Some aspects of the metabolism of mandelic acid by pseudomonas convexa, Ph.D. Thesis

Abstract

The metabolism of mandelic acid (phenylglycolic acid) has been studied in detail in several bacteria, and it has been shown that the side chain of mandelic acid is first degraded, resulting in the formation of benzoic acid, which is then oxidized to catechol before aromatic ring cleavage. The author has isolated an organism from soil by enrichment on mandelic acid and identified it as Pseudomonas convexa. The pattern of degradation of mandelic acid in P. convexa is strikingly different from all other known cases. This microorganism brings about hydroxylation at the 4?position of the aromatic ring of mandelic acid as the first step in its oxidative degradation, producing 4?hydroxymandelic acid. This is further degraded to give rise to 4?hydroxybenzaldehyde and 4?hydroxybenzoic acid. 4?Hydroxybenzoic acid is then hydroxylated at the 3?position to form 3,4?dihydroxybenzoic acid, which in turn is oxygenated, leading to the formation of 3?ketoadipic acid and entering the ??ketoadipate cycle. All these intermediates of the pathway were isolated and identified by paper chromatography, thin?layer chromatography, and UV and IR spectroscopy. L(+)-Mandelate?4?hydroxylase, which catalyzes the conversion of mandelic acid to 4?hydroxymandelic acid, has been partially purified (26?fold), and its properties were studied. The enzyme has a molecular weight of about 91,000. The requirements for the mandelate hydroxylation reaction are tetrahydropteridine, NADPH, Fe²?, and molecular oxygen. The enzyme was optimally active at pH 5.4 and at 38°C. The Km values for L(+)-mandelic acid and NADPH were calculated to be 1 × 10?? M and 1.9 × 10?? M respectively. Sulfhydryl groups are essential for the activity of L(+)-mandelate?4?hydroxylase, as indicated by inhibition studies with thiol inhibitors. The inhibition by p?HMB was reversed by thiol compounds. Neither substrates nor cofactors protected the enzyme from inhibition by p?HMB. The enzyme was sensitive to denaturing agents and was inhibited by methotrexate and aminopterin (specific inhibitors of pteridine?requiring enzymes), and by o?phenanthroline and 2,2??dipyridyl (specific iron chelators). L(+)-4?Hydroxymandelate oxidase, which catalyzes the conversion of 4?hydroxymandelic acid to 4?hydroxybenzaldehyde, is present in the particulate fraction (100,000 × g pellet). This enzyme was solubilized by treatment with n?butanol and purified 575?fold. It was optimally active at pH 6.6 and 55°C, and required FAD and Mn²? for its activity. The partially purified enzyme showed an absorption spectrum characteristic of a simple protein without visible absorption, suggesting that FAD is not present in the purified enzyme. The role of FAD as a prosthetic group was supported by:

its absolute requirement for catalytic activity, inhibition by atabrine (a specific inhibitor of flavoproteins), and equilibrium dialysis experiments demonstrating affinity between the enzyme and FAD.

These studies indicate that the flavin prosthetic group may be loosely bound to the enzyme and lost during purification. Another interesting property of the enzyme is its stability pattern. The unsolubilized enzyme (particulate fraction) is quite stable, whereas the solubilized enzyme was very labile during purification. When the solubilized enzyme was frozen at –20°C for at least 10 hours and later thawed, full enzyme activity was restored. This suggests involvement of conformational changes in activation/inactivation, or that freezing mimics a membrane?like environment that stabilizes the enzyme. The Km values for DL?4?hydroxymandelic acid and FAD were calculated to be 4.4 × 10?? M and 3.8 × 10?? M respectively. The enzyme is completely inactivated by thiol compounds but not by thiol inhibitors, suggesting that intact disulfide bridges are necessary for activity. The enzyme is inactivated by denaturing agents, heavy metal ions, and chelating agents. The presence of other enzymes of the pathway has also been demonstrated in the supernatant fraction of P. convexa:

mandelate racemase two 4?hydroxybenzaldehyde dehydrogenases (one NAD??dependent and one NADP??dependent) 4?hydroxybenzoate?3?hydroxylase 3,4?dihydroxybenzoate oxygenase

The two dehydrogenases (NAD? and NADP? dependent) were separated from each other. The metabolism of mandelic acid has been studied in many microorganisms. In all previously known cases, the biochemical steps of the pathway are the same. However, they differ with respect to enzyme complement and regulation. The results presented in this thesis suggest that even the biochemical sequence of mandelic acid degradation can differ among microorganisms.