Publications

• Genetic characterization and expression in heterologous hosts of the 3-(3-hydroxyphenyl)propionate catabolic pathway of Escherichia coli K-12.

  Ferrandez A., Garcia J.L., Diaz E.

  We report the complete nucleotide sequence of the gene cluster encoding the 3-(3-hydroxyphenyl)propionate (3-HPP) catabolic pathway of Escherichia coli K-12. Sequence analysis revealed the existence of eight genes that map at min 8 of the chromosome, between the lac and hemB regions. Six enzyme-en ... >> More

  We report the complete nucleotide sequence of the gene cluster encoding the 3-(3-hydroxyphenyl)propionate (3-HPP) catabolic pathway of Escherichia coli K-12. Sequence analysis revealed the existence of eight genes that map at min 8 of the chromosome, between the lac and hemB regions. Six enzyme-encoding genes account for a flavin-type monooxygenase (mhpA), the extradiol dioxygenase (mhpB), and the meta-cleavage pathway (mhpCDFE). The order of these catabolic genes, with the sole exception of mhpF, parallels that of the enzymatic steps of the pathway. The mhpF gene may encode the terminal acetaldehyde dehydrogenase (acylating) not reported previously in the proposed pathway. Enzymes that catalyze the early reactions of the pathway, MhpA and MhpB, showed the lowest level of sequence similarity to analogous enzymes of other aromatic catabolic pathways. However, the genes mhpCDFE present the same organization and appear to be homologous to the Pseudomonas xyl, dmp, and nah meta-pathway genes, supporting the hypothesis of the modular evolution of catabolic pathways and becoming the first example of this type of catabolic module outside the genus Pseudomonas. Two bacterial interspersed mosaic elements were found downstream of the mhpABCDFE locus and flank a gene, orfT, which encodes a protein related to the superfamily of transmembrane facilitators that might be associated with transport. All of the genes of the 3-HPP cluster are transcribed in the same direction, with the sole exception of mhpR. Inducible expression of the mhp catabolic genes depends upon the presence, in the cis or trans position, of a functional mhpR gene, which suggests that the mhpR gene product is the activator of the 3-HPP biodegradative pathway. The primary structure of MhpR revealed significant similarities to that of members of the IclR subfamily of transcriptional regulators. A 3-HPP catabolic DNA cassette was engineered and shown to be functional not only in enteric bacteria (E. coli and Salmonella typhimurium) but also in Pseudomonas putida and Rhizobium meliloti, thus facilitating its potential application to improve the catabolic abilities of bacterial strains for degradation of aromatic compounds. << Less

  J. Bacteriol. 179:2573-2581(1997) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Purification, characterization, and stereochemical analysis of a C-C hydrolase: 2-hydroxy-6-keto-nona-2,4-diene-1,9-dioic acid 5,6-hydrolase.

  Lam W.W., Bugg T.D.

  2-Hydroxy-6-keto-nona-2,4-diene-1,9-dioic acid 5,6-hydrolase (MhpC) from Escherichia coli has been purified to near homogeneity from an overexpressing strain of E. coli. The purified enzyme is a 29 kDa dimeric protein requiring no cofactors for catalytic activity. The enzyme has a Km of 2.1 microM ... >> More

  2-Hydroxy-6-keto-nona-2,4-diene-1,9-dioic acid 5,6-hydrolase (MhpC) from Escherichia coli has been purified to near homogeneity from an overexpressing strain of E. coli. The purified enzyme is a 29 kDa dimeric protein requiring no cofactors for catalytic activity. The enzyme has a Km of 2.1 microM and a kcat of 36 s-1 for its natural substrate and shows high selectivity for the propionate side chain of the substrate. The stereochemical course of the MhpC reaction was elucidated by conversion of protiosubstrate in 2H2O and conversion of deuteriated substrate in 1H2O, revealing that the reaction proceeds with overall replacement of a succinyl moiety by a proton from water in the H-5E position, with retention of regiochemistry. Isotope exchange was also observed in the H-5Z position of the product, which was rationalized by enzyme-catalyzed exchange of 2H into C-5 of the substrate from 2H2O. These data are consistent with a reversible keto-enol tautomerization taking place as the first step of the enzyme mechanism. << Less

  Biochemistry 36:12242-12251(1997) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Catabolism of phenylpropionic acid and its 3-hydroxy derivative by Escherichia coli.

