Characterization of the hca cluster encoding the dioxygenolytic pathway for initial catabolism of 3-phenylpropionic acid in Escherichia coli K-12

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. García, and E. Díaz, 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.

FIG. 1

Convergent pathways for the catabolism of PP (CI) and 3HPP (3HCI) in E. coli. (A) Physical and genetic map of the chromosomal hca region. The locations of the genes are shown relative to those of some relevant restriction endonuclease sites, i.e., BamHI (B), ClaI (C), KpnI (K), PstI (P), and SphI (Sp). Arrows indicate the directions of gene transcription. The boxed plus sign indicates stimulation of gene expression by the hcaR gene product in the presence of PP. Genes with similar shadings encode subunits of the same protein. (B) Proposed biochemistry of the PP (3HPP) and CI (3HCI) catabolic pathways. HcaA1A2CD and HcaB are the enzymes encoded by the corresponding hca structural genes. MhpA to MhpF are the enzymes for the catabolism of 3HPP (3HCI) and DHPP (DHCI). The metabolites are PP (compound I), cis-3-(3-carboxyethyl)-3,5-cyclohexadiene-1,2-diol (compound II), DHPP (compound III), 3HPP (compound IV), 2-hydroxy-6-ketononadienedioate (compound V), CI (compound VI), cis-3-(3-carboxyethenyl)-3,5-cyclohexadiene-1,2-diol (compound VII), DHCI (compound VIII), 3HCI (compound IX), and 2-hydroxy-6-ketononatrienedioate (compound X). Enzymes: HcaA1A2CD, 3-phenylpropionate dioxygenase; HcaB, 3-phenylpropionate-dihydrodiol dehydrogenase; MhpA, 3-(3-hydroxyphenyl)propionate hydroxylase; MhpB, 3-(2,3-dihydroxyphenyl)propionate 1,2-dioxygenase; MhpC, 2-hydroxy-6-ketonona-2,4-dienedioate hydrolase; MhpD, 2-keto-4-pentenoate hydratase; MhpE, 4-hydroxy-2-ketovalerate aldolase; MhpF, acetaldehyde dehydrogenase (acylating).

FIG. 2

Schematic representation of the subcloning and expression of the regulatory and catabolic hca genes. The subcloning strategies are described in detail in Materials and Methods. The relevant elements and restriction sites are indicated. The thick line represents the DNA fragment whose sequence is shown in Fig. 3. Vector-derived sequences are indicated by dashed lines. The Plac and the Ptrc promoters and direction of transcription are indicated (arrows). Δ, truncated gene. T₁ and T₂ are the transcriptional terminators of the E. coli rrnB operon (41). 1 and 2, oligonucleotides HCAR and HCA3, which were used as primers for the PCR to construct plasmid pCKER. The region encoding the replication (ori) function is also indicated. B, BamHI; Bg, BglII; E, EcoRI; H, HindIII; K, KpnI; N, NotI; P, PstI; S, SmaI; Sp, SphI; X, XhoI. Ap^r and Cm^r, genes conferring resistance to AP and CM, respectively.

FIG. 3

Nucleotide and derived amino acid sequences of the PP dioxygenolytic pathway. The sequence data in uppercase letters appear in the GenBank/EMBL data bank under accession numbers Y11071 (nucleotides 1 to 1433) and Y11070 (nucleotides 3948 to 7259). The sequence from nucleotide 1434 to 3947 (lowercase letters) was taken from the GenBank/EMBL data bank (accession number AE000340). Only the sequences of the 5′- and 3′-end-coding regions of the hca genes and orfX are shown. The 3′ end of the csiE gene is also shown. Short arrows, direction of gene transcription; asterisks, stop codons. Potential Shine-Dalgarno sequences are boldfaced. Inverted repeats are marked with facing arrows underneath the sequence. The putative binding motif of LTTRs (49) is doubly underlined, and the characteristic T and A residues are boxed. The nucleotide sequence present in oligonucleotide HCAR, used for PCR amplification of hcaA1, is italicized.