Biological Synthesis and Transformation of Nitriles Item Type text; Electronic Dissertation Authors Nelp, Micah Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 10/10/2021 06:33:53 Link to Item http://hdl.handle.net/10150/621560 BIOLOGICAL SYNTHESIS AND TRANSFORMATION OF NITRILES by Micah Taylor Nelp _____________________ A Dissertation Submitted to the Faculty of the DEPARTMENT OF CHEMISTRY AND BIOCHEMISTRY In Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY WITH A MAJOR IN CHEMISTRY In the Graduate College THE UNIVERSITY OF ARIZONA 2016 1 As members of the Dissertation Committee, we certify that we have read the dissertation prepared by Micah Taylor Nelp entitled Biological Synthesis and Transformation of Nitriles and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy ______________________________________________________ Date: April 1, 2016 Vahe Bandarian ______________________________________________________ Date: April 1, 2016 Matthew Cordes ______________________________________________________ Date: April 1, 2016 Indraneel Ghosh ______________________________________________________ Date: April 1, 2016 John Jewett ______________________________________________________ Date: April 1, 2016 Elisa Tomat Final approval and acceptance of this dissertation is contingent upon the candidate’s submission of the final copies of the dissertation to the Graduate College. I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement. ___________________________________________________ Date: April 1, 2016 Dissertation Director: Indraneel Ghosh 2 STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. 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SIGNED: Micah Taylor Nelp 3 TABLE OF CONTENTS LIST OF FIGURES ........................................................................................................ 5 ABSTRACT ...................................................................................................................... 6 CHAPTER I: A Review of Nitrile Biosynthesis and Degradation............................... 8 CHAPTER 2: Summary of the Present Study............................................................ 32 CHAPTER 3: Characterizing the Post-Translational Synthesis of the Active Site Complex of Nitrile Hydratase ...................................................................................... 43 CHAPTER 4: Development of an Assay to Determine the Means of Metal Specificity in Nitrile Hydratase ...................................................................................................... 66 REFERENCES ................................................................................................................74 Appendix A: A Single Enzyme Transforms a Carboxylic Acid into a Nitrile through and Amide Intermediate ............................................................................................... 83 Appendix B: The Alpha Subunit of Nitrile Hydratase is Sufficient for Catalytic Activity and Post-Translational Modification ........................................................... 101 Appendix C: A Protein Derived Oxygen is the Source of the Amide Oxygen of Nitrile Hydratase ...........................................................................................................119 4 List of Figures Figure 1.1 A summary of the enzymatic pathways herein described for the synthesis and transformation of nitriles. 9 Figure 1.2 The biosynthetic pathway for the production of cyanogenic glycosides. 10 Figure 1.3 Mechanism of aldoxime dehydratase. 13 Figure 1.4 Mechanism of nitrile synthetase, ToyM. 16 Figure 1.5 Mechanism of nitrilase. 19 Figure 1.6 (A) Structure of the active site of cobalt-type nitrile hydratase (PDB 1IRE). (B) Mechanism of nitrile hydratase. 22 Figure 1.7 Mechanism of rhodanese. 25 Figure 1.8 Mechanism of cyanase. 28 Figure 1.9 Mechanism of nitrile reductase. 30 Figure 2.1 The structure of cobalt type nitrile hydratase. 35 Figure 2.2 Possible mechanisms of nitrile hydratase. 39 Figure 3.1 Possible series of intermediates in the route from apoTNH to holoTNH. 48 Figure 3.2 A comparison of the increase in the A) enzyme activity, B) ratio of abundance of holo enzyme to apo enzyme, and C) absorbance at 300 nm over the course of the activation assays. 56 Figure 3.3 Mass spectrum of TNH showing multiple charge states for the subunit of interest, ToyJ, as well as ToyK. 59 Figure 3.4 Mass spectra of TNH at various times throughout the activation process. 60 Figure 3.5 UV-visible spectra of apo-TNH activation assay over time. 62 Figure 3.6 EPR spectra of apo-TNH activated in vitro compared to that of enzyme expressed in cobalt-containing media and purified in holo form. 64 Figure 4.1 Structures of A) iron and B) cobalt type nitrile hydratases with the predictive amino acid pairs highlighted in red. 67 5 Abstract Nitrile-containing natural products are rare in Nature, and there have been very few studies on the mechanisms by which they are synthesized and utilized. The biosynthesis of 7-deazapurine containing natural products is a unique case whereby both formation of a nitrile and its conversion to an amide are documented. The overall theme of this work is to interrogate the biosynthesis of the nitrile intermediate in the pathway and its subsequent hydration to an amide. The biosynthesis 7-cyano-7-deazaguanine (preQ0), the key intermediate in the biosynthesis of the hypermodified base queuosine and the toyocamycin natural product, is accomplished by preQ0 synthetase through a series of unprecedented reactions whereby the carboxylate moiety of the substrate, 7-carboxy-7-deazaguanine (CDG), is successively activated by adenylation, reacted with ammonia, and dehydrated to produce the nitrile. This one-enzyme synthesis of a nitrile is unique as the only other known route to nitriles proceeds through at least two enzymes. Nitrile hydratases are metalloenzymes that selectively hydrate nitriles to the amide and are used industrially to produce acrylamide and nicotinamide. These enzymes use a trivalent iron or cobalt complex comprised of two backbone amidate ligands and three cysteine thiolate ligands of which two are modified to the sulfenato and sulfinato form. This work describes aspects of a particular nitrile hydratase, toyocamycin nitrile hydratase (TNH). Whereas most nitrile hydratases are heterodimeric, TNH is heterotrimeric, and yet what was discovered is that only the subunit containing the active site metal complex is required for activity. This single subunit analog of the protein was used for single turnover assays in 18O-labeled water to show with high resolution mass 6 spectrometry that the source of the product amide oxygen is actually the enzyme itself and likely the sulfenato ligand oxygen acting as a nucleophile. The mechanism of the active site complex synthesis is described showing that this is self-catalytic in the presence of cobalt(II) and molecular oxygen. 7 CHAPTER I A Review of Nitrile Biosynthesis and Degradation Abstract - The rarity of nitriles in biology is belied by the rich chemistry life has evolved to create and transform them. Nitrile containing compounds represent a small fraction of all those published, but reports of newly discovered nitrile containing compounds suggest the unique chemistry of this functional group has not gone unnoticed by evolution and may prove to have undiscovered importance. 1 Indeed the use of nitriles in pharmaceuticals for their incredibly small steric bulk and polarity is, like most advanced chemical inventions, probably already being used by life, and that importance is underscored by the presence of this functional group in essential modified RNA bases used in all kingdoms of life. 2,3,4 This chapter will focus on the varied means by which enzymes are utilized in biology to produce and transform nitriles. Nitrile Biosynthesis - Of the known nitrile containing natural products, there have only been two routes to their synthesis that have been described in mechanistic detail. The aldoxime pathway is the best characterized of those and is present in at least 2500 species of plants as well as some arthropods. 1, 5 It involves multiple enzymes and produces cyanogenic glycosides that can act as a defense mechanism. 6 These cyanogenic glycosides are normally stored in vacuoles but are released into the cytoplasm upon herbivory where they encounter glycosidases that convert them into α-hydroxy