Abstract
Cell-free extracts of Pseudomonas putida, grown with syringic acid as carbon source, catalyzed the oxidation of 1 mole of gallate (3,4,5-trihydroxybenzoate) by 1 mole of oxygen to give 2 moles of pyruvate and 1 mole of carbon dioxide. Oxaloacetate, formed as an intermediate in reactions, could be reduced quantitatively when NADH and sufficient malate dehydrogenase were added, despite the presence in extracts of oxaloacetate decarboxylase. With these conditions of assay it was shown that oxaloacetate was the precursor of half of the pyruvate formed from gallate. Similarly, when 1 mole of gallate was first oxidized by protocatechuate 4,5-oxygenase purified from Pseudomonas testosteroni and was then degraded by cell-free extracts, 1 mole of oxaloacetate and 1 mole of pyruvate were again formed. Although gallate oxygenase proved too labile to isolate, it was concluded from these results that the enzyme gave the same ring fission product as that obtained by the action of protocatechuate 4,5-oxygenase on gallate, namely 4-carboxy-2-hydroxy-cis,cis-muconic acid. This compound appeared to undergo enzymic hydration to give 4-carboxy-4-hydroxy-2-oxoadipate which was then cleaved to oxaloacetate and pyruvate. Cell-free extracts oxidized 3-O-methylgallate, but protocatechuate (3,4-dihydroxybenzoate), 5-carboxy-3,4-dihydroxy benzoate and 3,4-dihydroxy-5-methylbenzoate were not attacked. Syringic acid was oxidized only in the presence of substrate amounts of NADH, when 2 moles of oxygen were taken up and 1 mole of pyruvate appeared; additional pyruvate was formed by a much slower reaction. There was no requirement for added NADH when concentrated cell extracts were used to oxidize syringate. These and other observations indicate that the C-5 methoxyl group of syringate was oxidized by an O-demethylase. The benzene nucleus of the resulting 3-O-methylgallate was then cleaved between C-3 and C-4 to give, as final products, pyruvate and a compound presumed to be a monomethyl ester of oxaloacetic acid. The substrate specificity of syringate O-demethylase was studied in experiments with intact cells.
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