| dc.description.abstract | Synopsis of the thesis entitled “Metabolism of Phenylalanine by Aspergillus niger” by Mr. G. Kishore for the award of the Ph.D. degree in the Faculty of Science of Indian Institute of Science, Bangalore.
Microorganisms possess a unique biochemical capacity to metabolize a diverse variety of aromatic compounds. The microbial degradation, and in particular the fungal catabolism of aromatic compounds, has been subjected to extensive research during the past two decades. Studies on the metabolism of DL tryptophan by the fungus Aspergillus niger had revealed that this aromatic amino acid is converted to 2,3 dihydroxybenzoic acid via anthranilic acid.
Similar studies on the metabolism of mandelic and phenylacetic acids had revealed the diversity of pathways employed by microbes for the dismutation of the aromatic nucleus. For a systematic investigation into the various aspects of microbial degradation of aromatic compounds, it was worthwhile initially to examine the various aspects of the catabolism of the parent aromatic compound, phenylalanine, in Aspergillus niger. The thesis describes some of the interesting features of such a study.
Initial studies revealed that the mode of degradation of phenylalanine by a strain of A. niger is distinct from those described previously in literature. The pathway, to summarise, consists of the following events:
DL Phenylalanine
Phenylpyruvic acid
Phenylacetic acid
2 Hydroxyphenylacetic acid
Homogentisic acid
Ring cleavage
4 Hydroxyphenylacetic acid
4 Hydroxymandelic acid
4 Hydroxybenzoylformic acid
4 Hydroxybenzaldehyde
4 Hydroxybenzoic acid
3,4 Dihydroxybenzoic acid
The following enzyme activities could be demonstrated in the cell free extracts of A. niger:
D Amino acid oxidase
L Phenylalanine aminotransferase
Phenylpyruvate decarboxylase
Phenylpyruvate oxygenase
Phenylacetate hydroxylase
4 Hydroxymandelate dehydrogenase
Benzoylformate decarboxylase
4 Hydroxybenzaldehyde dehydrogenase
4 Hydroxybenzoate 3 hydroxylase
Protocatechuate oxygenase
Homogentisate oxygenase
The following conversions could be demonstrated in vivo but not in the cell free systems:
4 Hydroxyphenylacetate oxygenase (hydroxylating)
2 Hydroxyphenylacetate 5 hydroxylase
The metabolism of 2 hydroxyphenylacetate to homogentisate occurred only in the sporulating phase of the organism and could not be demonstrated in the vegetative phase. Homogentisate was metabolized by the well established “animal” pathway for phenylalanine degradation.
4 Hydroxyphenylacetate was converted to 4 hydroxymandelate presumably by an enzyme system analogous to p hydroxylases of bacterial systems (Peterson, J.A. & M.J. Coon, J. Biol. Chem. 243, 329 (1968)).
In view of the extreme complexity of the enzymatic machinery, it is not surprising that such an activity could not be established in vitro. Nevertheless, the formation of 4 hydroxymandelate from 4 hydroxyphenylacetate has been demonstrated by replacement, radioactive and spectral data. Subsequent fate of 4 hydroxymandelate was analogous to the previously reported pathway for mandelate metabolism by A. niger (Jamaluddin, Ph.D. thesis, IISc, 1972).
To unravel the regulatory mechanisms of the pathway, a detailed study of one of the first enzymes of the pathway was undertaken and the results obtained are discussed below.
The enzyme, D amino acid oxidase of A. niger, catalyses the reaction:
RCH(NH )COOH + O RCOCOOH + NH + H O
The enzyme was purified using conventional protein purification techniques. One of the activities was poorly adsorbed to DEAE cellulose and was designated isozyme I, and the other strongly bound fraction as isozyme II. In the absence of phenylalanine in the growth medium of the mould, isozyme II activity was absent, suggesting that the enzyme was inducible, while the other was constitutive.
Both enzymes had a molecular weight of ~200,000; were maximally active with D phenylalanine as substrate, and could be readily resolved by electrophoresis on polyacrylamide gels. The substrate specificity of the two isozymes was slightly different, with isozyme I having a broader substrate specificity than isozyme II.
A new reaction was developed to follow the D amino acid oxidase reaction continuously, based on complexing phenylpyruvate with phenazine methosulfate (PMS). A brilliant bluish green complex with max at 600 nm is formed rapidly at alkaline pH values, and the enzyme can be easily assayed by monitoring the increase in absorption at 600 nm.
Only phenylpyruvate and 4 hydroxyphenylpyruvate gave this complex with PMS.
Enzyme assays could therefore be conveniently performed by:
measuring oxygen uptake (oxygen electrode),
following keto acid appearance (2,4 DNPH method),
PMS method for phenylalanine substrates,
ammonia formation (phenol hypochlorite method),
hydrogen peroxide formation (peroxidase o dianisidine system).
Kinetic studies revealed:
(a) The D amino acid oxidase reaction is not inhibited by L amino acids.
(b) Both oxidases are inhibited competitively by aromatic substrate analogues such as phenylpropionic acid and cinnamic acid.
Isozyme II additionally shows non competitive inhibition by 4 hydroxymandelate.
(c) 5 AMP shows dual effects-inhibition at low concentrations and activation at high concentrations-indicating two binding sites on the enzyme.
(d) Inhibition by thiol reagents is unusual:
N ethylmaleimide and DTNB show no inhibition,
PHMB and iodoacetate inhibit strongly,
GSH, L cysteine, and mercaptoethanol inhibit to varying degrees.
(e) Metal ions: Only Ag and Hg² inhibit strongly.
(f) No decrease in activity occurred after extensive dialysis.
(g) No stimulation by cofactors (NAD, NADP, FMN, FAD, folate, etc.).
(h) No visible region absorbance; tryptic digestion did not release known redox carriers.
Thus, the nature of the redox carrier is uncertain.
To summarise:
A new pathway for the degradation of phenylalanine in fungal systems has been established. The first enzyme of the pathway has been purified and studied, and its regulatory characteristics elucidated. | |
