A non-substrate effector of p-hydroxybenzoate hydroxylase

doi:10.1016/0006-291X(70)90986-1

Biochemical and Biophysical Research Communications

Volume 40, Issue 4, 24 August 1970, Pages 887-893

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Abstract

A substrate analogue, 6-hydroxynicotinate (6-OHNA), facilitates reduction of the enzyme bound flavin by TPNH but is not hydroxylated in the reoxidation of the enzyme by oxygen. Binding of 6-OHNA to oxidized p-hydroxybenzoate hydroxylase is accompanied by a perturbation of the visible spectrum and enhancement of the flavin fluorescence. Titrations using these properties indicate the formation of a 1:1 complex with a dissociation constant of 2.5 × 10⁻⁴ M. At high concentrations of 6-OHNA, binding of 6-OHNA to additional site(s) has been observed. The 6-OHNA binding is competitive with p-hydroxybenzoate binding as indicated by the reversal of p-hydroxybenzoate quenching of flavin fluorescence on addition of 6-OHNA. The presence of the effector, 6-OHNA, is necessary for the catalytic oxidation of TPNH.

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Cited by (22)

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1990, Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Salicylate hydroxylase from Pseudomonas putida (EC 1.14.13.1, salicylate, NADH:oxygen oxidoreductase) is an FAD-containing monooxygenase, which catalyzes decarboxylative hydroxylation of salicylate to produce catechol in the presence of NADH and O₂. By chemical treatment of the enzyme with dicarbonyl reagents, such as glyoxal, the original oxygenase activity was converted to the salicylate-dependent NADH-dehyrogenase activity with free FAD as electron acceptor. One of twenty arginine residues of this enzyme is concerned with this alteration of activity, as shown by the result of its modification at pH 6.9. This result is further supported by the isolation of one arginine-modified enzyme by chromatographic methods on DEAE-Sephadex, A-50 columns. It exhibits the dehydrogenase activity predominantly. This modified enzyme is spectrophotometrically and electrophoretically characterized by a minute conformational change around the active site, and kinetically by a 7-fold increase in an apparent K_m for NADH and a decrease of more than 5-fold in an apparent K_m for FAD as electron acceptor, with an apparent V_max of 22 s⁻¹ for the dehydrogenase activity. Flow kinetics also showed a marked decrease in the rate for oxygenation of the reduced enzyme-salicylate complex from 21 s⁻¹ (native enzyme) to 3.3 s⁻¹ (modified enzyme). These facts suggest that one arginine residue of the enzyme is responsible for the NADH binding site, and chemical modification of one arginine residue of the enzyme induces some conformational change around the active site to alter the catalytic activity from oxygenation to dehydrogenation.
p-Hydroxybenzoate Hydroxylase and Melilotate Hydroxylase
1978, Methods in Enzymology
This chapter focuses on the p-hydroxybenzoate hydroxylase and melilotate hydroxylase. Crystalline preparations of p-hydroxybenzoate hydroxylase have been obtained from four different species of Pseudomonas: P. desmolytica, P. putida A 3.12, putida M-6, and P. fluorescens. The enzymes from both P. putida species are extremely unstable undergoing rapid inactivation unless protected by substrates and other stabilizing agents, thereby making any detailed mechanistic investigation difficult. In contrast, the P. florescens enzyme described in the chapter is stable for months even in the absence of stabilizing agents. Several of these hydroxylases have been purified and characterized. Those obtained in pure stable form and studied include salicylate hydroxylase, p-hydroxybenzoate hydroxylase, and melilotate hydroxylase. A number of microorganisms capable of utilizing phenolic molecules as a sole source of carbon contain flavoprotein hydroxylases. The activity of the enzyme is determined spectrophotometrically by following the rate of substrate-dependent oxidation of NADPH at 340 nm. The enzyme activity can also be assayed by measuring the rate of oxygen consumption using an oxygen electrode.
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1978, Methods in Enzymology
This chapter provides information on enzyme salicylate hydroxylase. Salicylate hydroxylase is a flavoprotein hydroxylase, a member of a class of enzymes elaborated by some soil bacteria, typically pseudomonads. These enzymes are induced by the presence of phenolic compounds. The activity of the enzyme was determined spectrophotometrically by following the rate of substrate-dependent oxidation of NADH in a Cary 14 or Gilford 2400 spectrophotometer at 340 nm. Salicylate hydroxylate is established as a flavoprotein containing flavin adenine dinucleotide (FAD) as the sole prosthetic group. The purified hydroxylase migrates as one homogeneous and symmetrical peak in the Model E analytical untracentrifuge with 0.1 M KCl as solvent. Enzyme activity can also be assayed by measuring the rate of oxygen consumption using an oxygen electrode. This enzyme, in addition to its FAD prosthetic group, has three substrates: salicylate or other aromatic compound, pyridine nucleotide, and oxygen. Its kinetics, therefore, can be expected to be complex.
4 Flavin and Pteridine Monooxygenases
1975, Enzymes
The reactivity with oxygen of reduced flavins and flavoenzymes is one of the most interesting problems in the field of flavin chemistry. In the case of many flavoproteins, which are converted to the reduced form by the specific substrates whose oxidation they catalyze, the high rate of reaction with O₂, may be retained or even enhanced in which case they are classified as oxidases. The route of flavin reoxidation appears to be different in the two classes, involving the intermediate production of O₂^-,- and flavin semiquinone in the case of the dehydrogenases and direct two electron oxidation in the case of the oxidases, the product of reaction with O₂ in both classes, as with the free flavins, is H₂O₂. The flavoprotein monooxygenases can be conveniently subclassified on the basis of the electron donor involved in the catalytic reaction. In one group of enzymes, the substrate itself serves as the electron donor. This group is therefore referred to as internal monooxygenases. In addition to the numerous flavoprotein monooxygenases, there is a small group of enzymes that requires tetrahydropteridines as cofactors. In view of the similar chemistry of pteridines and flavins, it is widely considered that the flavoprotein and pteridine-linked monooxygenases may function by basically similar mechanisms.
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3-Hydroxybenzoate 4-hydroxylase has been purified to homogeneity from extracts from extracts pf Ps. testosteroni. It is a flavoprotein (FAD) which catalyzes the transformation of 3-hydroxybenzoate to protocatechuate with equimolar consumption of NADPH and O₂. NADH is a poor substitute for NADPH. Several analogues of 3-hydroxybenzoate substituted in the 2,4,5 and 6 positions, act as effectors and substrates for NADPH oxidation but with varying efficiencies of hydroxylation. 2,3-, 2,5-, 3,5-dihydroxybenzoates, 3-hydroxyanthranilate, 2-fluoro-5-hydroxybenzoate and 4-fluoro-3-hydroxybenzoate are competent substrates.

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^☆: Supported by grants GM 25736 and GM 11106 from the U.S. Public Health Service.

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A non-substrate effector of p-hydroxybenzoate hydroxylase☆

Abstract

J. Biol. Chem

Biochem. Biophys. Res. Comm

Biochem. Biophys. Res. Comm

J. Biol. Chem

Anal. Biochem

Biochem. Biophys. Res. Comm