The metal- and substrate-dependences of 2,4'-dihydroxyacetophenone dioxygenase

Abstract

While the enzyme, 2,4'-dihydroxyacetophenone dioxygenase (DAD), has been known for decades, very little has been characterized of the mechanism of the DAD-catalyzed oxidative cleavage of its reported substrate, 2,4'-dihydroxyacetophenone (DHA). The purpose of this study was to identify the active metal center and to characterize the substrate-dependence of the kinetics of the reaction to lay the foundation for deeper mechanistic investigation. To this, the DAD V1M mutant (bDAD) was overexpressed, purified, and reconstituted with various metal ions. Kinetic assays evaluating the activity of the reconstituted enzyme as well as the substrate- and product-dependences of the reaction kinetics were performed. The results from reconstitution of the apoprotein with a variety of metal ions support the requirement for an Fe³⁺ center for enzyme activity. Reaction rates showed simple saturation kinetics for DHA with values for k_cat and K_DHA of 2.4 s^-1 and 0.7 μM, respectively, but no significant dependence on the concentration of O₂. A low-level inhibition (K_I = 1100 μM) by the 4HB product was observed. The results support a minimal kinetic model wherein DHA binds to resting ferric enzyme followed by rapid addition of O₂ to yield an intermediate complex that irreversibly collapses to products.

Keywords: 2,4′-dihydroxyacetophenone dioxygenase; Dioxygenase; Non-heme iron; Steady-state kinetics; Substrate-dependence.

Fig. 1.

UV–visible absorbance spectra for DAD-Fe reconstitutions. Samples consisted of the anaerobic mixing of 20 μM DAD in 50 mM MES, pH 7.0 with varying equivalents of Fe²⁺ in 2mM HCl followed by overnight incubation in air. Spectra were determined from 10-fold dilutions of the reconstitution mixtures with 50 mM MES, pH 7.0. DAD-Fe mixtures: apoDAD (red crosses); apoDAD:Fe 1:1 (orange exes); apoDAD:Fe 1:2 (yellow diamonds); apoDAD:Fe 1:5 (green triangles); apoDAD:Fe 1:10 (blue circles); and bDAD (gray dashes). Inset: Reconstitution of apoDAD-Fe 1:10. Mixtures: apoDAD (red crosses), anaerobically-prepared apoDAD-Fe 1:10 (purple squares), and apoDAD-Fe 1:10 incubated in air overnight (blue circles). Spectra were determined without dilution. Each spectrum represents the average of at least four replicates. Spectra are offset for clarity. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2.

The consumption of DHA by DAD reconstituted with Fe²⁺. Reaction samples consisted of adding aerobically-incubated DAD-Fe reconstitutions to 83 μM DHA in 50 mM MES, 100 mM NaCl, pH 6.0 at 20.0 °C. The final enzyme concentration in each assay was 100 nM. DAD-Fe mixtures: apoDAD (red crosses); apoDAD:Fe 1:1 (orange exes); apoDAD:Fe 1:2 (yellow diamonds); apoDAD:Fe 1:5 (green triangles); apoDAD:Fe 1:10 (blue circles); bDAD (gray dashes), bDAD:Fe 1:5 (black solid line). Inset: The effect of incubation time on DHA consumption by apoDAD:Fe 1:5 reconstitutions incubated aerobically or anaerobically prior to assay. Incubation time: none (red dashes), 2 h anaerobic (black circles), 2 h aerobic (green dotted line), overnight aerobic (blue solid line). Each trace represents the average of four to five replicate traces. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3.

The dependence of the observed rate constant in the oxidation of DHA by bDAD on the concentrations of the substrates. Reactions samples consisted of mixtures containing varying concentrations of DHA and 12, 260, or 1200 μM O₂ in 50 mM MES, 100 mM NaCl, pH 6.0 at 20.0°C. Reactions were initiated with bDAD (4.2, 8.3, 17, 25, 33, or 41 nM). Each point represents the average of three to four replicates. The lines indicate the global fit of the concentration-dependent data with equation (2) with best-fit values for the kinetic parameters given in Table 2.

Fig. 4.

Product inhibition by 4HB in the oxidation of DHA by bDAD. Reaction samples consisted of 4.2 nM bDAD added to varying concentrations of DHA and 0 (red circles), 300 (orange squares), 600 (yellow diamonds), 900 (green triangles), or 1250 μM 4HB (blue exes) in 50 mM MES, 100 mM NaCl, pH 6.0 at 20.0 °C. Each point represents the average of three to six replicates. The dashed curves indicate the global fit of the concentration-dependent data with equation (3). Best-fit values for the kinetic parameters are given in Table 2. Inset: Double-reciprocal plot of the concentration-dependent data with the dashed lines representing the globally fit curves. Some points omitted for clarity. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Scheme 1.

The oxidative cleavage reactions catalyzed by DAD, ARD′, and the intradiol dioxygenases.

Scheme 2.

Proposed mechanisms for the DAD-catalyzed oxidative cleavage of DHA. (A) Adapted from Enya et al. [4] (B) Adapted from Keegan et al. [5].

Scheme 3.

Rapid-equilibrium ordered binding model with equilibrium formation of a DAD·O₂ complex followed by DHA binding.

Scheme 4.

Minimal kinetic model for the addition of O₂ to a DAD·DHA binary complex resulting in irreversible product formation.