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.
The gene queD encoding quercetinase of Streptomyces sp. FLA, a soil isolate related to S. eurythermus (T), was identified. Quercetinases catalyze the 2,4-dioxygenolytic cleavage of 3,5,7,3',4'-pentahydroxyflavone to 2-protocatechuoylphloroglucinol carboxylic acid and carbon monoxide. The queD gene was expressed in S. lividans and E. coli, and the recombinant hexahistidine-tagged protein (QueDHis(6)) was purified. Several flavonols were converted by QueDHis(6), whereas CO formation from the 2,3-dihydroflavonol taxifolin and the flavone luteolin were not observed. In contrast to bicupin quercetinases from Aspergillus japonicus and Bacillus subtilis, and bicupin pirins showing quercetinase activity, QueD of strain FLA is a monocupin exhibiting 35.9% sequence identity to the C-terminal domain of B. subtilis quercetinase. Its native molecular mass of 63 kDa suggests a multimeric protein. A queD-specific probe hybridized with fragments of genomic DNA of four other quercetin degrading Streptomyces strains, but not with DNA of B. subtilis. Potential ORFs upstream of queD probably code for a serine protease and an endoribonuclease; two ORFs downstream of queD may encode an amidohydrolase and a carboxylesterase. This arrangement suggests that queD is not part of a catabolic gene cluster. Quercetinases might play a major role as detoxifying rather than catabolic enzymes.
Merkens
Hedda
H
Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstr. 3, 48149 Münster, Germany.
Sielker
Sonja
S
Rose
Karsten
K
Fetzner
Susanne
S
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002248:Carbon Monoxide / Q000378:metabolism; D049308:Dioxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D044948:Flavonols / Q000378:metabolism*; D008969:Molecular Sequence Data; D011794:Quercetin / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D048170:Streptomyces lividans / Q000201:enzymology / Q000235:genetics
D001426:Bacterial Proteins; D044948:Flavonols; D011994:Recombinant Proteins; D002248:Carbon Monoxide; D011794:Quercetin; D049308:Dioxygenases; C003281:quercetin 2,3-dioxygenase