Utah State University DigitalCommons@USU All Graduate Theses and Dissertations Graduate Studies 12-2010 Kinetic, Mechanistic, and Structural Investigation of Features Controlling Stereoselectivity of (R)- and (S)-Hydroxypropyl CoM Dehydrogenases from Xanthobacter autrophicus Strain Py2 Dariusz Adam Sliwa Utah State University Follow this and additional works at: https://digitalcommons.usu.edu/etd Part of the Biochemistry Commons, Chemistry Commons, and the Microbiology Commons Recommended Citation Sliwa, Dariusz Adam, "Kinetic, Mechanistic, and Structural Investigation of Features Controlling Stereoselectivity of (R)- and (S)-Hydroxypropyl CoM Dehydrogenases from Xanthobacter autrophicus Strain Py2" (2010). All Graduate Theses and Dissertations. 755. https://digitalcommons.usu.edu/etd/755 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@USU. It has been accepted for inclusion in All Graduate Theses and Dissertations by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. KINETIC, MECHANISTIC, AND STRUCTURAL INVESTIGATION OF FEATURES CONTROLLING STEREOSELECTIVITY OF (R)- AND (S)-HYDROXYPROPYL COM DEHYDROGENASES FROM XANTHOBACTER AUTOTROPHICUS STRAIN PY2 by Dariusz Adam Sliwa A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Biochemistry Approved: ____________________________ ___________________________ Scott A. Ensign, Ph.D. Lance C. Seefeldt, Ph.D. Major Professor Committee Member ___________________________ ____________________________ Sean J. Johnson, Ph.D. John L. Hubbard, Ph.D. Committee Member Committee Member ___________________________ ____________________________ Jeff R. Broadbent, Ph.D. Byron R. Burnham, Ph.D. Committee Member Dean of Graduate Studies UTAH STATE UNIVERSITY Logan, Utah 2010 ii Copyright © Dariusz Adam Sliwa 2010 All Rights Reserved iii ABSTRACT Kinetic, Mechanistic, and Structural Investigation of Features Controlling Stereoselectivity of (R)- and (S)-Hydroxypropyl CoM Dehydrogenases from Xanthobacter autrophicus Strain Py2 by Dariusz Adam Sliwa, Doctor of Philosophy Utah State University, 2010 Major Professor: Dr. Scott A. Ensign Department: Chemistry and Biochemistry Enantiopure alcohols are valuable intermediates in fine organic synthesis, in particular for preparation of biologically active compounds. The necessity of preparing single enantiomer drugs in an optically pure form has triggered much research, especially in the pharmaceutical industry. The biocatalytical production of chiral alcohols by alcohol dehydrogenase enzymes is characterized by the asymmetric reduction of the corresponding ketones, usually with high degree of stereoselectivity. The commercial value of the enzymes as stereoselective biocatalysts has been a significant driving force in understanding features that control their mechanism of catalysis and stereoselectivity. This work focuses on two enantiocomplementary dehydrogenase enzymes ((R)- and 2-(S)-hydroxypropyl-CoM (HPC) dehydrogenases (DH)) of the epoxide carboxylation pathway in Xanthobacter autotrophicus strain Py2. The main goal of this dissertation is to kinetically, mechanistically and structurally characterize S-HPCDH and through the comparison studies with R-HPCDH reveal the basis for high degree of stereoselectivity exhibited by both enzymes. Analysis of the molecular structure of R-HPCDH and the homology model of S-HPCDH suggests a mechanism of substrate specificity in which the binding of the iv substrate sulfonate moiety at distinct sites on each stereoselective enzyme directs the orientation of the appropriate substrate enantiomer for the hydride abstraction. The positively charged residues responsible for binding the CoM moiety of the substrate were identified in R-HPCDH (Arg152 and Arg196), and in S-HPCDH (Arg211 and Lys214). Site-directed mutagenesis confirmed their importance in binding and orienting physiological substrates, but not the substrates lacking the CoM moiety. Extensive kinetic and mechanistic characterization of S- HPCDH reveals its key catalytic features similar to those of R-HPCDH, but also points out a few important differences. Furthermore, the role of the methionine residues flanking the substrate in the active site of both dehydrogenases was investigated. Substitution of these residues to alanine resulted in enzymes with significantly altered catalytic parameters and suggested their importance in binding and catalysis. Additionally, the X-ray crystal structures of the Met187Ala and Met192Ala mutants of R-HPCDH have revealed their role as "gate keepers," protecting the active site from the surrounding solvent. Kinetic analysis of Met187Leu and Met192Leu mutants implied a structural, rather than catalytic function of the methionines. It is proposed