Arthrobacter nicotinovorans pAO1 Why do we need its proteome? Marius Mihășan, PhD. Faculty of Biology Alexandru Ioan Cuza University of Iaşi, Romania E-mail: [email protected] 4/2/17 Arthrobacter nicotinovorans - Why do we need its proteome? Slide 1 / 26 A bit about my home country Romania Alexandru Ioan Cuza University of Iasi Area: 92,043 sq mi Population: 20,121,641 Transylvania Flight history: Chemistry: Biology: Tr aian Vu ia Vintilă Ciocâlteu Victor Babeș first airplane to take off on its own power co-developed the Folin-Ciocalteu reagent more than 50 types of bacteria Physics: Costin Nenițescu Emil Racoviță Ştefan Procopiu many new classes of inorganic compounds founder of biospeleology the Bohr-Procopiu magneton Nicolae Teclu Emil Palade the Teclu burner Nobel Prize for contributions to cell biology 4/2/17 Arthrobacter nicotinovorans - Why do we need its proteome? Slide 2 / 26 Alexandru Ioan Cuza University of Iaşi Facts and figures ü The oldest Romanian university ü Diplomas recognized all over Europe Sciences Social Sciences & Humanities ü15 departments ü Biology ü Economics and Business Administration ü Chemistry ü History ü 93 Bachelor programs ü Computer Science ü Law ü Geography and Geology ü Letters ü Mathematics ü Orthodox Theology ü 176 Master programs ü Physics ü Philosophy and Social – Political Sciences ü Physical Education and Sports ü 26 PhD programs ü Psychology and Education Sciences ü Roman – Catholic Theology ü Center for European Studies 4/2/17 Arthrobacter nicotinovorans - Why do we need its proteome? Slide 3 / 26 Department of Biology Bulevardul Carol I nr. 20A, Iaşi, Romania, 700505 Tel.:+40(0)232201072 Fax: 40(0)232201472 www.bio.uaic.ro Founded in 1948 ü 758 students: • 20 PhD students ü46 full-time faculty members ü 16 technicians and administrative staff 4/2/17 Arthrobacter nicotinovorans - Why do we need its proteome? Slide 4 / 26 Programs of Study Biology BACHELOR Biochemistry semesters Environmental science Molecular genetics MASTER Biology Cellular and microbial biotech. Biodiversity Conservation Environment Counceling PhD 4/2/17 Arthrobacter nicotinovorans - Why do we need its proteome? Slide 5 / 26 Study Programs Why I am here in Potsdam? Sponsor: U.S. Department of State Romanian-U.S. Fulbright Commission Exchange Visitor Program Number G-1-00005 • Observe and learn the dynamics of a research group here in US • Attend lectures and understand the US academic system • Identify all the nicotine-induced proteins in Arthrobacter nicotinovorans by means of mass-spectrometry 6 months in Darie’s Biochemistry & Proteomics Group Department of Chemistry & Biomolecular Science, Clarkson University 4/2/17 Arthrobacter nicotinovorans - Why do we need its proteome? Slide 6 / 26 Life is a collection of highly orchestrated chemical reactions catalyzed by enzymes 37 trillion (1012) reactions in one sec. in the human body Biological catalysts = Enzymes with only a few exceptions are all proteins High selectivity - Stereo-, Regio-, Chemo-selectivity High catalytic efficiency - acetylcholinesterase turnover is around 10 000 reactant molecules/min Mild operational conditions - work at “normal” temperature, pressure, pH David L. Nelson, Michael M. Cox, LehningerPrinciples of Biochemistry, 4th Edition, W. H. Freeman, pg. 27 Johannes T., Simurdiak MR, Zhao H., Biocatalysis in Encyclopedia of Chemical Processing DOI: 10.1081/E-ECHP-120017565 4/2/17 Arthrobacter nicotinovorans - Why do we need its proteome? Slide 7 / 26 Enzymes are cool, but are useful in our every day life? • Food and beverages • Forensic investigations industry • Early diagnostic and treatment of diseases • Biological detergents • Production of novel drugs break down protein, starches and fat and advanced materials in dirt and stains - food stains, sweat and mud • Remediation of polluted environments 200 out of the 4000 currently known enzymes, only 1% of existing enzymes in nature The trick is to find the right enzyme for the needed job Jemli S et. al. 2016, Biocatalysts: application and engineering for industrial purposes Critical Reviews In Biotechnology, 36(2):246-258 4/2/17 Arthrobacter nicotinovorans - Why do we need its proteome? Slide 8 / 26 What is Arthrobacter nicotinovorans? Why do we need its proteome? 