Frithjof-Hans Bernhardt

4-Methoxybenzoate monooxygenase from Pseudomonas putida: Isolation, biochemical properties, substrate specificity, and reaction mechanisms of the enzyme components

Publisher Summary Studies on the mechanism of biological degradation of lignin or lignin model substances by fungi showed that degradation of lignin down to vanillic acid followed the pathway involving successively the intermediates: α-guaiacyl glycerolconiferyl ether, 4-hydroxy-3-methoxyphenylpyruvic acid, or 4-hydroxy-3-methoxycinnamic acid and vanillin. This chapter describes purification of the components of 4-methoxybenzoate monooxygenase. The enzyme activity of the cell-free crude extract is not proportional to protein concentration in the assay mixture, especially at low protein concentrations. This behavior shows that 4-methoxybenzoate monooxygenase is a dissociable enzyme system—that is, it consists of several components. This is confirmed by the isolation of two components, a reductase and a dioxygen-activating protein. The reconstitution of these two components reveals full enzymatic activity. The chapter also describes the properties of putidamonooxin (PMO). PMO, the dioxygen-activating component of the 4-methoxybenzoate monooxygenase, has a molecular weight of 126,000, as derived from ultracentrifugation and gel filtration.

Reactivation studies on putidamonooxin — The monooxygenase of a 4-methoxybenzoate o-demethylase from Pseudomonas putida

Abstract Reduced putidamonooxin from a stock solution loses about 63 % of its activity within seconds when exposed to oxidizing conditions. This inactivation is prevented by the presence of substrate. Strongly inactivated putidamonooxin is reactivated, from 37 % to 66 % of its original activity, when preincubated with ferrous ions. The presence of thiol compounds in addition to ferrous ions leads to the enzymes complete reactivation. From these results the following conclusions are drawn: The reactivation of putidamonooxin (i) depends on Fe (II) ions and (ii) involves a sulfhydryl group. In the absence of the additional ferrous ion, reduced putidamonooxin is only partially oxidized by dioxygen. This finding indicates that the additional iron ion, possibly tightly bound to a mercaptide group, functions both as the dioxygen binding site and as the mediator of electron flow from an iron-sulfur centre to dioxygen.

A 4-methoxybenzoate monooxygenase system from Pseudomonas putida. Circular dichroism studies on the iron—sulfur protein

A monooxygenase system (0-demethylating) was isolated from P. puti& grown on 4methoxybenzoate as the sole carbon source and could be separated into two components: an iron-containing flavoprotein with FMN as flavin component (= a conjugated iron-sulfur protein, electron paramagnetic resonance (EPR) at g> 1.95) and a second iron-sulfur protein [l-4]. The iron-containing flavoprotein acts as the NADHiron-sulfur protein oxidoreductase, whereas the ironsulfur protein probably acts as the terminal oxidase capable of hydroxylating various substrates. Both the aliphatic CH-bond of different paraand meta-substituted benzoic acid derivatives and the aromatic ring are attacked by this enzyme [5]. The physicochemical properties of the iron-sulfur protein differ considerably from those of other well-known iron-sulfur proteins of the ferredoxin type, like adrenodoxin or putidaredoxin, which function as intermediate electron carriers between a flavin-dependent reductase and a cytochrome P-450 containing terminal oxidase [6,7]. The main difference is the following: the ironsulfur protein of the 4-methoxybenzoate monooxygenase shows an EPR spectrum in the reduced state with a mean g-value? of g= 1.90 [8] which is distinctly lower than that of the iron-sulfur proteins of the ferredoxin type. For the latter proteins mean d-values of g> 1.95 are characteristic [9,10]. Differences also exist with regard to the redox potential. While the iron-sulfur proteins of the ferredoxin type possess low redox potentials, i.e. less than -235 mV [ 111, the redox potential of the iron-sulfur protein of the 4-methoxybenzoate monooxygenase was determined to be about t5 mV at pH 7.8. In this communication we report on interactions of the iron-sulfur protein of the 4-methoxybenzoate monooxygenase with different substrates of this enzyme system. These findings confirm our hypothesis that this iron-sulfur protein is the terminal oxidase within this enzyme system.

Oxygen Reaction of 4-hydroxybenzoate Monooxygenase

4-hydroxybenzoate hydroxylase from Ps. putida has been studied with regard to the oxygen interaction with the reduced flavin. By rapid scanning spectrophotometry and EPR-measurements of rapidly frozen mixtures no intermediate could be established either in the presence of 4-hydroxybenzoate or the dead-end inhibitor 6-hydroxynicotinic acid.