Characterization of the hca Cluster Encoding the Dioxygenolytic Pathway for Initial Catabolism of 3-Phenylpropionic Acid in Escherichia coli K-12

Abstract

ABSTRACT We have identified, cloned, and sequenced the hca cluster encoding the dioxygenolytic pathway for initial catabolism of 3-phenylpropionic acid (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 putative operon ( hcaA1A2CBD ) and two additional genes transcribed in the opposite direction that encode a potential permease ( hcaT ) and a regulator ( hcaR ). Sequence comparisons revealed that while hcaA1A2CD genes encode the four subunits of the 3-phenylpropionate dioxygenase, the hcaB gene codes for the corresponding cis -dihydrodiol dehydrogenase. This type of catabolic module is homologous to those encoding class IIB dioxygenases and becomes the first example of such a catabolic cluster in E. coli . The inducible expression of the hca genes requires the presence of the hcaR gene product, which acts as a transcriptional activator and shows significant sequence similarity to members of the LysR family 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 derivative. Further catabolism of DHPP requires the mhp -encoded meta fission pathway for the mineralization of 3-hydroxyphenylpropionate (3HPP) (A. Ferrández, J. L. Garcı́a, and E. Dı́az, 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 recombinant bacteria in 3-hydroxycinnamic acid (3HCI) and CI, respectively. Thus, the convergent mhp - and hca -encoded pathways are also functional in S. typhimurium , and they are responsible for the catabolism of different phenylpropanoid compounds (3HPP, 3HCI, PP, and CI) widely available in nature.