Matabolism of phenylacetic acid by aspergillus niger

Abstract

The metabolism of phenylacetic acid by Aspergillus niger was studied. The culture filtrates of A. niger grown on phenylacetate for 40 hours exhibited the presence of 2? and 3?hydroxyphenylacetic acids, 3?hydroxybenzyl alcohol, 3?hydroxybenzoic acid, and protocatechuic acid. While 3?hydroxyphenylacetate and its derivatives are utilized during the stationary phase of the fungus, 2?hydroxyphenylacetate continues to accumulate in the medium until the onset of sporulation, during which it is rapidly utilized and homogentisic acid is excreted into the medium. Replacement culture studies corroborated these results and additionally led to the identification of m?cresol as an intermediate during phenylacetate catabolism. Activities of the following enzymes were demonstrated in the cell?free extracts:

Phenylacetate monooxygenase 3?Hydroxybenzyl alcohol dehydrogenase 3?Hydroxybenzaldehyde dehydrogenase 3?Hydroxybenzoate?4?monooxygenase Protocatechuate dioxygenase Homogentisate?1,2?dioxygenase Maleylacetate isomerase Fumaryl?acetoacetate hydrolase

??Oxoadipate was identified as the end product of protocatechuate oxidation, and fumarate and acetoacetate were found to be the end products of homogentisate oxidation. Based on these results, the following pathway is proposed for phenylacetate degradation by A. niger: Phenylacetate ? (2? or 3?hydroxylation) ? 3?Hydroxyphenylacetate ? m?Cresol ? 3?Hydroxybenzyl alcohol ? 3?Hydroxybenzaldehyde ? 3?Hydroxybenzoate ? Protocatechuate ? ??Oxoadipate and 2?Hydroxyphenylacetate ? Homogentisate ? Maleylacetate ? Fumarylacetate ? Fumarate + Acetoacetate

Phenylacetate Monooxygenase Phenylacetate monooxygenase occurs in the particulate fraction of A. niger and hydroxylates both phenylacetate and phenoxyacetate at the 2? and 3?positions. It is an inducible enzyme, induced by phenylalanine, phenyllactate, phenoxyacetate, and phenylacetate. Attempts to solubilize the enzyme were unsuccessful; therefore, all studies were conducted using the particulate fraction.

Requires NADPH and molecular oxygen Optimal activity at pH 7.8 and 30°C Km for phenylacetate = 0.49 mM Ortho : meta product ratio = 93 : 7 Hydroxylation proceeds through an arene oxide mechanism Exhibits properties of a cytochrome P?450 enzyme

Light reversal of CO inhibition, inhibition by cytochrome?c (reversed by cyanide), and the effect of monooxygenase inhibitors support the P?450 nature. The enzyme also generates superoxide anion (O??) in the particulate fraction when NADPH is added. Superoxide reacts with sulfhydryl groups of the enzyme causing inhibition; this inhibition is reversed by added sulfhydryl compounds or by superoxide dismutase. Deuterium?labelled substrates showed no primary kinetic isotope effect, supporting the arene?oxide mechanism. Mass spectral studies demonstrated formation of both 1,2? and 2,3?epoxides during phenoxyacetate hydroxylation, and negligible NIH shift during 2?hydroxylation.

Homogentisate Dioxygenase Based on competitive inhibition by 2?hydroxyphenylacetate, an affinity purification system was developed using homogentisate conjugated to benzyl?Sepharose. Homogentisate?1,2?dioxygenase binds strongly to this matrix in the presence of Fe²? at neutral/slightly acidic pH and can be specifically eluted with 0.05 M Tris–HCl, pH 8.0. Using this method, homogentisate?dioxygenase from A. niger was purified to homogeneity. It is:

A typical Fe²??requiring dioxygenase Molecular weight ~ 200,000 Optimal pH 6.4 in Tris?maleate buffer Km (homogentisate) = 0.6 mM Km (FeSO?) = 0.33 mM

Quinone acetate, 2?hydroxyphenylacetate, and 3?hydroxyphenylacetate inhibit the enzyme competitively. Catechol, protocatechuate, and homoprotocatechuate do not inhibit, suggesting that the enzyme requires a carboxymethylene group and an ortho oxygen substituent for binding—a specificity different from mammalian homogentisate dioxygenase, which requires a 1,4?quinol group. Sulfhydryl reagents strongly inhibit the enzyme; p?HMB acts as a parabolic non?competitive inhibitor with respect to homogentisate and competitive with respect to Fe²?, indicating involvement of SH groups in metal binding. Evidence was also presented for the participation of superoxide (O??) in the reaction.

Scope for Future Studies Some enzymes of the phenylacetate pathway in A. niger (e.g., m?cresol hydroxylase, 3?hydroxyphenylacetate decarboxylase, 2?hydroxyphenylacetate?5?monooxygenase) could not be isolated, necessitating further studies. Important future directions include:

Solubilizing phenylacetate monooxygenase for detailed mechanistic studies Synthesizing 2? and 3?deuterated substrates for isotope experiments Investigating subunit structure of homogentisate?dioxygenase Determining whether multiple pathways provide ecological/metabolic advantages Studying secondary metabolites formed by the alternative protocatechuate pathway

Although the homogentisate pathway is shorter and more economical, A. niger uses the protocatechuate pathway during early growth, possibly due to secondary metabolite formation.