Quercetinase QueD of Streptomyces sp. FLA, a monocupin dioxygenase with a preference for nickel and cobalt.

Quercetinase (QueD) of Streptomyces sp. FLA is an enzyme of the monocupin family and catalyzes the 2,4-dioxygenolytic cleavage of the flavonol quercetin. After expression of the queD gene in Escherichia coli, high specific QueD activity was found in crude cell extracts when the growth medium was supplemented with NiCl 2 or CoCl 2, but not when Mn (2+), Fe (2+), Cu (2+), or Zn (2+) was added. The metal occupancy of Ni- and Co-QueD purified from these cells was </=50%, presumably due to strong overproduction of QueD in E. coli. Circular dichroism spectroscopy indicated the same folded structure with a high content of beta-sheet for the Ni and Co protein. The apparent kinetic constants for quercetin of Ni-QueD ( k cat = 40.1 s (-1), and K m = 5.75 microM) and Co-QueD ( k cat = 7.6 s (-1), and K m = 0.96 muM) indicate similar catalytic efficiencies; however, the approximately 5-fold lower apparent K m value of Ni-QueD for dioxygen suggests that the nickel enzyme performs better under physiological conditions. The pH dependence of k cat,app indicates that an ionizable group with a p K a near 6.8 has to be deprotonated for catalysis. Electron paramagnetic resonance spectra of resting Co-QueD are indicative of a high-spin ( S = (3)/ 2) Co (2+) species in a tetrahedral or trigonal-bipyramidal coordination geometry. Anoxic binding of quercetin to QueD drastically altered the hyperfine pattern at g approximately 6 without changing the valence state of the Co(II) center and elicited a hypsochromic shift of UV-vis absorption band I of quercetin. On the basis of spectroscopic data, and considering the organic chemistry of flavonols, a nonredox role of the metal center in catalysis is discussed.