The isophthalate (IPA) degradation gene cluster (iphACBDR) responsible for the conversion of IPA into protocatechuate (PCA) was isolated from Comamonas sp. strain E6, which utilizes phthalate isomers as sole carbon and energy sources via the PCA 4,5-cleavage pathway. Based on amino acid sequence similarity, the iphA, iphC, iphB, iphD, and iphR genes were predicted to code for an oxygenase component of IPA dioxygenase (IPADO), a periplasmic IPA binding receptor, a 1,2-dihydroxy-3,5-cyclohexadiene-1,5-dicarboxylate (1,5-DCD) dehydrogenase, a reductase component of IPADO, and an IclR-type transcriptional regulator, respectively. The iphACBDR genes constitute a single transcriptional unit, and transcription of the iph catabolic operon was induced during growth of E6 on IPA. The iphA, iphD, and iphB genes were expressed in Escherichia coli. Crude IphA and IphD converted IPA in the presence of NADPH into a product which was transformed to PCA by IphB. These results suggested that IPADO is a two-component dioxygenase that consists of a terminal oxygenase component (IphA) and a reductase component (IphD) and that iphB encodes the 1,5-DCD dehydrogenase. Disruption of iphA and iphB resulted in complete loss of growth of E6 on IPA. Inactivation of iphD significantly affected growth on IPA, and the iphC mutant did not grow on IPA at neutral pH. These results indicated that the iphACBD genes are essential for the catabolism of IPA in E6. Disruption of iphR resulted in faster growth of E6 on IPA, suggesting that iphR encodes a repressor for the iph catabolic operon. Promoter analysis of the operon supported this notion.Phthalate isomers (o-phthalate [OPA], terephthalate [TPA], and isophthalate [IPA]) and their esters have been largely used as plasticizers. Moreover, they are considered potential starting compounds for the production of 2-pyrone-4,6-dicarboxylic acid (PDC), an intermediate metabolite in the protocatechuate (PCA) 4,5-cleavage pathway (28). PDC is a useful compound for synthesis of biodegradable and highly functional polymers, such as powerful adhesive agents (22,29,30).OPA degradation has been reported for many bacteria, including Burkholderia cepacia DBO1 (9), Mycobacterium vanbaalenii PYR-1 (43), Arthrobacter keyseri 12B (14), Terrabacter sp. DBF63 (19), Rhodococcus sp. DK17 (10), and Rhodococcus jostii RHA1 (32). Degradation of OPA is initiated by dihydroxylation of the aromatic ring by OPA 3,4-dioxygenase (10, 14), which is found mostly in Gram-positive bacteria, or by OPA 4,5-dioxygenase (9), which is found mostly in Gramnegative bacteria, which generates OPA dihydrodiols. These products are then transformed by rearomatization and decarboxylation by a dehydrogenase and a decarboxylase, respectively, yielding PCA. OPA dioxygenase from DBO1 has been purified (5). This enzyme is a multicomponent dioxygenase composed of a dioxygenase component and a reductase component. OPA dioxygenase requires Fe 2ϩ for activity and shows narrow substrate specificity with OPA. Microbial degradation of TPA has been characteriz...

The isophthalate (IPA) catabolic operon (iphACBDR) of Comamonas sp. strain E6 responsible for the conversion of IPA into protocatechuate is negatively regulated by an IclR-type transcriptional regulator, IphR. Promoter analysis showed that the region sufficient for the IPA-dependent induction of the iphA promoter was located within the 87 bp region upstream from the iphA start codon. The transcription start site of the iph operon was mapped at a cytosine located 49 bp upstream of the iphA start codon. Two inverted repeat sequences IR1 (positions -21 to -7 relative to the iphA transcription start site) and IR2 (-2 to +10) were found in the binding region of IphR identified by electrophoretic mobility shift assays (EMSA) using purified IphR. Mutation analysis of each half-site of these inverted repeat sequences indicated that both IR2 and a downstream half-site of IR1 are necessary for the binding of IphR. EMSA in the presence of IPA or its analogous substrates demonstrated that IPA had the ability to inhibit the binding of IphR to this operator region. In conclusion, the iph operon is negatively autoregulated by the binding of IphR to the operator region, and this repression is released by the presence of IPA.