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Kinetic and Spectroscopic Studies on the Quercetin 2,3-Dioxygenase from Bacillus Subtilis† Matthew R

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Kinetic and Spectroscopic Studies on the Quercetin 2,3-Dioxygenase from Bacillus Subtilis† Matthew R Biochemistry 2006, 45, 1009-1016 1009 Kinetic and Spectroscopic Studies on the Quercetin 2,3-Dioxygenase from Bacillus subtilis† Matthew R. Schaab, Brett M. Barney,‡ and Wilson A. Francisco* Department of Chemistry and Biochemistry, Arizona State UniVersity, Tempe, Arizona 85287-1604 ReceiVed August 8, 2005; ReVised Manuscript ReceiVed NoVember 16, 2005 ABSTRACT: Quercetin 2,3-dioxygenase from Bacillus subtilis (QueD) converts the flavonol quercetin and molecular oxygen to 2-protocatechuoylphloroglucinolcarboxylic acid and carbon monoxide. QueD, the only known quercetin 2,3-dioxygenase from a prokaryotic organism, has been described as an Fe2+- dependent bicupin dioxygenase. Metal-substituted QueDs were generated by expressing the enzyme in Escherichia coli grown on minimal media in the presence of a number of divalent metals. The addition of Mn2+,Co2+, and Cu2+ generated active enzymes, but the addition of Zn2+,Fe2+, and Cd2+ did not increase quercetinase activity to any significant level over a control in which no divalent ions were added to the media. The Mn2+- and Co2+-containing QueDs were purified, characterized by metal analysis and EPR spectroscopy, and studied by steady-state kinetics. Mn2+ was found to be incorporated nearly stoichiometrically to the two cupin motifs. The hyperfine coupling constant of the g ) 2 signal in the EPR spectra of the Mn2+-containing enzyme showed that the two Mn2+ ions are ligated in an octahedral coordination. The turnover number of this enzyme was found to be in the order of 25 s-1, nearly 40-fold higher than that of the Fe2+-containing enzyme and similar in magnitude to that of the Cu2+-containing quercertin 2,3-dioxygenase from Aspergillus japonicus. In addition, kinetic and spectroscopic data suggest that the catalytic mechanism of QueD is different from that of the Aspergillus quercetinases but similar to that proposed for the extradiol catechol dioxygenases. This study provides evidence that Mn2+ might be the preferred cofactor for this enzyme and identifies QueD as a new member of the manganese dioxygenase family. The flavonol quercetin, isolated from numerous plants, is Scheme 1: Reaction Catalyzed by Quercetin used in traditional medicine for its antioxidant and antimi- 2,3-Dioxygenase crobial properties (1). Various strains of the filamentous fungus Aspergillus can utilize quercetin and its 3-O-glycoside (rutin) as their only carbon source via an extracellular enzyme system (2, 3). The first enzyme in the metabolism of quercetin by this organism is quercetin 2,3-dioxygenase or quercetinase, which catalyzes the oxidative decomposition of quercetin to 2-protocatechuoylphloroglucinolcarboxylic acid and carbon monoxide, as shown in Scheme 1. The quercetinases from Aspergillus flaVus (4), Aspergillus niger (5), and Aspergillus japonicus (6) have been characterized, and the crystal structure of the A. japonicus quercetinase has been reported (6). Recently, the first example of a bacterial structure (10). The cupin domain comprises two conserved quercetinase was reported (7, 8), corresponding to the YxaG motifs with the following characteristic conserved se- protein of Bacillus subtilis. quences: G(X)5HXH(X)3,4E(X)6G and G(X)5PXG(X)2H- While the sequences of the quercetinases from A. japonicus (X)3N, separated by a variable loop of ∼20 amino acids (10). (6) and B. subtilis share only a 19% sequence identity and These two motifs have been found to ligate a number of 39% similarity, their three-dimensional structures are highly divalent metal ions (e.g., Mn2+,Cu2+, and Fe2+), which are similar (9). Both enzymes belong to the cupin superfamily, ligated by two histidines and glutamic acid from motif 1 a family of proteins characterized by their â-barrel tertiary and a histidine residue from motif 2 (11). While the Aspergillus quercetinases are Cu2+-containing enzymes, the † This work was supported by NSF Grant MCB-0317126 (to W.A.F.). quercetinase from B. subtilis (QueD)1 has been reported to B.M.B. was supported in part by a fellowship through the Research + Training Group in Optical Biomolecular Devices provided under a grant contain two atoms of Fe2 per subunit, occupying the two from the NSF (DB1-9602258-003). cupin-binding domains present in this protein (9). Glu 69, * To whom correspondence should be addressed. Telephone: 480- His 62, His 624, and His 103 coordinate the first Fe2+ ion at 965-7480. Fax: 480-965-2747. E-mail: [email protected]. ‡ Present address: Department of Chemistry and Biochemistry, Utah the N-terminal cupin motif, and Glu 241, His 234, His 236, State University, 300 Old Main Hill, Logan, UT 84322-0300. and His 275 coordinate