CHARACTERIZATION OF THE HCA CLUSTER ENCODING THE DIOXYGENOLYTIC PATHWAY FOR INITIAL CATABOLISM OF 3-PHENYLPROPIONIC ACID IN ESCHERICHIA-COLI K-12

We have identified, cloned, and sequenced the hca cluster encoding the dioxygenolytic pathway for initial catabolism of 3-phenylpropionic ac id (PP) in Escherichia coli K-12. This cluster maps at min 57.5 of the chromosome and is composed of five catabolic genes arranged as a puta tive operon (hcaAlA2CBD) and two additional genes transcribed in the o pposite direction that encode a potential permease (hcaT) and a regula tor (hcaR), Sequence comparisons revealed that while hcaA1A2CD genes e ncode the four subunits of the 3-phenylpropionate dioxygenase, the hca B gene codes for the corresponding cis-dihydrodiol dehydrogenase. This type of catabolic module is homologous to those encoding class IIB di oxygenases and becomes the first example of such a catabolic cluster i n E. coli, The inducible expression of the hca genes requires the pres ence of the hcaR gene product, which acts as a transcriptional activat or and shows significant sequence similarity to members of the LysR fa mily of regulators. Interestingly, the HcaA1A2CD and HcaB enzymes are able to oxidize not only PP to 3-(2,3-dihydroxyphenyl)propionate (DHPP ) but also cinnamic acid (CI) to its corresponding 2,3-dihydroxy deriv ative, Further catabolism of DHPP requires the mhp-encoded meta fissio n pathway for the mineralization of 3-hydroxyphenylpropionate (3HPP) ( A. Ferrandez, J. L. Garcia, and E. Diaz, J. Bacteriol, 179:2573-2581, 1997), Expression in Salmonella typhimurium of the mhp genes alone or in combination with the hca cluster allowed the growth of the recombin ant bacteria in 3-hydroxycinnamic acid (3HCI) and CI, respectively. Th us, the convergent mhp and hca-encoded pathways are also functional in S. typhimurium, and they are responsible for the catabolism of differ ent phenylpropanoid compounds (3HPP, 3HCI, PP, and CI) widely availabl e in nature.