85% of the usefully enzymes are from microorganisms Arthrobacter Photo by: T. Tamura, T. Nishii & K. Hatano l Ubiquitous in polluted and toxic soil samples l Extremely high metabolic versatility Are able to degrade highly toxic, highly stable or novel, man-made chemicals Microorganism isolated from tobacco cultivated soil Dana Krempels, Introduction to Evolution, http://www.bio.miami.edu/dana/107/107F10_5print.html 4/2/17 Arthrobacter nicotinovorans - Why do we need its proteome? Slide 9 / 26 Arthrobacter nicotinovorans and nicotine degradation Nicotine • Produced by the tobacco plant to prevent consumption by herbivores • Responsible for smoking addiction • Potentially lethal to humans (LD50 0.5 – 1.0 mg/kg) Arthrobacter nicotinovorans can grow on media containing up to 6 g/L nicotine pAO1 plasmid Brandsch group Albert Ludwigs University of Freiburg, Germany 4/2/17 Arthrobacter nicotinovorans - Why do we need its proteome? Slide 10 / 26 The nicotine degrading program on pAO1 – the nic gene cluster kdhS ndhM transp coxF coxD coxG pkc mobA coxE nit dhph kdhL ponH kdhM 6hlno ndhLndhS IS1473 II TRu TRd I Tn554 nepA perm transp Tn554 nbor mao sad nepB folD abo purU 49500 pmfR III IV moaD moeAmodA modC lacI hdno trsp moaA moaC modB Nicotine catabolism, experimental Nicotine catabolism, putative hdnoR V VI 118000 Transcription factors, experimental or putative Putative ORFs, no function Transposons and insertion elements 4/2/17 Arthrobacter nicotinovorans - Why do we need its proteome? Slide 11 / 26 The nicotine break-down pathway in A. nicotinovorans I VI II IV III Mihasan M. Arthrobacter nicotinovorans – new insights into its metabolism, molecular genetics and biotechnological applications, Habilitation thesis, Iasi, 2016 4/2/17 Arthrobacter nicotinovorans - Why do we need its proteome? Slide 12 / 26 Upper nicotine pathway in A. nicotinovorans Nicotine 6-OH, L-Nicotine oxidase dehydrogenase 6-OH, D-Nicotine oxidase Nicotine 6-OH NicotineN 6-OH, CH3-Myosmine 6HNic Ketone dehydrogenase 6-OH PseudooxinicotineN 2,6-diOH Pseudooxinicotine N I VI NDH – nicotine dehydrogenase 6-OH L–Nicotine oxidase KDH – ketone dehydrogenase and heterotrimeric (αβγ)2 enzymes FAD , MCD and FeS clusters 6-OH D–Nicotine oxidase absolute stereospecificity, genetically and structurally unrelated flavoenzymes V Kachalova et al., 2010, J Molec Biol., 396(3):785-799 4/2/17 Arthrobacter nicotinovorans - Why do we need its proteome? Slide 13 / 26 The nicotine break-down pathway in A. nicotinovorans I VI II IV III 4/2/17 Arthrobacter nicotinovorans - Why do we need its proteome? Slide 14 / 26 Pyridine ring metabolism in A. nicotinovorans II 2,6-diOH PseudooxinicotineN An α/β fold C-C bond hydrolase 2,6-diOH Pseudooxinicotine hydrolase no sequence similarity to known bacterial meta-cleavage c-c bond hydrolases Methyl-γ-aminobutyricN acid 2,6-diOHN Pyridine MGABA 2,6-diOH Pyridine hydroxylase 2,3,6-triOHN Pyridine Nicotine-blue Oxidoreductase Schleberger et al., 2006, J Molec Biol., 367(2):409-418 Mihasan et al. 2007 Appl Environ Microbiol 73 (8), 2479-2485. 4/2/17 Arthrobacter nicotinovorans - Why do we need its proteome? Slide 15 / 26 The nicotine break-down pathway in A. nicotinovorans I VI II IV III 4/2/17 Arthrobacter nicotinovorans - Why do we need its proteome? Slide 16 / 26 Side chain metabolism in A. nicotinovorans Methyl-γ-aminobutyric acid Oxidoreductase Amine IV Oxidase Genetic or γ-aminobutyric acid allosteric Monoamine regulation probably Oxidase switches between the two variants III Dehydrogenase 4/2/17 Arthrobacter nicotinovorans - Why do we need its proteome? Slide 17 / 26 Blank spots in nicotine metabolic pathway in A. nicotinovorans 1. Is Nicotine-Blue (NB) the final product of the pathway? A. A ω-amidase for α-ketoglutaramate is part of the nic-gene cluster nit 2,3,6-triOH 3,6-diOH α-keto- II Pyridine 2-Pyridinon glutamarate Flexible targeted docking ω-amidase α-keto- glutaramate can be docked into the NIT active site Cobzaru, C., et al. (2011) Research in Microbiology, 162(3), 285–91. Petra G. Nicotine-blue α-keto-glutarate B. Nicotine-blue is consumed in old cultures A pyridine ring cleaving enzyme must exist Razavn B., B.Sc Doina G. B.Sc Unknown enzyme? Chromosomal gene? Andreea A., M.Sc 4/2/17 Arthrobacter nicotinovorans - Why do we need its proteome? Slide 18 / 26 Blank spots in nicotine metabolic pathway of A. nicotinovorans 2. How is nicotine transported across the cell membrane? L-nicotine uptake in A. nicotinovorans: facilitated diffusion • Dependent on nicotine degradation but not on pAO1 driven by the nicotine gradient due • Energy-independent to its intracellular degradation • Saturable with a K(m) of 6.2 microM and a V(max) of => 0.70 micromol/ min/mg protein) by a unknown Ganas & Brandsch, 2009, Microbiology, 155: 1866–77 chromosomal permease 3. Several other bacteria have the nic gene cluster, but not a functional nicotine catabolism pAO1 AK-YN10 99% sequence identity between key nic-genes on A. nicotinovorans and AK-YN10. Victor B., M.Sc. France Razvan B, M.Sc Same plasmid but no nic-genes in AK-YN10 AK-YN10 contains a plasmid are on the plasmid nicotine metabolism Some chromosomal genes must be directly/indirectly involved in nic catabolism 4/2/17 Arthrobacter nicotinovorans - Why do we need its proteome? Slide 19 / 26 Potential