Characterization of the genes encoding the 3-carboxy-cis,cis-muconate-lactonizing enzymes from the 4-sulfocatechol degradative pathways of Hydrogenophaga intermedia S1 and Agrobacterium radiobacter S2

Abstract

Hydrogenophaga intermedia strain S1 and Agrobacterium radiobacter strain S2 form a mixed bacterial culture which degrades sulfanilate (4-aminobenzenesulfonate) by a novel variation of the b-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-cis, cis-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-cis,cis-muconate. In the present study the genes for two CMLEs (pcaB2S1 and pcaB2S2) were cloned from H. intermedia S1 and A. radiobacter 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 pcaB2S1 and pcaB2S2 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-cis,cis-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 Pseudomonas putida PRS2000 for the conversion of 3-carboxy-cis,cis-muconate and 3-sulfomuconate. The CMLEs from the 4-sulfocatechol pathway converted 3-sulfomuconate with considerably higher activities than 3-carboxy-cis,cis-muconate. Also the CMLE from P. putida converted 3-sulfomuconate, but this enzyme demonstrated a clear preference for 3-carboxy-cis,cis-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.

4-Sulfomuconolactone Hydrolases from Hydrogenophaga intermedia S1 and Agrobacterium radiobacter S2

“…The genes coding for the protocatechuate 3,4-dioxygenase type II and 3-carboxy-cis,cis-muconate-lactonizing enzyme type II from A. radiobacter (ArCMLE2), which in A. radiobacter S2 are responsible for the conversion of 4-sulfocatechol to 4-sulfomuconolactone, had been previously identified on plasmid pMCS2-2. Downstream of the gene encoding the A. radiobacter CMLE2, a truncated gene encoding a putative hydrolase was identified (open reading frame [ORF] 4) (10,17). The missing part of ORF 4 was obtained using partial inverse PCR (32).…”

“…The characteristics of all plasmids used are given in Table 1. The isolation of genomic DNA of H. intermedia S1 and A. radiobacter S2, the amplification of DNA by PCR, and all recombinant DNA work was performed as described previously (17).…”

“…The abilities of the 4-sulfocatechol-and 3-sulfomuconate-transforming enzymes to also convert their carboxylated structural counterparts (protocatechuate and 3-carboxy-cis,cis-muconate) and sequence comparisons clearly demonstrated that both enzymes are related to the corresponding enzymes of the protocatechuate branch of the ␤-ketoadipate pathway. These enzymes were therefore designated as "type II enzymes" (8,10,15,17,18,19). The product formed enzymatically from 3-sulfomuconate in the cycloisomerization reaction has been identified as 4-carboxymethylen-4-sulfo-but-2-en-olide ("4-sulfomuconolactone" [4SL]) (15,17).…”

“…These enzymes were therefore designated as "type II enzymes" (8,10,15,17,18,19). The product formed enzymatically from 3-sulfomuconate in the cycloisomerization reaction has been identified as 4-carboxymethylen-4-sulfo-but-2-en-olide ("4-sulfomuconolactone" [4SL]) (15,17). This metabolite structurally resembles its equivalent from the protocatechuate pathway (4-carboxymethylen-4-carboxy-but-2-enolide ["4-carboxymuconolactone"]) (Fig.…”

Genome Sequence of Hydrogenophaga sp. Strain PBC, a 4-Aminobenzenesulfonate-Degrading Bacterium

“…PBC strains were auxotrophic for biotin and p-aminobenzoic acid, which may explain the roles of their respective helper strains in the biodegradation of 4-ABS. To date, the molecular mechanism of 4-ABS degradation has been studied in great detail in this genus via an approach involving transposon mutagenesis and enzymology (2,4,(7)(8)(9). The recent identification of the genetic components for the oxidation of 4-ABS to 4-sulfocatechol from Hydrogenophaga sp.…”