By Nicole M. Gaudelli

MECHANISTIC AND STRUCTURAL STUDIES OF THE THIOESTERASE DOMAIN IN THE TERMINATION MODULE OF THE NOCARDICIN NRPS By Nicole M. Gaudelli A dissertation submitted to The Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, MD 2013 © Nicole M. Gaudelli 2013 All Rights Reserved Abstract The nocardicins are monocyclic -lactam antibiotics produced by the actinomycete Nocardia uniformis subsp., tsuyamanensis ATCC 21806. In 2004 the gene cluster responsible for the biosynthesis of the flagship antibiotic, nocardicin A, was identified. This gene cluster accommodates a pair of non- ribosomal peptide synthetase (NRPS) whose five modules are indispensible for antibiotic production. In accordance with the prevailing co-linearity model of NRPS function, a linear L,L,D,L,L pentapeptide was predicted to be synthesized. Contrary to expectation and precedent, however, a stereodefined series of synthesized potential peptide substrates for the nocardicin thioesterase (NocTE) domain failed to undergo hydrolysis. The stringent discrimination against peptide intermediates was dramatically overcome by prior monocyclic -lactam formation at an L-seryl site to render now facile substrates for C-terminal epimerization and hydrolytic release. It was concluded through biochemical and kinetic experimentation that the TE domain acts as a gatekeeper to hold the assembling peptide on an upstream domain until -lactam formation takes place and then rapidly catalyzes epimerization and hydrolysis to discharge a fully-fledged pentapeptide -lactam harboring nocardicin G, the simplest member of the nocardicin family. An x-ray crystal structure of the TE domain revealed a catalytic center containing the expected Asp, His, Ser triad. Mutational analysis of these catalytic residues along with a proximal His established that the His of the catalytic triad was likely responsible for the epimerization activity rendered by the domain. ii The in vitro reconstitution of the termination module, or module 5, established that the -lactam core present in the nocardicins is formed within module 5 of the NRPS. This discovery has implications regarding the formation of the azetidinone moiety present in the entire class of monobactam antibiotics. Thesis Advisor: Professor Craig A. Townsend Dissertation Committee: Professor John D. Tovar Professor Rebekka Klausen iii Dedication This dissertation is dedicated to: Papa You have inspired and cultivated my curiosity and taught me to dream big, love deep and give more than I can take. Because of you, the sun always shines just “behind the clouds”. iv Acknowledgements First, I would like to express my deep gratitude to Prof. Townsend for not only accepting me into his group, but also supporting me through these past years both personally and scientifically. I have grown and changed since I first entered this program and through the failures and triumphs that have impacted my own personal and scientific evolvement he has guided me. He has been exceedingly supportive of my research endeavors, and was always ready and willing to discuss experiments and future goals. I truly believe that I could not have achieved success in the ways that I have with any other advisor. He has not only nurtured my scientific abilities but has in the process given me the confidence I needed to succeed. For this, I will be eternally grateful. To my family – my mother, my father, my brothers - I certainly could not have achieved anything without you and I hope you are as proud of me as I am of you. To my father – ever since I was a little girl you told me how proud you were of me and gave me all that you had. You were there for every ballet recital, band concert and awards ceremony since kindergarden. During the hard times in my life, when I thought I should just give up, you dropped everything to make it all better and reassured me that I could make it through. If it weren’t for you, I would be a shadow of who I am today. Though I may have grown, I will always be Daddy’s little girl. To my labmates – there are so many to thank. Rongfeng – you have been a constant presence and positive force in my life since I first joined the lab. You have been very helpful, instructive and kind when I needed help and have v become a great friend to me. You have always made yourself available and have supported me all the way. Thank you for all that you do. Jesse Li and Courtney Hastings – you two have been great sounding boards for my ideas and have given me valuable suggestions – thank you. Evan Lloyd, Daniel Marous and Katherine Belecki – thanks so much for all your support throughout my time here – you have become dear friends and I appreciate every time you were able to lend an ear or provide a shoulder to cry on. To Darcie Long – you are the best “little grasshopper” anyone could ever hope for. You have been a joy to work with and teach and I deeply appreciate all the support you’ve provided me during this time in my life. You have a bright future ahead of you. To my Kristos. Thank you for everything (there are far too many things to list). I cannot imagine my life without you in it. You bring me more joy than words can express and everyday the sun shines when I am with you. vi Publications Gaudelli, N.M. & Townsend, C.A. Stereocontrolled Syntheses of Peptide Thioesters Containing Modified Seryl Residues as Probes of Antibiotic Biosynthesis. J. Org. Chem. 78, 6412-6426 (2013). Manuscripts in Preparation Gaudelli, N.M. & Townsend, C.A. Epimerization and High Substrate Gating by a Non-Ribosomal Peptide Synthetase TE Domain in Monocyclic β-Lactam Antibiotic Biosynthesis. (Submitted) Gaudelli, N.M., Buller, A.R., Labonte, J.W. & Townsend, C.A. Structural Basis for the Selection and C-Terminal Epimerization of the Monocyclic -Lactam Antibiotic Nocardicin A by NocTE vii Table of Contents Abstract ......................................................................................................................... ii Dedication .....................................................................................................................iv Acknowledgements ...................................................................................................... v Publications .................................................................................................................vii Introduction to Non-Ribosomal Peptide Synthetases and -Lactam Antibiotics ..... 1 1.1 Non-ribosomal peptide synthetases ................................................................. 1 1.1.1 NRPS catalytic domains ................................................................................. 1 1.1.2 NRPS editing domains ................................................................................... 7 1.2 -Lactam Antibiotics........................................................................................... 9 1.2.1 Bacterial cell wall biosynthesis ......................................................................11 1.2.2 Penicillins ......................................................................................................14 1.2.3 Clavams ........................................................................................................17 1.2.4 Carbapenems ...............................................................................................19 1.2.4 Monobactams ...............................................................................................20 1.3 References .........................................................................................................28 Stereocontrolled Syntheses of Peptide Thioesters Containing Modified Seryl Residues as Probes of Antibiotic Biosynthesis ........................................................36 2.1 Introduction ........................................................................................................36 2.2 Results and Discussion ....................................................................................38 2.2.1 Synthesis of potential tri- and pentapeptides in nocardicin biosynthesis ........38 2.2.2 Synthesis of potential O-phosphoryl and O-acetyl substrates........................43 2.2.3 Synthesis of potential -lactam containing thioester substrates. ....................46 2.3 Experimental ......................................................................................................55 2.3.1 Syntheses .....................................................................................................56 2.3.3 Uncatalyzed Noc G-SNAC to epi-Noc G-SNAC epimerization ......................95 2.4 References .........................................................................................................96 Epimerization and High Substrate Gating by a Non-Ribosomal Peptide Synthetase TE Domain in Monocyclic -Lactam Antibiotic Biosynthesis ...................................99 3.1 Introduction ........................................................................................................99 3.2 Results and Discussion .................................................................................. 105 3.2.1 In vitro reconstitution of TE activity .............................................................. 105 3.2.2 NocTE domain does not form the -lactam ring .......................................... 114 viii 3.2.3 1H-NMR studies of NocTE epimerization activity ......................................... 118 3.2.4 Epimerase function of NocTE.....................................................................

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