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Alcohol Dehydrogenase) from Cylindrospermopsis Raciborskii T3 by Zymography and Mass Spectrometry

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Alcohol Dehydrogenase) from Cylindrospermopsis Raciborskii T3 by Zymography and Mass Spectrometry Investigation of the Putative Enzyme Function of SxtL (GDSL-lipase) and SxtU (Alcohol dehydrogenase) from Cylindrospermopsis raciborskii T3 by Zymography and Mass Spectrometry by Kulbhushan N Ugemuge z3274021 Supervisor: Prof. Brett A. Neilan Co supervisors: Dr. Sohail Siddiqui and Dr. Michelle Gehringer UNSW 21 April 2011 A.D. In Partial Fulfilment of the Requirements for the Award of Master of Philosophy (Research) School of Biotechnology and Biomolecular Sciences Centre for Cyanobacteria and Astrobiology The University of New South Wales, Sydney, Australia i ii Acknowledgement I would like to thank my supervisor Brett Neilan for giving me this wonderful opportunity to learn and prosper in the field of cyanobacterial research as an MPhil student. I couldn’t have gotten a better supervisor. Long back I dreamed of becoming a scientist and was seeking a role model. Thank you for being that inspiration for me. I am thankful for your support and guidance during the research, your knowledge in the field of cyanobacteria is extraordinary. I am grateful for your friendship during the ups and downs of my life and also for guiding me throughout. Thank you so much. I am grateful to my co-supervisors Michelle Gehringer and Sohail Siddiqui who have been the heart of my project. This thesis wouldn’t have been the same without your tremendous knowledge and expertise in the field. Thank you for all the discussions and valuable inputs while trying to solve the mysteries in the project. Your ideas and suggestions have been remarkable. Thank you for all the technical support and also for making this thesis presentable. I would like to present a special appreciation to Sohail sir, who has been a fatherly figure for me. Thank you so much for being there for me during my fall backs and bringing alive the hopes in me from time to time in this project. Things took a boost when you joined the project for protein work. One idea led to another, and we never knew how things were going to end up. You have always inspired me to find the true researcher in me. You have not only been the soul behind some of the important findings in this project, but also made the environment so friendly and exciting while working with you. Thank you for being the support during the funny as well as intense moments during my thesis. I have never come across a mind in my life which is so active in research.. Having you around has always reminded me of my father. Peptide mass fingerprinting was an important need for this project. I would like to appreciate Anne Poljak from The Bioanalytical Mass Spectrometry Facility, UNSW, who has patiently analyzed all the protein samples used in this study. I would also like to thank Menuk Jayawardena from The Centre for Marine Bio-innovations, UNSW, for his expert inputs in esterase activity assays. A special thanks to Young Jae Jeon, Troco Kaan Mihali, Leanne Pearson, Sarah Ongley and Julia Muenchhoff for sharing your experiences and timely contributions. Thank you so much Sarah, for sharing your knowledge in cloning experiments. We went through difficult times trying to make the cloning work. You share a special contribution in helping me understand the background in cloning which will help me the next time I try it again. I will miss the long and funny discussions. Thank you very much. This thesis has been based on the PhD contributions of Ralf Kellman and Kenlee Nagasuki in cyanobacterial research. I am grateful for knowing their work. iii Last but not least, thank you Chris Marquis for your generosity while helping me get settled in BABS and marking important points during my reviews. A special thanks to my special friends from BGGM group - Jeffrey Noro, Alfonsus Alvin, John Kalaitzis, Maria Wiese, Rati Sinha, Gurjeet Singh Kohli, Alper Yasar, Jamal al Tebrineh, Rocky Chau, Ivan Wong, Bhumika Azad, Toby Mills, Angela Chilton, Marie Dahl, Nathan Dunn, Ashish Srivastav, Bhargavi Veppalla and Sreekanth Dasari for being there for me. Jeff bro, you have an iconic personality. Alvin, thanks for sharing so many chocolates. John, you are a supercool post-doc. Maria, you have always looked after your trouble maker. Jamal, your skills in soccer are amazing. Alper, thanks for the nice pictures and helping me get things around in the lab. Ivan, thanks for helping me with endnote. Gurjeet, your cooking lessons and other moments were fun times. Ashish sir, I missed you a lot. You made me laugh and also stood by me. Rocky, you are the best bench buddy; sorry for leaving the bench messy at times!! Angie, Rati, Bhumika, Bhargavi and Marie, I hope you were not annoyed by my pranks. Rati, you have been a lovely sister. Toby and