ENZYMOLOGY OF OXIDATION OF TROPIC ACID TO PHENYLACETIC ACID IN METABOLISM OF ATROPINE BY PSEUDOMONAS SP STRAIN AT3

Pseudomonas sp. strain AT3 grew with dl-tropic acid, the aromatic comp onent of the alkaloid atropine, as the sole source of carbon and energ y, Tropic acid-grown cells rapidly oxidized the growth substrate, phen ylacetaldehyde, and phenylacetic acid, Crude cell extracts, prepared f rom dl-tropic acid-grown cells, contained two NAD(+)-linked dehydrogen ases which were separated by ion-exchange chromatography and shown to be specific for their respective substrates, dl-tropic acid and phenyl acetaldehyde. Phenylacetaldehyde dehydrogenase was relatively unstable , The stable tropic acid dehydrogenase was purified to homogeneity by a combination of ion-exchange, molecular-sieve, and affinity chromatog raphy. It had a pH optimum of 9.5 and was equally active ce with both enantiomers of tropic acid, and at this pH, phenylacetaldehyde was the only detectable product of tropic acid oxidation, The formation of ph enylacetaldehyde from tropic acid requires, in addition to dehydrogena tion, a decarboxylation step, By analogy with NAD(+)-specific isocitra te and malate dehydrogenases, phenylmalonic semialdehyde, a 3-oxoacid, would be expected to be the precursor of phenylacetaldehyde. Other wo rkers have established that isocitrate and malate dehydrogenases catal yze the decarboxylation of enzyme-bound or added 3-oxoacid intermediat es, a reaction that requires Mn2+ or Mg2+ ions, Studies with tropic ac id dehydrogenase were hampered by lack of availability of phenylmaloni c semialdehyde, but in the absence of added divalent metal ions, both enantiomers of tropic acid were completely oxidized and we ha re not, by a number of approaches, found any evidence for the transient accumu lation of phenylmalonic semialdehyde.