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Biosynthesis of Pyrrolysine, the 22Nd Amino Acid Dissertation Presented

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Biosynthesis of Pyrrolysine, the 22Nd Amino Acid Dissertation Presented Biosynthesis of pyrrolysine, the 22nd amino acid Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Marsha Ann Gaston, M.S. Graduate Program in Microbiology The Ohio State University 2011 Dissertation Committee: Dr. Joseph Krzycki Dr. Karin Musier-Forsyth Dr. John Reeve Dr. F. Robert Tabita Copyright by Marsha Ann Gaston 2011 Abstract Pyrrolysine is the 22nd genetically encoded amino acid, co-translationally inserted into methylamine methyltransferases as directed by in-frame amber (UAG) codons in methanogenic Archaea. UAG is interpreted as pyrrolysine with a devoted tRNA that is directly aminoacylated with pyrrolysine by a dedicated aminoacyl-tRNA synthetase (encoded by pylS). The pyrrolysine gene cluster, pylTSBCD, encodes three additional proteins bearing homology with biosynthetic enzymes (by pylBCD). The expression of pylB, pylC, and pylD from Methanosarcina acetivorans is necessary and sufficient for pyrrolysine biosynthesis in Escherichia coli, suggesting that the precursors of pyrrolysine are common between the metabolism of an enterobacterioacaea species and methanogenic archaea. However, the identity of those metabolic precursors of pyrrolysine have remained unknown. Here we present a thin layer chromatography methodology that separates pyrrolysine from other E. coli metabolites. A series of isotopic labeling experiments using thin layer chromatography and mass spectrometry indicate two molecules of lysine serve as the entire carbon and nitrogen skeleton of pyrrolysine. All carbons between the two molecules of lysine are retained in ii pyrrolysine, where an epsilon-nitrogen of one lysine molecule is lost during pyrrolysine biosynthesis. The biosynthesis of a pyrrolysine analog, desmethylpyrrolysine, is also described. This analog is produced in E. coli expressing pylCD when cultured in the presence of D-ornithine. The presence of D-ornithine circumvents PylB, acting as an analog of the pyrrolysine ring precursor. These studies allow us to place PylB as the first enzyme in the pyrrolysine biosynthetic pathway as well as propose the product of the reaction it catalyzes. Additional studies using D-ornithine have allowed us to order PylC as the first enzyme and PylD as the second enzyme to act in the desmethylpyrrolysine biosynthetic pathway. Furthermore, deuterium labeling studies presented here provide insights into the mechanism of PylB. Finally, an ordered pathway for pyrrolysine biosynthesis is proposed. iii Dedication Dedicated to my husband and parents for their endless support and encouragement iv Acknowledgements Many professional and personal thanks need to be extended to those folks who have made this work possible. First and foremost I would like to thank my advisor, Dr. Joseph Kryzcki. His knowledge, patience, and guidance have contributed significantly to my scientific upbringing. I am grateful that he allowed me to dive into such a fascinating problem. I am also appreciative of my committee members, Dr. Karin Musier-Forsyth, Dr. John Reeve, and Dr. Robert Tabita for their insights and suggestions. Much of the work presented here would not have been possible without the expertise of Dr. Kari Green-Church and Dr. Liwen Zhang of The Ohio State University Mass Spectrometry and Proteomics Facility of the Campus Chemical Instrument Center. Dr. Zhang’s dedication to our project and her willingness to answer my many questions have been very much appreciated. My labmates Sherry Blight, Dave Longstaff, Anirban Mahapatra, Jitesh Soares, Ross Larue, and Ruisheng Jiang have been a phenomenal source of training, encouragement, scientific discussion, and friendship. I have had a phenomenal amount of support from friends, old and new. I am also thankful to Dr. Juan Alfonzo, Dr. Mary Anne Rubio, and the members of the Alfonzo lab. Their enthusiasm for science and generosity have contributed greatly to the work presented here. I am appreciative for the kindness and generosity of Michael Carter and Stacia Kock, whose hospitality has allowed v me to see my degree to fruition. My parents, Ron and Carol Thalhofer fostered my curiosity early in life and have been an endless source of encouragement for which I am immensely thankful. Finally, I am infinitely grateful for the support, inspiration, and understanding of my husband Kirk. vi Vita 2005............................................. B.S. Biology, Saint Vincent College 2008............................................. M.S. Microbiology, The Ohio State University 2005 to present............................ Graduate Research and Teaching Associate, The Ohio State University Publications 1. Gaston MA, Zhang L, Green-Church KB, Krzycki JA. The complete biosynthesis of the genetically encoded amino acid pyrrolysine from lysine. Nature. 