp. 240B. We used aiiA for our studies as a positive QQ enzyme control because of its broad-spectrum vi AHL-degrading ability. For therapeutic purposes, prokaryotic enzymes are very likely to generate adverse immune responses. Subsequently, it would be desirable to have a therapeutic quality enzyme that is as close as possible to the native human protein. Currently, no such enzyme meets all these requirements. Human paraoxonases (huPONs) have been discovered to have AHL- lactonase-like activity, hydrolyzing lactones of various modifications, and carbon chain lengths. HuPONs in particular modulates the stress response of endothelial cells to oxidized phospholipids as well as a bacterial quorum-sensing molecule. However, huPONs are difficult to express in soluble forms, and when heterogeneously expressed, the yield is considerably low. Thus, we intend to enhance the performance of huPON2 by improving its solubility, yield, and activity. By replacing the hydrophobic helices of huPON2 with degenerate short peptide linkers, we isolated human PON2 variants exhibiting high levels of soluble expression. Engineered huPON2s are biologically functional in P. aeruginosa swimming, swarming motility and biofilm formation tests and have lactone hydrolysis activities toward a spectrum of QS molecules. Finally, I introduced random mutations to the soluble expressed huPON2s to improve its catalytic activity. This engineered huPON2 as a quorum quenching enzyme shed light on a novel selection pressure-free selection approach to fight against pathogenic infections. vii Table of Contents 1 Chapter 1 Introduction ....................................................................................... 1 1.1 THREAT OF ANTIBIOTIC RESISTANCE ........................................................................... 1 1.2 MOLECULAR MECHANISMS OF ANTIBIOTICS RESISTANCE ............................................ 3 1.2.1 PREVENTION OF ACCESS TO TARGET ....................................................................... 4 1.2.2 TARGETS PROTECTION ............................................................................................. 7 1.2.3 DIRECT MODIFICATION OF ANTIBIOTIC MOLECULE..................................................... 8 1.3 DEVELOPMENT OF NOVEL ANTIBIOTICS ..................................................................... 10 1.3.1 PEPTIDOMIMETIC ANTIMICROBIALS .......................................................................... 11 1.3.2 AMINOGLYCOSIDES AND DERIVATIVES .................................................................... 13 1.3.3 NANOPARTICLES..................................................................................................... 14 1.3.4 PROBIOTICS APPROACHES ...................................................................................... 15 1.4 QUORUM SENSING QUENCHING ................................................................................. 17 1.4.1 QUORUM SENSING SYSTEM .................................................................................... 17 1.4.2 QUORUM QUENCHING............................................................................................. 25 1.4.3 HUMAN PARAOXONASES ......................................................................................... 30 1.5 OUTLINES 32 1.6 REFERENCES 34 2 Chapter 2 Engineering Soluble Human Paraoxonase 2 for Quorum Quenching 49 2.1 ABSTRACT 49 2.2 INTRODUCTION 50 2.3 METHODS 54 viii 2.3.1 DESIGN AND CONSTRUCTION OF THE HUPON2 LIBRARY. ........................................ 54 2.3.2 SELECTION OF SOLUBLE EXPRESSED HUPON2 VARIANTS....................................... 56 2.3.3 CLONING, EXPRESSION, AND PURIFICATION OF MBP FUSED HUPON2 VARIANTS. ... 56 2.3.4 LACTONASE ACTIVITY ASSAYS................................................................................. 57 2.3.5 BIOLUMINESCENCE QUORUM SENSING BIOASSAY. .................................................. 59 2.3.6 P. AERUGINOSA PAO1 SWARMING AND SWIMMING MOTILITY TESTS........................ 60 2.4 RESULTS AND DISCUSSION ........................................................................................ 60 2.4.1 GENERATION OF SOLUBLE EXPRESSED HUPON2 VARIANTS. ................................... 60 2.4.2 QUORUM QUENCHING ACTIVITY OF HELIX-FREE SOLUBLE HUPON 2S. .................... 72 2.4.3 INHIBITION OF P. AERUGINOSA SWIMMING AND SWARMING MOTILITIES. ................... 75 2.4.4 PROMISCUOUS HYDROLYSIS ACTIVITIES OF SOLUBLE HUPON2. ............................. 81 2.5 CONCLUSION 86 2.6 REFERENCE 88 3 Chapter 3 Engineering Human Paraoxonase 2 for Enhanced Quorum- Quenching Activity 95 3.1 ABSTRACT 95 3.2 INTRODUCTION 96 3.3 MATERIALS AND METHODS ........................................................................................ 97 3.3.1 GENERATION OF HUPON2 RANDOM MUTAGENESIS LIBRARY .................................. 97 3.3.2 BIOLUMINESCENCE QUORUM QUENCHING SCREENING ASSAY ................................. 98 3.3.3 EXPRESSION AND PURIFICATION OF HUPON2 VARIANTS ......................................... 98 3.3.4 SDS-PAGE AND WESTERN BLOTTING .................................................................... 99 3.3.5 LACTONASE ACTIVITY ASSAYS............................................................................... 100 ix 3.3.6
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  • Springer Handbook of Enzymes

