Structure and function of benzoyl-coenzyme A reductases
Final Report Abstract
Aromatic compounds are widely abundant in nature and their biodegradation is essential for the global carbon cycle and for the elimination of aromatic pollutants. In aerobic, aromatic compound degrading microorganisms, oxygenases are involved in all key steps of aromatic degradation. In contrast, anaerobic bacteria have to employ a completely different, oxygen-independent strategy to use aromatic growth substrates. In the last decades, benzoyl-coenzyme A (benzoyl-CoA) was established as key intermediate in the degradation of the vast majority of monocyclic aromatic compounds; it serves as substrate for dearomatizing benzoyl-CoA reductases (BCRs). These key enzymes of anaerobic aromatic degradation break aromaticity by reduction to a cyclic dienoyl-CoA. Surprisingly, two different, non-related BCR classes exist in nature that accomplish this mechanistically demanding redox reaction in different manners. The class I BCRs contain three [4Fe- 4S] cluster cofactors and couple the endergonic benzoyl-CoA reduction to a stoichiometric ATP hydrolysis. The class II BCRs reduce the aromatic ring at a W-cofactor most possibly driven by flavinbased electron bifurcation. In the project, the structure and function of representative members of both BCR classes should be studied by numerous methodologies including X-ray structural analyses of protein crystals, spectroscopic, kinetic, and computational analyses. With the development of a heterologous production platform, previously unknown members of class I BCRs were produced in Escherichia coli, isolated and characterized. A catalytically versatile enzyme from T. chlorobenzoica was identified that converts a number of methylated and halogenated substrate analogues. Moreover, the ATP-binding subunits of the class I BCR from hyperthermophilic archaeon Ferroglobus placidus, that degrades aromatic compounds at 85 °C was isolated and characterized. The heterologous production platform allowed for obtaining anaerobically grown protein crystals that opened the door for solving the first X-ray structure of a class I BCR at 1.9 Å resolution. The structure supports the proposed Birch-like mechanism of enzymatic dearomatization by class I BCRs. An active site [4Fe-4S] cluster binds the aromatic substrate via the thioester carbonyl suggesting spatially separated single electron and proton transfer events. Unexpectedly, a previously unknown, mechanistically demanding defluorination activity of class I BCR was identified. The extremely oxygen-sensitive class I BCRs are protected from oxygendamage by a newly identified detoxifying enzyme: the 1,5-dienoyl-CoA oxidase catalyses the rearomatization of the cyclic dienoyl-CoA formed by BCR in order to reduce toxic O2 to water. The crystal structure of the active site subunits of a prototypical class II BCR from the Fe(III)-respiring Geobacter metallireducens was solved providing first insights into the catalytic mechanism. Here, benzoyl-CoA reduction is accomplished at an active site W-cofactor that transfers single electrons via an inorganic ligand directly to the aromatic ring. Based on the structure obtained, a sophisticated computational analysis revealed a plausible mechanism of class II BCR catalysis, in which hydrogen atom transfer from an aqua ligand at the W-cofactor to the aromatic ring initiates the reaction. We isolated and characterized the 1 MDa class II BCR complex from a Fe(III) reducing and a sulfatereducing model organism, respectively. With six W-cofactors, six flavins, two selenocysteines and more than 50 FeS cluster it represents probably the most complex electron transfer machinery in nature. In Fe(III)-respiring organisms, this complex appears to be even more complicated as evidenced by the membrane association of the complex. Plausible scenarios for a flavin-based electron bifurcation driven aromatic ring reduction where deduced. In summary, the project revealed a number of insights into the function of the two classes of dearomatizing BCRs that in spite of their marked differences dearomatize their substrate by reduction to the same product.
Publications
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(2015) Structural basis of enzymatic benzene ring reduction. Nat Chem Biol 11:586-91
Weinert, T, Huwiler, SG, Kung, JW, Weidenweber, S, Hellwig, P, Cotelesage, JH, George, GN, Ermler, U and Boll, M
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(2017) ATP-Dependent C-F Bond Cleavage Allows the Complete Degradation of 4-Fluoroaromatics without Oxygen. MBio 7(4)
Tiedt O, Mergelsberg M, Boll K, Müller M, Adrian L, Jehmlich N, von Bergen M, Boll M
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(2017) Breaking Benzene Aromaticity-Computational Insights into the Mechanism of the Tungsten-Containing Benzoyl-CoA Reductase. J Am Chem Soc 139:14488-14500
Culka M, Huwiler SG, Boll M, Ullmann GM
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(2018) A catalytically versatile benzoyl-CoA reductase, key enzyme in the degradation of methyl- and halobenzoates in denitrifying bacteria. J Biol Chem 293:10264-10274
Tiedt O, Fuchs J, Eisenreich W, Boll M
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(2019) One megadalton metalloenzyme complex in Geobacter metallireducens involved in benzene ring reduction beyond the biological redox window. Proc Natl Acad Sci 116, 2259-2264
Huwiler, SG Löffler, C., Anselmann, S.E.L., Stärk, H.-J., von Bergen, M., Flechsler, J., Rachel, R. & Boll, M
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(2019) The class II benzoyl-coenzyme A reductase complex from the sulfate-reducing Desulfosarcina cetonica. Environ Microbiol 21:4241-4252
Anselmann SEL, Löffler C, Stärk HJ, Jehmlich N, von Bergen M, Brüls T, Boll M
