Functional genomics by NMR spectroscopy. Phenylacetate catabolism in Escherichia coli.

Aerobic metabolism of phenylalanine in most bacteria proceeds via oxidation to phenylacetate. Surprisingly, the further metabolism of phenylacetate has not been elucidated, even in well studied bacteria such as Escherichia coli. The only committed step is the conversion of phenylacetate into phenylacetyl-CoA. The paa operon of E. coli encodes 14 polypeptides involved in the catabolism of phenylacetate. We have found that E. coli K12 mutants with a deletion of the paaF, paaG, paaH, paaJ or paaZ gene are unable to grow with phenylacetate as carbon source. Incubation of a paaG mutant with [U-13C8]phenylacetate yielded ring-1,2-dihydroxy-1,2-dihydrophenylacetyl lactone as shown by NMR spectroscopy. Incubation of the paaF and paaH mutants with phenylacetate yielded delta3-dehydroadipate and 3-hydroxyadipate, respectively. The origin of the carbon atoms of these C6 compounds from the aromatic ring was shown using [ring-13C6]phenylacetate. The paaG and paaZ mutants also converted phenylacetate into ortho-hydroxyphenylacetate, which was previously identified as a dead end product of phenylacetate catabolism. These data, in conjunction with protein sequence data, suggest a novel catabolic pathway via CoA thioesters. According to this, phenylacetyl-CoA is attacked by a ring-oxygenase/reductase (PaaABCDE proteins), generating a hydroxylated and reduced derivative of phenylacetyl-CoA, which is not re-oxidized to a dihydroxylated aromatic intermediate, as in other known aromatic pathways. Rather, it is proposed that this nonaromatic intermediate CoA ester is further metabolized in a complex reaction sequence comprising enoyl-CoA isomerization/hydration, nonoxygenolytic ring opening, and dehydrogenation catalyzed by the PaaG and PaaZ proteins. The subsequent beta-oxidation-type degradation of the resulting CoA dicarboxylate via beta-ketoadipyl-CoA to succinyl-CoA and acetyl-CoA appears to be catalyzed by the PaaJ, PaaF and PaaH proteins.

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