Metall-Spezifität und Katalysemechanismus der Quercetinase QueD
Zusammenfassung der Projektergebnisse
Quercetinases (flavonol 2,4-dioxygenases) are metal-dependent dioxygenases of the cupin superfamily. While fungal quercetinases are copper proteins, recombinant Streptomyces quercetinase (QueD) was previously described to be capable of incorporating Ni2+ and some other divalent metal ions. The molecular basis and the mechanistic implications of such “metal promiscuity” are not well understood and were addressed in this project. Metal occupancies of heterologously produced QueD proteins followed the order Ni > Co > Fe > Mn. This ranking is similar to the Irving-Williams series, which describes the stability of divalent transition-metal complexes as Mn2+ < Fe2+ < Co2+ < Ni2+ < Cu2+ > Zn2+. In terms of activity, we observed the ranking Ni > Co > Mn. Iron, in contrast to the other metals, did not support catalytic activity. QueD isolated from the wild-type Streptomyces sp. strain FLA contained mainly nickel and zinc, and its catalytic activity likely can be attributed to a nickel center. In vitro synthesis of QueD in a cell-free transcription-translation system yielded catalytically active protein when Ni2+ was present, and comparison of the circular dichroism spectra of in vitro produced proteins suggested that Ni2+ ions support correct folding. Ni2+ most likely is the physiologically relevant cofactor of QueD of Streptomyces sp. FLA. Replacement of individual amino acids of the 3His/1Glu metal binding motif by alanine drastically reduced or abolished quercetinase activity and affected its structural integrity. Only substitution of the glutamate ligand (E76) by histidine resulted in Ni- and Co-QueD variants that retained the native fold and showed residual activity. Thus, heterologous formation of catalytically active, native QueD holoenzyme requires Ni2+, Co2+ or Mn2+, i.e., metal ions that prefer an octahedral coordination geometry, and an intact 3His/1Glu motif or a 4His environment of the metal. EPR studies and crystallographic data gave important insights into the reaction mechanism of Streptomyces quercetinase. Soaking experiments of NiQueD crystals with quercetin verified the formation of enzyme-substrate complex under anoxic conditions. Monodentate metal binding of quercetin followed by substrate deprotonation was proposed as the first reaction step, which was promoted by a “flip-in” conformation change of E76 upon substrate binding. In the complex of NiQueD with quercetin and O2, the dioxygen was “side-on” coordinated to the metal with an almost perpendicular orientation towards the substrate. However, it remained unclear whether the C2 atom of the bound quercetin shows an sp character indicative of substrate activation via radical formation. EPR analysis of Ni- and CoQueD proved conservation of the divalent valence state for both metal centers and of the high-spin state in case of Co2+ center after substrate addition and oxygen exposure. Evidence for presence of an intermediate radical state was derived for CoQueD variants Y117A and I66A which allow for interaction with reducing CMH which is obviously hindered in the wild type protein. Modification of Co ligand sphere in the E76H variant leads to one (or two radicals) close to the Co-center showing spin coupling. Here, activation of substrate is occurring without participation of oxygen. This is apparently not the case for NiQueD-E76H, albeit it has a very similar structure as the Co form, which confirms the importance of electronic properties of the metal ion in affecting the activation mechanism. The metal ion, coordinating both O2 and the quercetin anion (deprotonated by the conserved glutamate ligand), may act as Lewis acid and facilitate single electron transfer from the bound flavonolate to O2. The higher Lewis acidity of nickel over cobalt or manganese should make it more favorable for catalysis. A non-redox role of the metal ion in catalysis is proposed.
Projektbezogene Publikationen (Auswahl)
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(2012) Characterisation of the cambialistic quercetinase of Streptomyces sp. FLA. BIOspektrum Sonderausgabe zur VAAM-Jahrestagung 2012, S. 161-162, OTP112
Nianios D, Fetzner S
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(2012) Ring-cleaving dioxygenases with a cupin fold. Appl. Environ. Microbiol. 78: 2505-2514
Fetzner S
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(2013) Variation of the 3His-1Glu-motif in quercetinase from Streptomyces sp. FLA, a monocupin dioxygenase with preference for Ni2+ ions. FEMS Congress 2013, July 21-25, Leipzig
Nianios D, Fetzner S
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(2014) IX Conference of European Federation of EPR Societies, EFEPR, 7-11 September 2014, Marseille, France: Cyclic Hydroxylamines for EPR Detection of Transient Radicals and Reactive Oxygen Species
N.N. Bui, B. Pasieka, E.J. Slowik, I. Bogeski, M. Hoth, S. Thierbach, D. Nianios, S. Fetzner, R. Kappl
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(2015) Nickel quercetinase, a "promiscuous" metalloenzyme: Metal incorporation and metal ligand substitution studies. BMC Biochemistry 16:10
) Nianios D, Thierbach S, Steimer L, Lulchev P, Klostermeier D, Fetzner S
