Abstract
3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is an NADH-specific flavoprotein monooxygenase that catalyzes the para-hydroxylation of 3-hydroxybenzoate (3HB) to form 2,5-dihydroxybenzoate (2,5-DHB). Based on results from stopped-flow spectrophotometry, the reduced enzyme-3HB complex reacts with oxygen to form a C4a-peroxy flavin with a rate constant of 1.13 ± 0.01 × 10(6) m(-1) s(-1) (pH 8.0, 4 °C). This intermediate is subsequently protonated to form a C4a-hydroperoxyflavin with a rate constant of 96 ± 3 s(-1). This step shows a solvent kinetic isotope effect of 1.7. Based on rapid-quench measurements, the hydroxylation occurs with a rate constant of 36 ± 2 s(-1). 3HB6H does not exhibit substrate inhibition on the flavin oxidation step, a common characteristic found in most ortho-hydroxylation enzymes. The apparent kcat at saturating concentrations of 3HB, NADH, and oxygen is 6.49 ± 0.02 s(-1). Pre-steady state and steady-state kinetic data were used to construct the catalytic cycle of the reaction. The data indicate that the steps of product release (11.7 s(-1)) and hydroxylation (36 ± 2 s(-1)) partially control the overall turnover.
Highlights
Monooxygenases or hydroxylases are useful biocatalysts for catalyzing regio-specific oxygenation by molecular oxygen under mild conditions
The overall structure of 3-Hydroxybenzoate 6-hydroxylase (3HB6H) is similar to other single-component flavoprotein aromatic hydroxylases, the arrangement of the active site residues is quite different from ortho-hydroxylation enzymes such as p-hydroxybenzoate 3-hydroxylase (PHBH)
This work reports on the kinetics of the oxidative half-reaction of 3HB6H from R. jostii RHA1 using various transient kinetic and mechanistic tools
Summary
Reagents—NADH (purity Ն95%) and FAD (purity Ն95%) were purchased from Sigma. 3-Hydroxybenzoic acid (3HB) and 2,5-dihydroxybenzoic acid (2,5-DHB) were purchased from Merck. Steady-state Kinetics—To determine an apparent catalytic constant (kcat) at 4 °C, a solution of the enzyme at 1 M in the presence of 10 mM 3HB and 2 M FAD in air saturation (0.26 mM oxygen) was mixed with an oxygenated buffer (1.92 mM oxygen) plus 10 mM 3HB, 10 mM NADH, and 2 M FAD using a stopped-flow spectrophotometer. The reduced enzyme solution was mixed with buffers containing various oxygen concentrations in the stopped-flow spectrophotometer. To prepare enzyme in D2O buffer, the concentrated enzyme solution (700 l, A452 ϳ 5.1) was equilibrated inside the anaerobic glove box for 30 min to remove oxygen. The solution of reduced enzyme-3HB complex was transferred into a tonometer and left overnight (ϳ18 h) at 4 °C prior to the stopped-flow experiment This preparation process was to assure that the enzyme was fully equilibrated in D2O and that all of the exchangeable sites on the reduced enzyme had incorporated deuterium. The concentrations of product formed were estimated based on a standard curve in the range of 2– 40 M 2,5-DHB
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