Sreekanth, we shared great friendship. All of you have inspired me a lot, not just to be a better researcher but also to be a better person. Ismah Kamil, Lennon Bk Lim and Ameen Kamal, we shared good friendship. My best wishes for your future endeavours. I would like to thank everyone else from BBGM group for sharing all the moments in the lab as well as fun times on the beach. Thank you all for cheering up, when I climbed a tree once. Penny Hamilton and Kylie Jones, thanks for your quick inputs towards official formalities in the university and also towards the submission of this dissertation. I would like to dedicate this thesis to my family, which has sacrificed a lot in the process of getting me up to this stage. My love and friends from India, this is a special gift for you. Thank you everyone for being so patient with me. It has been a great journey so far with so many emotions attached to this thesis. I wish I had a time machine to go back in life and rectify some of the things. Nevertheless, I have a whole new beginning ahead. These 2 years of my experience, will be treasured in my life forever. iv Abstract The recent discovery of the saxitoxin (sxt) gene cluster and studies involving the in vitro production of paralytic shellfish toxins (PSTs) has helped predict the complex mechanism of the biosynthesis of saxitoxin (STX) in particular strains of toxic bloom forming fresh water cyanobacteria like Cylindrospermopsis raciborskii T3, Anabaena circinalis AWQC131C and Aphanizomenon sp. NH-5. The sxt gene cluster which spans up to a region of about 40000 base pairs, consists of around 26 open reading frames, each representing a bioinformatically deduced putative enzyme. This gene cluster attributes the involvement of some putatively identified tailoring enzymes which govern the formation of STX and its analogues in these organisms. SxtL is a putative GDSL lipase which is predicted to be involved in the hydrolytic conversion of STX and its other carbamoylated analogues to form decarbamoylated compounds (dcSTX). Two commercially available substrates, 4-methylumbilleferyl butyrate and 1-naphthyl acetate, were used to investigate the esterase activity of E. coli BL21 overexpressed SxtL protein in zymography studies. SxtU is a putative short chain alcohol dehydrogenase, ideal for the reduction of the terminal aldehyde group at C-1 of the STX precursor to form the tricyclic alcohol intermediate in the post-PKS reactions during the biosynthesis of STX. The recombinant SxtU protein, obtained by overexpression in E. coli Rosetta cells was found to be active on ethanol and clavulanic acid in a zymography study involving the alcohol dehydrogenase activity assay. In addition to overexpression studies, zymography of both activity assays were also performed in order to detect these putative enzymes in crude protein extracts of C. raciborskii T3 and A. circinalis AWQC131C. In the zymogram of alcohol dehydrogenase activity, two proteins from the sxt gene cluster, SxtN and SxtC were identified in A. circinalis AWQC131C via peptide mass fingerprinting of bands in the zymogram. In case of C. raciborskii T3, a similar short-chain alcohol dehydrogenase was also detected. In a separate experiment, an integrative approach was also implemented to clone the sxtL gene from C. raciborskii T3 into a Synechocystis integrative vector with a final purpose of heterologous expression of soluble SxtL protein in Synechocystis PCC6803. v Table of Contents CHAPTER 1- A BIOCHEMICAL ACCOUNT OF SAXITOXIN 1.1 GENERAL INTRODUCTION .......................................................................................... 1 1.2 DEVELOPMENT OF SAXITOXIN RESEARCH.................................................................. 3 1.2.1 Adverse effects on marine environment ................................................... 4 1.2.2 Economical losses and health effects ........................................................ 5 1.2.3 Mechanism of action: Contributions in unlocking the sodium channel mystery ............................................................................................................... 6 1.2.3.1 Architecture of sodium channel as described by neurotoxins ...... 6 1.3 BIOCHEMICAL PERSPECTIVE OF SAXITOXIN .............................................................. 9 1.3.1 Putative biosynthetic pathway of Saxitoxin .............................................. 9 1.4 SAXITOXIN GENE CLUSTER ...................................................................................... 12 1.4.1 Tailoring enzymes ................................................................................... 16 1.4.1.1 SxtL (putative GDSL lipase) ...................................................... 16 1.4.1.2 SxtU (putative short-chain alcohol dehydrogenase) ................... 17 1.5 AIMS OF THE PROJECT ............................................................................................
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