Metabolism of protocatechnic acid in tecoma stans

Abstract

This thesis encompasses the studies on the biosynthesis and degradation of protocatechuic acid in Tecoma stans. In the introduction, a brief survey of the role and metabolism of phenolic compounds in plants is given.

In the course of studies on the metabolism of phenolic acids in the common Indian hedge plant, Tecoma stans L., it was found necessary to separate and identify the phenolic acids in this plant. A ethanolic extract of leaves of Tecoma stans was fractionated, and various fractions obtained were analysed by paper chromatography for the presence of phenolic acids. The following phenolic acids were identified by the above technique:

Protocatechuic acid

m-Hydroxybenzoic acid

Pyrocatechuic acid

Salicylic acid

Caffeic acid

Vanillic acid

Dihydrocaffeic acid

Ferulic acid

p-Hydroxybenzoic acid

Syringic acid

p-Hydroxyphenyllactic acid

Phloretic acid

p-Hydroxyphenylacetic acid

Sinapic acid

p-Coumaric acid

Gentisic acid

Protocatechuic acid occurred in the free form in the leaves of Tecoma stans. This phenolic acid is formed from phenylalanine via cinnamate, p-coumarate, and caffeic acid. It was possible to demonstrate the presence of phenylalanine ammonia-lyase (PAL), tyrosine ammonia-lyase (TAL), cinnamate-4-hydroxylase, and p-coumarate hydroxylase. PAL and TAL were present in the acetone powder from an extract of Tecoma leaves. The other two enzymes were present in the mitochondrial fraction of the leaf extracts.

PAL and TAL had pH optima in the alkaline range (8.2) in Tris-HCl buffer; cinnamate-4-hydroxylase was maximally active at pH 7.4 in sodium buffer, and p-coumarate hydroxylase had an optimum pH at 6.2 in citrate phosphate buffer. Km values for PAL, TAL, cinnamate-4-hydroxylase, and p-coumarate hydroxylase were 2.0 × 10?? M, 1.8 × 10?? M, 1.6 × 10?? M, and 1.4 × 10?? M respectively. Both cinnamate-4-hydroxylase and p-coumarate hydroxylase showed an absolute requirement for molecular oxygen. Cinnamate-4-hydroxylase required NADPH as a cofactor for its activity; p-coumarate hydroxylase required ascorbate for maximum activity.

The ?-oxidation of caffeic acid to protocatechuic acid also occurred in the mitochondrial fraction of Tecoma leaves. This oxidation did not require any added cofactor.

An enzyme, designated as protocatechuate 3,4-dioxygenase, catalyzed the aerobic oxidation of protocatechuic acid by an intradiol cleavage. The enzyme was present in the soluble fraction of the leaves of Tecoma stans L. The enzyme was purified 350-fold with a recovery of 33%. The purified enzyme was fairly stable and showed temperature stability up to 60°C for 5 minutes. On polyacrylamide gel electrophoresis, the purified enzyme showed two closely moving bands, one major band and one minor band. The spectrum of the purified enzyme exhibited a maximum absorption at 280 nm and a minimum at 255 nm, with no absorption in the visible region.

The enzyme required Fe²? and molecular oxygen for its activity. The dioxygenase was optimally active at pH 5.2 and at 40°C. The Km values for protocatechuic acid and Fe²? were calculated to be 3.3 × 10?? M and 4 × 10?? M respectively. Co²? and Cu²? did not inhibit the enzyme activity, whereas Fe³? and sulfhydryl reagents like p-chloromercuribenzoate and 5,5?-dithiobis-(2-nitrobenzoic acid) inhibited the enzyme activity. The Tecoma enzyme had an approximate molecular weight of 155,000. The enzyme was specific for protocatechuic acid as substrate; other dihydric phenols were not attacked. The dioxygenase was not inhibited by substrate analogues. Diurnal variation studies indicated that the activity of the enzyme in the leaves of Tecoma stans was maximum at 2 A.M.

The purified enzyme from the leaves of Tecoma stans oxidized one mole of protocatechuic acid with the consumption of one mole of oxygen, and there was no evolution of carbon dioxide. The fact that muconolactone, ?-ketoadipate, and carbon dioxide were not detected suggested that the finally purified enzyme contained only protocatechuate dioxygenase activity and was devoid of the other enzymes of the pathway. The enzymic product was identified as ?-carboxy-cis,cis-muconic acid by comparison of its properties with those of an authentic sample.

The enzyme necessary for the conversion of ?-carboxy-cis,cis-muconic acid to muconolactone and ?-ketoadipate was also present in the leaves of Tecoma stans. The lactonizing enzyme was partially purified 5-fold with 61% recovery from extracts of the leaves. The pH optimum of the lactonizing enzyme was 5.6, and it was heat stable up to 60°C for 5 min. The lactonizing enzyme also catalyzed the conversion of cis,cis-muconic acid to muconolactone.

Acetone fractionation (2.0% – 50%) of the manganese sulfate supernatant caused the removal of the lactonizing activity, leaving only the decarboxylase enzyme activity. The decarboxylase enzyme catalyzed the conversion of ?-carboxy-cis,cis-muconic acid to cis,cis-muconic acid. Incubation of protocatechuic acid with the heat-treated preparation resulted in the formation of ?-ketoadipic acid.

Based on the above evidence, the following pathway is proposed for the degradation of protocatechuic acid in Tecoma stans L.: