Metabolism of 4-hydroxy phynylaceticacid: studies on 4-hydroxy-phynylacetate-1-hydroxylase and homogentisate dioxygenase in Nocardia...

Abstract

1. Microorganisms are extraordinarily versatile in their ability to degrade a wide variety of organic compounds. Molecular oxygen plays a crucial role in the microbial degradation of aromatic compounds. An examination of the literature on biodegradation of aromatic compounds reveals a variety of pathways involving reactions such as oxygenations, decarboxylations, and dehydrogenations. The degradative pathways are usually initiated by the introduction of molecular oxygen into the substrate molecules. It is also evident that while different compounds are metabolized through diverse pathways, very often the same compound is metabolized through different pathways in different groups of microorganisms. 2. Phenylacetic acid and its derivatives occur as intermediates in the degradation of many xenobiotics and toxic compounds. The various pathways for the degradation of phenylacetic acids, mandelic acids, and benzoic acids and the enzymes involved therein have been reviewed (Fig. 1-4). A brief account of phenolic amines in some organisms has also been presented. The ring cleaving dioxygenases involved in the metabolism of aromatic compounds and their role in the mineralization of these compounds have also been discussed (Fig. 5-7). 3. The aim and scope of the study were to demonstrate the metabolic pathway of 4 hydroxyphenylacetic acid (4 HPA) in a new species of Nocardia, namely Nocardia DMI, and to study the enzymes involved in the degradation-4 hydroxyphenylacetate 1 hydroxylase (4 HPA hydroxylase) and homogentisate dioxygenase. 4 HPA hydroxylase, an inducible enzyme of the pathway, was chosen for detailed study because flavoprotein monooxygenases play an important role in the detoxification of toxic environmental pollutants. 4 HPA hydroxylase has so far been purified from only one source, and its properties have not been studied in detail. 4. The materials and methods used in the study-composition of media, isolation and identification of intermediates of the degradative pathways of various aromatic compounds, characterization of the enzymes, and relevant techniques-have been described. 5. Nocardia DMI, a new species isolated and identified in our laboratory, was used in this study. The organism could utilize several unrelated aromatic compounds as carbon sources. Good growth was observed on tyrosine, benzoic acid, phenolic amines, and nitrophenols (Table 2). It failed to utilize simple aromatic hydrocarbons such as benzene, toluene, and indole compounds. Compounds with a hydroxyl group at the C 4 position supported growth very well. Isolation of intermediates indicated: o 3 hydroxybenzoic acid, 4 hydroxybenzoic acid, and 4 hydroxymandelic acid were metabolized via the protocatechuate pathway o Benzoic acid via the catechol pathway o Salicylic acid via the gentisate pathway dl Synephrine and 4 HPA were metabolized via the homogentisate pathway. 4 HPA was an intermediate in dl synephrine metabolism (Fig. 22). dl Synephrine was also partly metabolized via formation of 4 hydroxymandelic acid 4 hydroxybenzoic acid protocatechuic acid. 6. Activities of 4 HPA hydroxylase and homogentisate dioxygenase were demonstrated in cell free extracts of Nocardia DMI grown on 4 HPA. Both enzymes were inducible and absent in glucose grown cells. 4 HPA hydroxylase was induced by its substrate and by dl synephrine. Induction occurred within 2-3 h (Fig. 23). 7. 4 HPA hydroxylase was purified using ammonium sulfate fractionation, DEAE Sephacel chromatography (Fig. 25), and hydrophobic interaction chromatography on phenyl Sepharose (Fig. 26). 8. Enzyme homogeneity was confirmed by PAGE (Fig. 27). The enzyme had a molecular weight of 90,000 (Sephacryl S 200, Fig. 29). SDS PAGE showed a single 45,000 Da band (Fig. 30), indicating a dimer of identical subunits. N terminal sequencing by dansylation confirmed a single N terminal L alanine (Fig. 31). 