that steric clashes of the terminal methyl group of the HPC substrates with the nicotinamide ring of NAD+ are a major determinant of the enantioselectivity in S-HPCDH. This research provides the first side-by-side characterization of a pair of short-chain dehydrogenase/reductase (SDR) enzymes expressed simultaneously to act on two enantiomers of the same alcohol produced in a metabolic pathway. The R-HPCDH and S-HPCDH enzymes are distinguished from all other known members of the SDR family in using the novel sulfonate functional group of coenzyme M as a handle for chiral discrimination. These results provide a standard for examining the molecular basis of stereoselectivity in other such enzyme pairs. (206 pages) v ACKNOWLEDGMENTS There are a lot of people who I owe a great deal of gratitude for their contribution to my success. Their kind help and support during the course of my Ph.D. work made this journey an enriching, meaningful and life-shaping experience. Without them any of these would not be possible and because of them all of these have sense and great value to me. For that I will always be grateful. First of all, I would like to thank my advisor, Prof. Scott Ensign, for the opportunity to work in his laboratory and for his financial support. He has always encouraged me to become an independent researcher and gave me freedom to take my projects in a direction that interested me. I am very grateful for his academic guidance, for help in improving my writing skills and for the water-skiing trips in the sub-zero temperatures, which almost made my heart stop beating. I also owe thanks to my supervisory committee: Prof. Lance C. Seefeldt, Prof. John L. Hubbard, Dr. Sean J. Johnson and Prof. Jeff R. Broadbent. I appreciate their time, patience, openness for questions and helpful discussions. Special thanks to Prof. John L. Hubbard for believing in me, for his continues encouragement and advice, for teaching me the “applied aspects of biochemistry” and for a great sense of humor. I want to express my sincere gratitude to all of the former and present students of the Ensign lab that I have been fortunate to work with. In particular, I have to thank Dr. Jeff Boyd for getting me started in the biochemistry lab. Also, I would like to acknowledge Missy Kofoed, Ameya Mashruwala, Brandon Russel, Ashwini Wagh and Chris Potter for being great lab mates and friends. I extend my thanks to Danyal Karamatullah for his help, support and inspiring discussions on science, photography and life. Ameya and Danyal, thank you for your company during the long nights and weekends, while working in the lab side by side. vi I must recognize my wonderful friends Jordan, Ellie, Shannon, Odessia, Malgorzata and Piotr for making me feel home far away from home. Undoubtedly without their help, encouragement and support in times of hardship I would not be where I am today. Special thanks to Dr. Jordan Ramilowski who has been on the path with me through collage and grad school. He’s been a great friend, roommate and a skiing partner. We’ve celebrated together the high points and commiserated the low points over the last six years in Logan. I extend my thanks to my departmental family, especially to Kasia R. and Kasia G., Slava, Jeremy, Brenda, Ryan and Russ. I will always be grateful for the love and support given to me by Mayra Iglesias. Your patience, understanding, hard work and optimism helped me more than the words can express. I’d like to give very special thanks to my family (my Mum, Ania and Krzys) for trusting in me and investing in me. They have been very supportive and always believed in me. Without your love and encouragement this would have never been finished. I apologize to my niece and nephews for not being a very good uncle, I promise to make up for it when back in Europe. Lastly, I would like to acknowledge Utah State University for giving me a great opportunity to persue my doctoral degree. Dariusz A. Sliwa vii To my mum Maria Sliwa I dedicate this dissertation. viii CONTENTS Page ABSTRACT .................................................................................................................................... iii ACKNOWLEDGMENTS ............................................................................................................... v LIST OF TABLES .......................................................................................................................... ix LIST OF FIGURES ........................................................................................................................ xi ABBREVIATIONS .....................................................................................................................