Carbon monoxide forming dioxygenases: Non-copper quercetinases from Streptomyces sp. and Actinoplanes missouriensis
Final Report Abstract
The aerobic microbial degradation of quercetin is initiated by 2,4-dioxygenolytic cleavage of the O-heterocyciic ring of the flavonol with formation of carbon monoxide. In order to further our understanding of how dioxygenases work at the molecular level, this project aimed at the comparison of oxygenases that are isofunctional, but contain different cofactors. Quercetinases (flavonol 2,4-dioxygenases) were chosen as model enzymes to address this question, because the fungal enzymes and a bacterial representative were known to contain different metal ions: Whereas quercetinases from Aspergillus spp. depend on a mononuclear Cu(ll) cofactor, quercetinase from Bacillus subtilis was reported to be most active with Mn(ll). Another goal was to compare metal-dependent flavonol 2,4-dioxygenases with cofactor-independent 3-hydroxy-4(1H)quinolone 2,4-dioxygenases, which catalyze a similar reaction, and with Ni(ll)-containing aci-reductone dioxygenase, another CO-forming enzyme. These CO forming dioxygenases may be mechanistically similar, or they may have evolved different ways to catalyze similar reactions. Whereas the cofactor-independent 2,4- dioxygenases are related to the o/ß-hydrolase fold enzymes, fungal as well as Bacillus quercetinases and aci-reductone dioxygenase belong to the cupin superfamily. In the flavonol degrading Sfreptomyces sp. strain FLA, we detected a quercetinase which appeared to not depend on Cu(ll) ions for catalytic activity. The queD gene encoding this novel quercetinase was identified. Sequence analysis indicated that queD codes for a protein of the monocupin family, whereas other known quercetinases are composed of bicupin subunits (each subunit comprising two cupin domains). Transcription of queD in Streptomyces sp. strain FLA is induced by quercetin and the quercetin glycoside rutin, whereas possible intermediates of the downstream metabolism and metal ions apparently did not increase queD expression. The queD gene presumably is transcribed from a a70- dependent promoter; an LmrA/YxaF-like represser protein (TetR family) may be involved in transcriptional regulation. Overexpression of queD in E. coli and purification of recombinant protein from cells grown in the presence of different metal salts revealed that QueD is most active with Ni(ll) as cofactor, followed by Co(ll), whereas Fe(ll) - the metal which is incorporated when recombinant E. coli was grown in complex medium - is a poor cofactor. Such preference for Ni(ll) or Co(ll) is very unusual for oxygenases. The Ni- and Co-QueD were purified, and characterized by metal analysis, CD spectroscopy, and steady-state kinetics. The pH dependence of kinetic parameters suggested that in the enzyme-substrate complex, an ionisable group with a pKa of about 6.8 has to be deprotonated for catalysis. The QueD proteins (as isolated) as well as anoxic enzyme-substrate complexes, were also characterized by UV/Vis and EPR spectroscopy. Taken together, the kinetic and spectroscopic data suggested a non-redox role of the metal cofactor of QueD in catalysis. We propose that the catalytic mechanisms of cofactor-independent 2,4-dioxygenases, Ni(ll)-dependent aci-reductone dioxygenase, and quercetinases may be more similar than previously thought.
Publications
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(2007) A new monocupin quercetinase of Streptomyces sp. FLA: Identification and heterologous expression of the queD gene and activity of the recombinant enzyme towards different flavonols. Arch. Microbiol. 187:475-487
Merkens H, Sielker S, Rose K, Fetzner S
