TY  - JOUR
AB  - Background: Quercetinases 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. This raises the questions of which factors determine metal selection, and which metal ion is physiologically relevant. Results: Metal occupancies of heterologously produced QueD proteins followed the order Ni > Co > Fe > Mn. Iron, in contrast to the other metals, does not support catalytic activity. QueD isolated from the wild-type Streptomyces sp. strain FLA contained mainly nickel and zinc. 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. 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 catalytic activity. Conclusions: 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. The observed metal occupancies suggest that metal incorporation into QueD is governed by the relative stability of the resulting metal complexes, rather than by metal abundance. Ni2+ most likely is the physiologically relevant cofactor of QueD of Streptomyces sp. FLA.
AU  - Nianios, Dimitrios
AU  - Thierbach, Sven
AU  - Steimer, Lenz
AU  - Steimer, Lenz Rudolf
AU  - Lulchev, Pavel
AU  - Klostermeier, Dagmar
AU  - Fetzner, Susanne
DA  - 2015-04-23
DO  - 10.1186/s12858-015-0039-4
KW  - Dioxygenase
KW  - Flavonol
KW  - Metalloprotein
KW  - Nickel
KW  - Coordination geometry
KW  - Cell-free protein synthesis
LA  - eng
N1  - BMC Biochemistry 16 (2015) 10, 1-11
N1  - Finanziert durch den Open-Access-Publikationsfonds 2015/2016 der Westfälischen Wilhelms-Universität Münster (WWU Münster).
PY  - 2015-04-23
SN  - 1471-2091
TI  - Nickel quercetinase, a “promiscuous” metalloenzyme: metal incorporation and metal ligand substitution studies
UR  - https://nbn-resolving.org/urn:nbn:de:hbz:6-09239698891
Y2  - 2024-11-22T04:56:37
ER  -