FOR 1220: Prosthetic Groups: Transport and Insertion - PROTRAIN
Chemistry
Final Report Abstract
Enzymatic reactions are the driving force of metabolism. Complex cofactors and prosthetic groups of enzymes mediate the catalysis of challenging chemical reactions. While the biosynthesis of many prosthetic groups is well studied, their transport and regulated insertion into target enzymes is only poorly understood. Based on our long standing background in biosynthesis und functionality of prosthetic groups, we focus in Braunschweig with nine projects on the investigation of the unknown molecular strategies for transport and insertion of molybdenum cofactors (Moco) and hemes into enzymes. The main objective of the Research Group is to fill the gap in our knowledge about (1) what happens with the prosthetic groups heme and Moco subsequent to their biosynthesis, and (2) how is the process of insertion of these groups into their cognate apo-enzymes catalyzed. On the basis of the results from the first funding period, we sharpened and focussed our aims for the second period and incorporated three more groups (TP8, TP10, TP11) that provided additional key technologies for the PROTRAIN research unit, thus broadening our scope. In summary, we reached our goals: For objective 1, we identified and characterized both for eukaryotic Moco and bacterial heme novel proteins that are involved in transport of these prosthetic groups beyond biosynthesis. For objective 2, namely insertion, we also reached the goal in the way that these novel proteins were shown to be able to insert the prosthetic group into the respective apo-enzyme. However, the detailed mechanism for insertion has to be studied in the future. To name only a few: • A family of 8 Moco-binding proteins has been identified, characterized and localized in the plant Arabidopsis thaliana. They interact both with the Moco-donor protein Cnx1 and with the target protein nitrate reductase. • The atomic mechanism for the insertion of molybdenum into molybdopterin catalyzed by the Moco-donor protein Cnx1 was uncovered. Here we won the front cover page of the Biochemical Journal (2016) • For xanthine oxidase, the sequence of insertion of its three types of prosthetic groups has been determined, and a mechanism for the insertion of its Moco has been put forward. • The novel bacterial heme-binding protein HemW and its eukaryotic orthologue RSAD1 were identified as novel radical SAM proteins with heme chaperone function. • The maturation complex for NirS was identified, consisting of NirF, NirN and NirS, all three proteins are interacting with each other in vivo. All these results were only made possible through a tight collaboration between the single project groups: TP6 has synthesized fluorescent linkers, substrates, heme derivatives and Moco-derivatives, TP10 produced local chemical probes for the study of heme transfer processes and won the front cover page of the European Journal of Inorganic Chemistry, TP11 provided the proteomics platform, and our monoclonal antibody facility (TP8) has generated 149 monoclonal antibodies for all projects. Among others, publications appeared in Nature, PNAS and J Biol Chem. PROTRAIN also helped to shape the long-term profile of our Faculty of Life Sciences (biochemistry became one out of four strategic research focusses), led to the appointment of new biochemistry-oriented professors, led to establish the new Masterstudiengang "Biochemie / Chemische Biologie" at our university, was the corner stone to set up the novel "Braunschweig Center for Integrated Systems Biology BRICS" and finally formed the basis to successfully set up our new DFG-Graduiertenkolleg "Protein Complex Assembly" in 2016, where 7 out of 9 former PROTRAIN groups participate.
Publications
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Lactococcus lactis HemW (HemN) is a heme-binding protein with a putative role in heme trafficking (2012) Biochem. J. 442:335-43
Abicht, H. K., Martinez, J., Layer, G., Jahn, D., and Solioz, M.
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(2013) Maturation of the cytochrome cd1 nitrite reductase NirS from Pseudomonas aeruginosa requires transient interactions between the three proteins NirS, NirN and NirF. Biosci. Rep., 33, art:e00048
Nicke, T., Schnitzer, T., Münch, K., Adamczack, J., Haufschildt, K., Buchmeier, S., Kucklick, M., Felgenträger, U., Jänsch, L., Riedel, K. and Layer, G.
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(2014) Molybdenum site structure of MOSC family proteins. Inorg Chem. 53(18): 9460-9462
Giles LJ, Ruppelt C, Yang J, Mendel RR, Bittner F, Kirk ML
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(2014) NirN from Pseudomonas aeruginosa is a novel electron-bifurcating dehydrogenase catalyzing the last step of heme d1 biosynthesis. J. Biol. Chem., 289, 30753-30762
Adamczack, J., Hoffmann, M., Papke, U., Haufschildt, K., Nicke, T., Bröring, M., Sezer, M., Weimar, R., Kuhlmann, U., Hildebrandt, P. and Layer, G.
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Fungal Genet Biol. 2014 Genetic characterization of the Neurospora crassa molybdenum cofactor biosynthesis 66:69-78
Probst C, Ringel P, Boysen V, Wirsing L, Alexander MM, Mendel RR, Kruse T
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(2015) Oxyl and hydroxyl radical transfer in mitochondrial amidoxime reducing component-catalyzed nitrite reduction. J Am Chem Soc. 137(16): 5276-5279
Yang J, Giles LJ, Ruppelt C, Mendel RR, Bittner F, Kirk ML
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(2015) Sulphur shuttling across a chaperone during molybdenum cofactor maturation. Nat Commun. 6:6148
Arnoux P, Ruppelt C, Oudouhou F, Lavergne J, Siponen MI, Toci R, Mendel RR, Bittner F, Pignol D, Magalon A, Walburger A
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(2015). A Periplasmic Complex of the Nitrite Reductase NirS, the Chaperone DnaK and the Flagellum Protein FliC is essential for Flagellum Assembly and Motility in Pseudomonas aeruginosa. J. Bacteriol. Vol. 197-19, 3066-3075
Borrero-de Acuña JM , Molinari G, Rohde M, Dammeyer T, Wissing J, Jänsch, L, Arias S, Jahn M, Timmis KN, Schobert M, Jahn D
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Enzymatic Characterization of Recombinant Nitrate Reductase Expressed and Purified from Neurospora crassa; Fung. Gen. Biol. 80, 10-18, 2015
P. Ringel, C. Probst, T. Dammeyer, S. Buchmeier, L. Jänsch, J. Wissing, P. Tinnefeld, R.R. Mendel, B.M. Jockusch, T. Kruse
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Linear Discriminant Analysis Identifies Mitochondrially Localized Proteins in Neurospora crassa. J Proteome Res. (2015) 14:3900-11
Wirsing L, Klawonn F, Sassen WA, Lünsdorf H, Probst C, Hust M, Mendel RR, Kruse T, Jänsch L
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(2016). Dimerization of the plant molybdenum insertase Cnx1E is required for synthesis of the molybdenum cofactor. Biochem. J.
Joern Krausze, Corinna Probst, Ute Curth, Joachim Reichelt, Sayantan Saha, David Schafflick, Dirk W. Heinz, Ralf R. Mendel, Tobias Kruse