9. The enzyme was identified as an external flavoprotein monooxygenase requiring reduced pyridine nucleotides. The bound chromophore was FAD, demonstrated by chromatographic, spectroscopic, and reconstitution evidence (Fig. 34-35, Table 11-12). 10. The enzyme required NADH, FAD, and O for activity. NADPH substituted with only 25% efficiency. Km for NADH = 32 M (Fig. 44). 11. The reaction product was homogentisic acid (Figs. 11-12, Table 15). Stoichiometry of reactants and products was 1:1 (Table 16). 12. Heavy metals (Cu², Hg²) and thiol reagents strongly inhibited activity, suggesting involvement of sulfhydryl groups (Tables 17, 19, 20). 13. Exogenous FAD was required for full activity (Fig. 36). Km for FAD = 3 M. Atebrin, a competitive inhibitor of flavoprotein monooxygenases, inhibited activity (Table 22). 14. The enzyme exhibited high substrate specificity-only 4 HPA was hydroxylated. Km for 4 HPA = 50 M (Fig. 43). 15. Several analogs inhibited the enzyme (Table 24). Strong inhibitors: • 4 hydroxybenzoic acid (Ki = 0.15 mM) • 4 hydroxymandelic acid (Ki = 0.27 mM) Lack of inhibition by analogs lacking the C 4 hydroxyl group revealed its importance for binding. 16. The absorption spectrum was characteristic of flavoproteins, with peaks at 278 nm, 380 nm, and 460 nm (Fig. 37). 17. Fluorescence studies showed emission at 335 nm (excitation 285 nm), indicating multiple tryptophan environments (Fig. 39). 18. Far UV CD spectrum revealed 41% helical structure (Fig. 41). 19. Substrate binding perturbed the visible spectrum, confirming ES complex formation (Fig. 42). 20. Denaturation caused loss of activity and exposure of buried tryptophans (Table 21, Figs. 48-50). 21. Fluorescence quenching with KI indicated that 25% of tryptophans are buried (Fig. 54). 22. NADH quenched enzyme fluorescence; Kd = 50 M (Figs. 57-58). 23. FAD binding caused major fluorescence changes and was essential for reconstitution (Table 12, Figs. 59-61). Kd for FAD = 20 M (Fig. 60). FAD induced structural and functional changes paralleled each other (Fig. 71). 24. FAD binding altered the enzyme’s backbone conformation: CD spectra showed increased sheet content (Fig. 62-63). 25. The induction of structure correlated with restored activity, suggesting its necessity (Fig. 72). 26. Cibacron Blue F3G A inhibited activity non competitively (Ki = 1.1 M) and quenched fluorescence (Kd = 1.3 M) (Figs. 65-70). 27. Phenylglyoxal completely inactivated the enzyme, indicating involvement of essential arginine residues (Figs. 73-74). Stoichiometry 1.8 molecules per active site. 28. Substrate and competitive inhibitors protected against phenylglyoxal inactivation, indicating modification near the active site (Figs. 75-76). Molecules with a C 4 hydroxyl group and appropriate side chain length provided strong protection (Table 26). 29. The terminal homogentisate dioxygenase was partially purified (Figs. 78-79). 30. Molecular weight of homogentisate dioxygenase = 120,000 Da (Fig. 81). 31. Product identified as maleylacetoacetate (Fig. 80). 32. Activity required O and Fe²; chelators inhibited activity (Tables 29-30). 33. Strict substrate specificity; Km for homogentisate = 1.2 mM (Fig. 84). 34. Protocatechuic acid was a competitive inhibitor (Ki = 1.5 mM) (Figs. 85-86). 35. Superoxide dismutase inhibited enzyme activity; protection by thiols (Fig. 87). 36. Comparison with homogentisate dioxygenases from other sources showed differences in molecular weight, pH optimum, substrate specificity, and inhibition pattern. 37. In conclusion, 4 HPA is metabolized via homogentisic acid and maleylacetoacetic acid in Nocardia DMI. 4 HPA hydroxylase, a dimeric flavoprotein monooxygenase requiring NADH, was purified and characterized structurally and functionally. FAD binding induced necessary conformational changes ( structure formation). Chemical modification suggested an essential arginine residue at the active site. Homogentisate dioxygenase differed significantly from known counterparts.