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Cloning, Purification, and Biochemical Characterization of PA1225, A View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by ScholarWorks @ Georgia State University Georgia State University ScholarWorks @ Georgia State University Chemistry Theses Department of Chemistry 12-11-2017 Cloning, Purification, and Biochemical Characterization of PA1225, a Novel FAD Containing NADPH:Quinone Reductase from Pseudomonas aeruginosa PAO1 Elias Flores [email protected] Follow this and additional works at: https://scholarworks.gsu.edu/chemistry_theses Recommended Citation Flores, Elias, "Cloning, Purification, and Biochemical Characterization of PA1225, a Novel FAD Containing NADPH:Quinone Reductase from Pseudomonas aeruginosa PAO1." Thesis, Georgia State University, 2017. https://scholarworks.gsu.edu/chemistry_theses/112 This Thesis is brought to you for free and open access by the Department of Chemistry at ScholarWorks @ Georgia State University. It has been accepted for inclusion in Chemistry Theses by an authorized administrator of ScholarWorks @ Georgia State University. For more information, please contact [email protected]. CLONING, PURIFICATION, AND BIOCHEMICAL CHARACTERIZATION OF PA1225, A NOVEL FAD CONTAINING NADPH:QUINONE REDUCTASE FROM Pseudomonas aeruginosa PAO1 by ELIAS FLORES Under the Direction of Giovanni Gadda, PhD ABSTRACT The gene product of pa1225 found in the opportunistic bacterium Pseudomonas aeruginosa strain PAO1 is currently annotated as a putative NAD(P)H dehydrogenase. Prior investigation of LysR (PA4203), which regulates the transcription of detoxifying enzymes, revealed PA1225 to be the most upregulated gene in the LysR deletion mutant in PAO1. A combination of steady-state and rapid kinetics was used to investigate the enzymatic properties of PA1225, which may play a potential role in the viability of P. aeruginosa PAO1 in toxic environments. In this study, it was experimentally determined that PA1225 catalyzes the oxidation of NADPH and NADH to reduce one- and two-ring quinones. PA1225 has a clear preference for NADPH as the reducing substrate as determined by a 50-fold difference in kcat/Km with respect to NADH at pH 6.0. The effect of pH was also investigated with NADPH and NADH to elucidate changes in kred and Kd. INDEX WORDS: PA1225, NADH, NADPH, Pseudomonas aeruginosa, reductive-half reaction, steady-state, quinone, LysR, detoxifying. CLONING, PURIFICATION, AND BIOCHEMICAL CHARACTERIZATION OF PA1225, A NOVEL FAD CONTAINING NADPH:QUINONE REDUCTASE FROM Pseudomonas aeruginosa PAO1 by ELIAS FLORES A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science in the College of Arts and Sciences Georgia State University 2017 Copyright by Elias Noel Flores 2017 CLONING, PURIFICATION, AND BIOCHEMICAL CHARACTERIZATION OF PA1225, A NOVEL FAD CONTAINING NADPH:QUINONE REDUCTASE FROM Pseudomonas aeruginosa PAO1 by ELIAS FLORES Committee Chair: Giovanni Gadda Committee: Dabney Dixon Donald Hamelberg Electronic Version Approved: Office of Graduate Studies College of Arts and Sciences Georgia State University December 2017 iv DEDICATION This thesis is dedicated in sweet, loving memory to Jakob Eli Camacho. v ACKNOWLEDGEMENTS I would like to thank everyone whom, in their unique way, made me into the scientist I am today. I would like to give a very special thank you to my mother, Maria Flores, for scarifying her younger years to raise me, for without her unconditional love, I would not be here writing this today. Thank you for raising me with a kind heart and for giving me the opportunity to go to a 4- year institution in a city I’ve always wanted to experience. Thank you for letting my imagination blossom as a child. I want to thank my late grandmothers Maria Flores and Serafina Vasquez. Maria for teaching me the importance of family and by showing me first hand that hard work and integrity pay off. Serafina for the beautiful memories I hold dear to this day; the midafternoon Super Mario Bros. we played together, the delicious food you prepared just for me, and the walks we would take in the garden. I want to thank Ryan Harmon, Lillian Greene, Jamie Camacho, Jennifer Maldonado, Ryan Day, Caitlin and Jake Krieger, Jess and Jasper Bloomingdale, Juan Carro, Raquel Puente, Yen- cheng chen, and Stephanie Thompson! I want to thank my second family: Chris Aguillon, Maria Vodovoz, Renata Reis, Archana Iyer, Daniel Ouedraogo, Jacob Ball, and Dan Su. I do not think I will ever find another lab with the same chemistry we all had. Thank you all for your insightful discussions and a very special thank you to Renata Reis for her work on the PA1225 crystal. Lastly, I would like to give special thanks to my advisor, Dr. Giovanni Gadda, for dedicating his time in mentoring me, valuable life advice, and for advice on the ins-and-outs of academia. If it were not for his mentorship, I would not be able to write this thesis. I would like to vi thank Dr. Dabney Dixon for the insightful discussions during our joint group meetings and the strides you take to teach young scientist. And I would also like to thank Dr. Donald Hamelberg for being on this committee and for your insightful discussions. vii TABLE OF CONTENTS ACKNOWLEDGEMENTS ............................................................................................ V LIST OF TABLES .......................................................................................................... IX LIST OF FIGURES ......................................................................................................... X 1 INTRODUCTION ...................................................................................................... 1 1.1 Purpose of the Study ........................................................................................... 2 2 EXPERIMENTAL PROCEDURES ......................................................................... 3 2.1 Materials .............................................................................................................. 3 2.2 Cloning ................................................................................................................. 3 2.3 Overexpression and Purification ....................................................................... 6 2.4 Cofactor Identification ........................................................................................ 7 2.5 Spectroscopic Properties..................................................................................... 8 2.6 Enzymatic Activity .............................................................................................. 9 2.7 Reductive Half-Reaction ................................................................................... 10 2.8 Steady-State Mechanism................................................................................... 11 2.9 Flavin-monitored Turnover ............................................................................. 12 2.10 Data Analysis.................................................................................................. 12 3 RESULTS .................................................................................................................. 14 3.1 Overexpression and Purification ..................................................................... 14 3.2 Cofactor Identification ...................................................................................... 15 viii 3.3 Reducing Substrate Identification ................................................................... 16 3.4 Oxidizing Substrate Identification ................................................................... 18 3.5 Steady-state Kinetic Mechanism with NADP+ ................................................ 20 3.6 Reductive Half-reaction at pH 6.0 ................................................................... 21 3.7 Effect of pH on the Reductive Half-reaction .................................................. 22 3.8 Flavin-monitored Turnover ............................................................................. 26 4 DISCUSSION ............................................................................................................ 27 REFERENCES ................................................................................................................ 33 ix LIST OF TABLES Table 1 Oligonucleotides used for cloning pa1225. ....................................................................... 6 Table 2: Apparent steady-state kinetic parameters of PA1225 at a fixed concentration of benzoquinone and varying concentrations of NADH or NADPH at pH 6.0. ............................... 17 Table 3: Apparent kcat values of PA1225 with various oxidizing substrates. ............................... 19 Table 4: Substrate inhibition pattern with NADP+ ....................................................................... 21 Table 5: Reductive half-reaction kinetic parameters at pH 6.0, 8.0, 9.0, and 10.0. ...................... 23 x LIST OF FIGURES Figure 1: SDS-Page of PA1225 .................................................................................................... 15 Figure 2: UV-visible absorption spectrum, fluorescence emission, and MALDI-TOF(-) spectra of PA1225 ......................................................................................................................................... 16 Figure 3: Concentration dependence of the initial rate of reaction of PA1225 with NADPH (A) and NADH (B) .............................................................................................................................
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