the second Fe2+ ion at the C-terminal 10.1021/bi051571c CCC: $33.50 © 2006 American Chemical Society Published on Web 12/22/2005 1010 Biochemistry, Vol. 45, No. 3, 2006 Schaab et al. cupin motif (9). The coordination of Fe2+ in QueD is unlike concentration of 50 mg/L and brought to a final concentration 2+ that observed for other Fe -dependent cupin dioxygenases, of 1 µM MnSO4 or CoCl2. The cultures were grown for an which are usually coordinated by two histidines and a additional4hat15°C and harvested by centrifugation at glutamic acid (12). The turnover number for this QueD is 10000g for 5 min. The cell paste was resuspended in 50 mM approximately 2 orders of magnitude lower than that of the Tris‚HCl, pH 7.5, and stored at -80 °C. quercetinase from A. flaVus (13), which suggests that Fe2+ Cells were thawed and ruptured by a French press at 7000 might not be the correct cofactor for this enzyme. Unfortu- psi in the presence of DNase (1 mg) and phenylmethane- nately, QueD has yet to be isolated from B. subtilis,sothe sulfonyl fluoride (1 mg). The cell debris was removed by natural cofactor is currently unknown. In previous reports, centrifugation at 20000g for 15 min. The supernatant was purified QueD overexpressed in Escherichia coli has been loaded onto a DEAE-Sephacel column (1 × 16 cm), shown to contain less than 2 equiv of Fe/subunit, and the equilibrated with 50 mM Tris‚HCl, pH 7.5, and eluted with presence of small amounts of various transition metals was a NaCl gradient (0-600 mM) in the same buffer. Fractions also detected in protein samples (7, 8). In addition, recon- were collected and assayed for quercetinase activity using stitution of the apoenzyme in the presence of Cu2+,Mn2+, the standard assay. The most active fractions were pooled Ni2+, and Co2+ resulted in reactivation of the enzyme (9). and brought to a 55% saturation level of ammonium sulfate. In this report, we describe the expression, purification, and The suspension was separated by centrifugation at 20000g characterization of Mn- and Co-containing QueDs, all with for 20 min. The supernatant was decanted, and the pellet higher activities than that of the Fe2+-containing QueD. We was resuspended in 50 mM Tris‚HCl, pH 7.5, and 100 mM present evidence that Mn2+ might be the correct cofactor NaCl. The protein solution was loaded onto an Ultrogel AcA for QueD and provide insights into the mechanism of this 34 column (2.5 × 120 cm) and eluted with 50 mM Tris‚ enzyme, in particular, the mechanism of oxygen activation. HCl, pH 7.5, and 100 mM NaCl. The most active fractions were analyzed for purity by SDS-PAGE, pooled, loaded MATERIALS AND METHODS onto a DEAE-Sepharose column (1 × 16 cm) equilibrated ‚ All chemicals were of the highest grade available and, with 50 mM Tris HCl, pH 7.5, and eluted with a gradient of NaCl (100-500 mM) in the same buffer. The most active unless otherwise stated, were purchased from Sigma-Aldrich. - All electrophoresis equipment and chemicals were purchased fractions were analyzed for purity by SDS PAGE, and the from Bio-Rad. Protein concentration was determined using most pure fractions were pooled and concentrated by the Bio-Rad DC assay using bovine serum albumin as a ultrafiltration using an Amicon Ultra-15 centrifugal filter unit standard. (10000 MWCO). The purified protein was frozen in liquid nitrogen and stored at -20 °C. Metal Dependence on the ActiVity of QueD. One liter Enzyme Assays. The standard activity assay was conducted cultures of M9 media were inoculated witha1mLovernight in 1 mL of 50 mM Tris‚HCl, pH 7.5, 100 mM NaCl, 50 µM starter culture (5 mL) of E. coli BL21(DE3) pQUER4 (8). quercetin, and 5% (v/v) DMSO, and the reaction was initiated Kanamycin was added to a final concentration of 30 µg/ by the addition of enzyme. The reaction was monitored by mL. The cultures were grown at 37 °C and 200 rpm for 6 h, the loss of absorbance of the substrate quercetin at 380 nm induced with isopropyl â-D-thiogalactopyranoside (IPTG) to ( ) 18500 M-1 cm-1) on a HP 8453 photodiode array a final concentration of 50 mg/L in the presence of 10 µM 380 spectrophotometer. One unit of enzyme activity is defined ZnSO , CuCl , CoCl , MnSO , FeCl , CdCl , or NiCl , and 4 2 2 4 2 2 2 as the amount of enzyme required to convert 1 µmol of allowed to grow for an additional4hatroom temperature quercetin to product in 1 min at 25 °C. (∼25 °C). The cells were harvested by centrifugation at Initial velocities were measured at varied quercetin and 10000g for 8 min. The cell paste was resuspended in 10 mL oxygen concentrations by the rate of oxygen consumption of 50 mM Tris‚HCl, pH 7.5, frozen at -80 °C, and thawed. using a Clark-type oxygen electrode. The decrease in oxygen The cells were ruptured using a French press at 7000 psi, concentration was measured with a YSI Model 5300 biologi- and the cell debris was removed by centrifugation at 20000g cal monitor.
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