1887

Abstract

strain S1 and strain S2 form a mixed bacterial culture which degrades sulfanilate (4-aminobenzenesulfonate) by a novel variation of the -ketoadipate pathway via 4-sulfocatechol and 3-sulfomuconate. It was previously proposed that the further metabolism of 3-sulfomuconate is catalysed by modified 3-carboxy-,-muconate-lactonizing enzymes (CMLEs) and that these ‘type 2’ enzymes were different from the conventional CMLEs (‘type 1’) from the protocatechuate pathway in their ability to convert 3-sulfomuconate in addition to 3-carboxy-,-muconate. In the present study the genes for two CMLEs ( and ) were cloned from S1 and S2, respectively. In both strains, these genes were located close to the previously identified genes encoding the 4-sulfocatechol-converting enzymes. The gene products of and were therefore tentatively identified as type 2 enzymes involved in the metabolism of 3-sulfomuconate. The genes were functionally expressed and the gene products were shown to convert 3-carboxy-,-muconate and 3-sulfomuconate. 4-Carboxymethylene-4-sulfo-but-2-en-olide (4-sulfomuconolactone) was identified by HPLC-MS as the product, which was enzymically formed from 3-sulfomuconate. His-tagged variants of both CMLEs were purified and compared with the CMLE from the protocatechuate pathway of PRS2000 for the conversion of 3-carboxy-,-muconate and 3-sulfomuconate. The CMLEs from the 4-sulfocatechol pathway converted 3-sulfomuconate with considerably higher activities than 3-carboxy-,-muconate. Also the CMLE from converted 3-sulfomuconate, but this enzyme demonstrated a clear preference for 3-carboxy-,-muconate as substrate. Thus it was demonstrated that in the 4-sulfocatechol pathway, distinct CMLEs are formed, which are specifically adapted for the preferred conversion of sulfonated substrates.

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2006-11-01
2024-04-27
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