Characterization of the 4-carboxy-2-hydroxymuconate hydratase and the 4-carboxy-4-hydroxy-2-oxoadipate/4-hydroxy-4-methyl-2-oxoglutarate aldolase from Pseudomonas putida by Scott Mazurkewich A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Doctor of Philosophy in Molecular and Cellular Biology Guelph, Ontario, Canada © Scott Mazurkewich, May, 2016 ABSTRACT CHARACTERIZATION OF THE 4-CARBOXY-2-HYDROXYMUCONATE HYDRATASE AND THE 4-CARBOXY-4-HYDROXY-2-OXOADIPATE/4-HYDROXY-4- METHYL-2-OXOGLUTARATE ALDOLASE FROM PSEUDOMONAS PUTIDA Scott Mazurkewich Advisor: University of Guelph, 2016 Dr. Stephen Y.K. G. Seah The protocatechuate and gallate 4,5-cleavage pathways are important bacterial catabolic pathways for environmental carbon cycling and xenobiotic remediation. This thesis focuses on the characterization of the 4-carboxy-2-hydroxymuconate (CHM) hydratase and the 4-hydroxy- 4-methyl-2-oxoglutarate (HMG)/4-carboxy-4-hydroxy-2-oxoadipate (CHA) aldolase which are the last two steps of the protocatechuate and gallate 4,5-cleavage pathways. HMG/CHA aldolases are class II pyruvate aldolases that have structural similarity to a group of proteins termed Regulators of RNase E activity A (RraA). The Escherichia coli RraA (EcRraA) binds to the regulatory domain of RNase E, inhibiting ribonuclease activity. Sequence and kinetic analyses of homologous RraA-like proteins identified minimal motifs, either a D- X20-R-D or a G-X20-R-D-X2-E/D motif, required for metal binding and aldolase activity amongst homologs. The EcRraA, which lacked sequence conservation to either motif, lacked detectable C-C lyase activity. Upon restoration of the G-X20-R-D-X2-E/D motif, the EcRraA was able to catalyze oxaloacetate decarboxylation. RraA-like gene products are found across all the domains of life with a large proportion containing one of the sequence motifs, implying that the proteins likely support a metal dependent enzyme function. iii The HMG/CHA aldolase is activated in the presence of inorganic phosphate (Pi), increasing its turnover rate >10-fold, which is unique for a class II aldolase. The HMG/CHA aldolases pyruvate methyl proton exchange rate was increased 300-fold in the presence of 1 mM Pi. Docking studies revealed a potential Pi binding site close to the proposed general acid water site. The residues which comprise the putative binding pocket were probed through mutagenesis studies with substitution of either of the residues leading to a reduction in Pi activation, supporting the mechanism of Pi activation through the general acid half reaction. The CHM hydratase (GalB) from the gallate degradation pathway of Pseudomonas putida KT2440 has only 12% sequence identity to a previously identified CHM hydratase (LigJ) from Sphingomonas sp. SYK-6. The structure of GalB was determined and found to be a member of the PIG-L N-acetyl glucosamine deacetylase family, and is structurally distinct from the amidohydrolase fold of LigJ. LigJ has the same stereo-specificity as GalB, providing an example of convergent evolution in bacterial aromatic degradation pathways. iv Acknowledgements Throughout my studies I have utilized several facilities, resources, and funding sources that made the projects possible and without whom the answers we sought may not have been obtainable. In relation to the investigation into the RraA proteins, I would like to thank Brian Bryska, Douglas Grahame and Dr. Rickey Yada for their help and support with the DSC experiments. In relation to the characterization of the CHM hydratase GalB, I would like to thank: Elyse Roach and Cezar Khursigara for their assistance and expertise with use of anaerobic chamber, Prof David Rose and colleagues from his lab at the University of Waterloo for providing an X-ray source and assistance in screening several crystals, and Robert Reed for his assistance with polarimetry assays. I would also like to thank the government of Ontario which provided funds that supported me through portions of my studies through the Ontario Graduate Scholarship (OGS) and Ontario Graduate Fellowship (OGF). I would also like to thank the University of Guelph, Graduate Student Association, department of Molecular and Cellular Biology, and other communities on campus for a number of research and academic awards. I would like to thank all of the past and present members of Stephen Seah’s lab group for all their support throughout the years. I have been truly blessed with the opportunity to work with and befriend several amazing people over these years. In particular I would like to thank Dr. Weijun Wang, Dr. Perrin Baker, and Dr. Jason Carere who served as mentors and colleagues throughout the early years of my studies. Additionally, I would like to thank Amanda Ruprecht, Alexander Sterling, and Stephanie Gilbert for creating a great work environment over last few years and who entertained me through the many breaks needed from writing. I also need to thank a very special person, Ashley Brott, who started in the lab as a volunteer and who became v undergraduate research project student. She put in countless hours on the GalB project and even lied to me to let her work late the day of her birthday. I would like to sincerely thank her for all the hard work she put into the project and for her continued friendship. She is a truly amazing person and scientist. Lastly, I would like to sincerely thank Dr. Stephen Seah for his all of his support throughout my studies. He is an amazing advisor who instilled in me a passion for science. None of this work would be possible without the love and support of my family and friends. In particular I would like to thank Ken and Erika Russell for their support throughout the years and for providing weekends of escape from Guelph and science. I would like to thank my sisters, Mandy and Amy, and the newer additions to our family, my brother-in-law Craig and my nephew Ronan, for their love and support. To my parents, who have supported through the many years of studies, I truly appreciate everything you have done for me and our whole family. You have been great parents and without your love and support none of this would have been possible: thank you. Lastly, I would like to thank my partner, Aurora Patchett. You are my everything. You inspire me to be a better person and I couldn’t have done this without you. I look forward to all the adventures that await us. Authors Declaration of Work Completed I declare that, unless otherwise stated, I have completed all the work presented in this thesis. vi Table of Contents Abstract ....................................................................................................................................... ii Acknowledgements .................................................................................................................... iv Authors Declaration of Work Completed ................................................................................... v Table of Contents ....................................................................................................................... vi List of Figures ............................................................................................................................. x List of Tables ............................................................................................................................ xii List of Equations ...................................................................................................................... xiv List of Abbreviations ................................................................................................................ xv Chapter 1: Introduction ............................................................................................................... 1 1.1 Aromatic compounds in the environment ............................................................................. 1 1.1.1 Lignin ............................................................................................................................. 1 1.1.2 Aromatic pollutants ........................................................................................................ 3 1.2 Bacterial aromatic metabolism ............................................................................................. 5 1.2.1 Protocatechuate 4,5-cleavage pathway .......................................................................... 8 1.2.2 Gallate degradation pathway ....................................................................................... 11 1.2.3 Comparison with other aromatic meta-cleavage pathways ......................................... 11 1.3 Hydratases from bacterial aromatic cleavage pathways ..................................................... 12 1.3.1 The CHM hydratases (LigJ and GalB) ........................................................................ 12 1.4 Aldolases ............................................................................................................................. 13 1.4.1 Classes ......................................................................................................................... 13 1.4.2 Pyruvate specific class II aldolases .............................................................................. 14 1.5 The HMG/CHA aldolase .................................................................................................... 15 1.5.1 Enzyme structure ........................................................................................................