Novel multi-site enzymes in the transformation of aliphatic and aromatic hydrocarbons
Final Report Abstract
Structure–function relationships of complex multi-site enzymes have been studied. These enzymes are involved in the transformation of aliphatic and aromatic hydrocarbons. We have focussed on the ThDP/FAD enzyme Cyclohexane-1,2- dione Hydrolase (CDH) which cleaves a cyclic α-diketone to 6-oxohexanoate followed by the NAD+ dependent oxidation to adipate, and the W-molybdopterin/Fe-S enzyme Acetylene Hydratase (AH) which converts acetylene to acetaldehyde. (1) CDH (1 ThDP, 1 FAD, 1 Mg2+/64.5 KDa) has been purified in large quantities which allows extensive studies both in solution and in crystallo. Phylogenetic analysis shows the ThDP enzymes acetohydroxyacid synthase, cytochrome pyruvate dehydrogenase, and pyruvate oxidase to be the closest relatives. Sequence analysis reveals the localisation of the ThDP binding motif and of the twofold Rossmann fold for FAD binding at the C-terminal end and central region of CDH, respectively. The 3D structure (1.26 Å, with inhibitors Cl- and 2-methyl-2,4-pentanediol bound) reveals a homotetramer; in solution CDH is a homodimer. Localization and V- conformation of ThDP, and the proximities of N1‟ and N4‟ of ThDP to a glutamate are highly conserved among ThDP enzymes, they are responsible for the formation of the catalytic ThDP-ylide. A congruent residue of Phe479, responsible for electron transfer from ThDP to FAD in pyruvate oxidase, is missing in CDH. Moreover, the isoalloxazine ring of FAD is planar. The active site funnel is rearranged in an unprecedented manner providing the structural basis for the specific binding and cleavage of alicyclic compounds. Crucial features include a new funnel entrance, a semi-circularly shaped loop segment preceding the C-terminal arm and the attachment of the C-terminal arm to other subunits of the CDH tetramer. The 3D structure, with CDO bound at the active site (1.1 Å), together with kinetic and spectroscopic studies, help to develop a first mechanism of the ring cleavage reaction. The substrate binding site is characterized by polar and non-polar moieties reflected in the structures of MPD and CDO and three histidine residues that most likely play a crucial role in substrate activation. The NAD+ dependent oxidation of 6- oxohexanoate remains enigmatic as the redox-active cofactor FAD seems not to participate in catalysis, and no obvious NAD+ binding site is found. The activity of a large set of compounds has been studied which are structurally related to CDO or 6-oxohexanoate. Surprisingly, 4-chloro-3-nitro-benzaldehyde is a substrate (15% activity of CDO), and CDH shows carboligase activity with pyruvate and benzaldehyde. Early attempts to produce variants of CDH failed as no soluble protein could be expressed. However, active CDH and variants have been obtained very recently by our colleagues at the Universität Freiburg using a synthetic gene. (2) AH (1 W, 2 MGD, 1 [4Fe-4S] has been studied extensively in crystallo through exposure of crystalline protein to various gases (C2H2, CO, N2, Xe). In two structures, C2H2 molecules can be detected; in one the carbon atoms of C2H2 are 5.10 Å and 5.16 Å away from W bound H2O. This is too far away for efficient catalysis, the position of this C2H2 molecule shows that it can pass the hydrophobic ring and can enter the active site cavity. Current computational approaches favor a direct coordination of the substrate acetylene to tungsten (not via the bound water) which appears unlikely in view of the structural data.
Publications
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(2009) Cyclohexane-1,2-dione hydrolase: A new tool to degrade alicyclic compounds. J. Molecular Catalysis B: Enzymatic 61, 47–49
S. Fraas, A. K. Steinbach, A. Tabbert, J. Harder, U. Ermler, K. Tittmannd, A. Meyer, P. M.H. Kroneck
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(2011) Cyclohexane-1,2-Dione Hydrolase from Denitrifying Azoarcus sp. Strain 22Lin, a Novel Member of the Thiamine Diphosphate Enzyme Family. J. Bacteriol. 193, 6760-6769
A. K. Steinbach, S. Fraas, J. Harder, A. Tabbert, H. Brinkmann, A. Meyer, U. Ermler, P.M. H. Kroneck
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(2011) Exploring the Active Site of the Tungsten, Iron-Sulfur Enzyme Acetylene Hydratase. J. Bacteriol. 193, 1229–1236
F. ten Brink, B. Schink, P.M.H. Kroneck
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(2011), Acetylene Hydratase. Handbook of Metalloproteins (A. Messerschmidt, ed). Chichester, UK. Vol. 4+5, pp. 541-548. (Online Article)
G.B. Seiffert, D. Abt, F. ten Brink, D. Fischer, O. Einsle, P.M.H. Kroneck
