title: Nickel quercetinase, a "promiscuous" metalloenzyme: metal incorporation and metal ligand substitution studies.
abstract: 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 Ni(2+) 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 Ni(2+) was present, and comparison of the circular dichroism spectra of in vitro produced proteins suggested that Ni(2+) 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 Ni(2+), Co(2+) or Mn(2+), 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. Ni(2+) most likely is the physiologically relevant cofactor of QueD of Streptomyces sp. FLA.
authors.affiliation: Institute of Molecular Microbiology and Biotechnology, University of Muenster, Corrensstrasse 3, Muenster, D-48149, Germany. d_nian01@uni-muenster.de.
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Nianios
authors.affiliation: Institute of Molecular Microbiology and Biotechnology, University of Muenster, Corrensstrasse 3, Muenster, D-48149, Germany. s.thierbach@uni-muenster.de.
authors.forename: Sven
authors.initials: S
authors.lastname: Thierbach
authors.affiliation: Institute of Physical Chemistry, University of Muenster, Corrensstrasse 30, Muenster, D-48149, Germany. lenz.steimer@gmx.de.
authors.forename: Lenz
authors.initials: L
authors.lastname: Steimer
authors.affiliation: Institute of Physical Chemistry, University of Muenster, Corrensstrasse 30, Muenster, D-48149, Germany. plulc_01@uni-muenster.de.
authors.forename: Pavel
authors.initials: P
authors.lastname: Lulchev
authors.affiliation: Institute of Physical Chemistry, University of Muenster, Corrensstrasse 30, Muenster, D-48149, Germany. dagmar.klostermeier@uni-muenster.de.
authors.forename: Dagmar
authors.initials: D
authors.lastname: Klostermeier
authors.affiliation: Institute of Molecular Microbiology and Biotechnology, University of Muenster, Corrensstrasse 3, Muenster, D-48149, Germany. fetzner@uni-muenster.de.
authors.forename: Susanne
authors.initials: S
authors.lastname: Fetzner
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D055162:Biocatalysis; D049308:Dioxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D009532:Nickel / Q000378:metabolism*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: D008024:Ligands; D011994:Recombinant Proteins; D009532:Nickel; D049308:Dioxygenases; C003281:quercetin 2,3-dioxygenase