2011 Mar 31;471(7340):647-50. 2. Gaston MA, Jiang R, Krzycki JA. Functional context, biosynthesis, and encoding of pyrrolysine. Current Opinion in Microbiology. 2011 Jun;14(3):342-9. Field of Study Major Field: Microbiology vii Table of Contents Abstract.................................................................................................................. ii Dedication............................................................................................................. iv Acknowledgements ............................................................................................... v Vita....................................................................................................................... vii List of Tables ........................................................................................................ xi List of Figures ...................................................................................................... xv Chapters: 1. Introduction .................................................................................................1 1.1 Archaea.................................................................................................1 1.1.1 Archaeal diversity .......................................................................1 1.1.2 Methanogens..............................................................................3 1.2 Amino acid biosynthesis .......................................................................5 1.3 Selenocysteine .....................................................................................7 1.4 Pyrrolysine, the 22nd genetically encoded amino acid ..........................9 1.4.1 Discovery of an amber codon read-through event in Methanosarcina spp. ..................................................................9 1.4.2 Genetic encoding of pyrrolysine ...............................................13 1.4.3 Pyrrolysine biosynthesis ...........................................................19 1.4.4 Pyrrolysine function ..................................................................28 1.5 Overview of thesis...............................................................................29 2. Pyrrolysine is biosynthesized entirely from lysine.....................................31 2.1 Introduction .........................................................................................31 2.2 Experimental procedures....................................................................32 2.2.1 Organisms and plasmids ..........................................................32 2.2.2 Growth conditions for recombinant biosynthesis of pyrrolysine ............................................................................33 2.2.3 Growth conditions for recombinant radiolabelled biosynthesis of pyrrolysine .......................................................34 2.2.4 Extraction of recombinantly expressed pyrrolysine using organic solvents ..............................................................35 2.2.5 Ninhydrin-based standardization of concentrations of cell extracts...........................................................................35 2.2.6 Thin layer chromatography separation of pyrrolysine...............36 viii 2.2.7 Pixel density correlation to pyrrolysine concentration in organic solvent extracts using TLC.......................................36 2.2.8 Growth conditions for MtmB(His)6 production for labelling of pyrrolysine with stable isotope ...............................37 2.2.9 Enrichment of MtmB(His)6 ........................................................38 2.2.10 Anti-MtmB immunoblot .............................................................39 2.2.11 Sample preparation and mass spectrometric analysis of MtmB(His)6 ...........................................................................39 2.3 Results................................................................................................40 2.3.1 TLC separation of recombinantly expressed pyrrolysine..........40 2.3.2 Quantification of pyrrolysine produced by recombinant E. coli using TLC.......................................................................47 2.3.3 Radiolabelled lysine is incorporated into recombinantly biosynthesized pyrrolysine .......................................................51 2.3.4 Expression and enrichment of MtmB(His)6 from M9-SAG........54 2.3.5 All carbon and all nitrogen in pyrrolysine are derived from two molecules of lysine ....................................................55 2.3.6 The epsilon-nitrogen from one lysine molecule is eliminated during pyrrolysine biosynthesis ...............................64 2.4 Discussion...........................................................................................70
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