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    Dietmar Schomburg Ida Schomburg (Eds.) Springer Handbook of Enzymes Alphabetical Name Index 1 23 © Springer-Verlag Berlin Heidelberg New York 2010 This work is subject to copyright. All rights reserved, whether in whole or part of the material con- cerned, specifically the right of translation, printing and reprinting, reproduction and storage in data- bases. The publisher cannot assume any legal responsibility for given data. Commercial distribution is only permitted with the publishers written consent. Springer Handbook of Enzymes, Vols. 1–39 + Supplements 1–7, Name Index 2.4.1.60 abequosyltransferase, Vol. 31, p. 468 2.7.1.157 N-acetylgalactosamine kinase, Vol. S2, p. 268 4.2.3.18 abietadiene synthase, Vol. S7,p.276 3.1.6.12 N-acetylgalactosamine-4-sulfatase, Vol. 11, p. 300 1.14.13.93 (+)-abscisic acid 8’-hydroxylase, Vol. S1, p. 602 3.1.6.4 N-acetylgalactosamine-6-sulfatase, Vol. 11, p. 267 1.2.3.14 abscisic-aldehyde oxidase, Vol. S1, p. 176 3.2.1.49 a-N-acetylgalactosaminidase, Vol. 13,p.10 1.2.1.10 acetaldehyde dehydrogenase (acetylating), Vol. 20, 3.2.1.53 b-N-acetylgalactosaminidase, Vol. 13,p.91 p. 115 2.4.99.3 a-N-acetylgalactosaminide a-2,6-sialyltransferase, 3.5.1.63 4-acetamidobutyrate deacetylase, Vol. 14,p.528 Vol. 33,p.335 3.5.1.51 4-acetamidobutyryl-CoA deacetylase, Vol. 14, 2.4.1.147 acetylgalactosaminyl-O-glycosyl-glycoprotein b- p. 482 1,3-N-acetylglucosaminyltransferase, Vol. 32, 3.5.1.29 2-(acetamidomethylene)succinate hydrolase, p. 287 Vol.
  • Discovery of an L‑Fucono-1,5-Lactonase from Cog3618 of the Amidohydrolase Superfamily † § ‡ ‡ Merlin Eric Hobbs, Matthew Vetting, Howard J

    Discovery of an L‑Fucono-1,5-Lactonase from Cog3618 of the Amidohydrolase Superfamily † § ‡ ‡ Merlin Eric Hobbs, Matthew Vetting, Howard J

    Article pubs.acs.org/biochemistry Discovery of an L‑Fucono-1,5-lactonase from cog3618 of the Amidohydrolase Superfamily † § ‡ ‡ Merlin Eric Hobbs, Matthew Vetting, Howard J. Williams, Tamari Narindoshvili, ‡ § § § Devon M. Kebodeaux, Brandan Hillerich, Ronald D. Seidel, Steven C. Almo,*, † ‡ and Frank M. Raushel*, , † Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843, United States ‡ Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States § Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, United States *S Supporting Information ABSTRACT: A member of the amidohydrolase superfamily, BmulJ_04915 from Burkholderia multivorans, of unknown function was determined to hydrolyze a series of sugar lactones: L-fucono-1,4-lactone, D-arabino-1,4-lactone, L-xylono- 1,4-lactone, D-lyxono-1,4-lactone, and L-galactono-1,4-lactone. The highest activity was shown for L-fucono-1,4-lactone with a −1 × 5 −1 −1 kcat value of 140 s and a kcat/Km value of 1.0 10 M s at pH 8.3. The enzymatic product of an adjacent L-fucose dehydrogenase, BmulJ_04919, was shown to be L-fucono-1,5- lactone via nuclear magnetic resonance spectroscopy. L- Fucono-1,5-lactone is unstable and rapidly converts non- enzymatically to L-fucono-1,4-lactone. Because of the chemical instability of L-fucono-1,5-lactone, 4-deoxy-L-fucono-1,5-lac- tone was enzymatically synthesized from 4-deoxy-L-fucose using L-fucose dehydrogenase. BmulJ_04915 hydrolyzed 4-deoxy-L- −1 × 6 −1 −1 fucono-1,5-lactone with a kcat value of 990 s and a kcat/Km value of 8.0 10 M s at pH 7.1.