  Burlingame R., Chapman P.J.

  A number of laboratory strains and clinical isolates of Escherichia coli utilized several aromatic acids as sole sources of carbon for growth. E. coli K-12 used separate reactions to convert 3-phenylpropionic and 3-(3-hydroxyphenyl)propionic acids into 3-(2,3-dihydroxyphenyl)propionic acid which, ... >> More

  A number of laboratory strains and clinical isolates of Escherichia coli utilized several aromatic acids as sole sources of carbon for growth. E. coli K-12 used separate reactions to convert 3-phenylpropionic and 3-(3-hydroxyphenyl)propionic acids into 3-(2,3-dihydroxyphenyl)propionic acid which, after meta-fission of the benzene nucleus, gave succinate, pyruvate, and acetaldehyde as products. Enzyme assays and respirometry showed that all enzymes of this branched pathway were inducible and that syntheses of enzymes required to convert the two initial growth substrates into 3-(2,3-dihydroxyphenyl)propionate are under separate control. E. coli K-12 also grew with 3-hydroxycinnamic acid as sole source of carbon; the ability of cells to oxidize cinnamic and 3-phenylpropionic acids, and hydroxylated derivatives, was investigated. The lactone of 4-hydroxy-2-ketovaleric acid was isolated from enzymatic reaction mixtures and its properties, including optical activity, were recorded. << Less

  J Bacteriol 155:113-121(1983) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

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

  Diaz E., Ferrandez A., Garcia J.L.

  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 (hcaA1 ... >> More

  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. << Less

  J. Bacteriol. 180:2915-2923(1998) [PubMed] [EuropePMC]

  This publication is cited by 10 other entries.

• Isolation and characterization of Escherichia coli mutants defective for phenylpropionate degradation.

  Burlingame R.P., Wyman L., Chapman P.J.

  Mutants of Escherichia coli defective in catabolism of 3-phenylpropionate, 3-(3-hydroxyphenyl)propionate, or both were isolated after mutagenesis with ethylmethane sulfonate. Nine phenotypically distinct classes of mutants were identified, including strains lacking each of the first five enzyme ac ... >> More

  Mutants of Escherichia coli defective in catabolism of 3-phenylpropionate, 3-(3-hydroxyphenyl)propionate, or both were isolated after mutagenesis with ethylmethane sulfonate. Nine phenotypically distinct classes of mutants were identified, including strains lacking each of the first five enzyme activities for the degradation of these compounds and mutants pleiotropically negative for some of these activities. Characterization of these mutants was greatly facilitated by the use of indicator media in which accumulation of 3-(2,3-dihydroxyphenyl)propionate or 2-hydroxy-6-ketononadienedioic acid led to the formation of dark red or bright yellow colors, respectively, in the medium. Assays with wild-type and mutant strains indicated that 3-phenylpropionate (or its dihydrodiol), but none of the hydroxylated derivatives tested, induced the synthesis of enzymes for its conversion to 3-(2,3-dihydroxyphenyl)propionate. The remaining enzymes were induced by the 2- or 3-hydroxy or 2,3-dihydroxy derivatives of 3-phenylpropionate, with the 2-hydroxy compound acting as an apparent gratuitous inducer. Metabolism to nonaromatic intermediates appeared to be unnecessary for full induction of any pathway enzyme. One unusual class of mutants, in which 2-keto-4-pentenoate hydratase appeared to be uninducible, indicated a level of control not previously shown in meta-fission catabolic pathways. << Less

  J Bacteriol 168:55-64(1986) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Catalytic mechanism of C-C hydrolase MhpC from Escherichia coli: kinetic analysis of His263 and Ser110 site-directed mutants.

  Li C., Montgomery M.G., Mohammed F., Li J.-J., Wood S.P., Bugg T.D.H.

  C-C hydrolase MhpC (2-hydroxy-6-keto-nona-1,9-dioic acid 5,6-hydrolase) from Escherichia coli catalyses the hydrolytic C-C cleavage of the meta-ring fission product on the phenylpropionic acid catabolic pathway. The crystal structure of E. coli MhpC has revealed a number of active-site amino acid ... >> More

  C-C hydrolase MhpC (2-hydroxy-6-keto-nona-1,9-dioic acid 5,6-hydrolase) from Escherichia coli catalyses the hydrolytic C-C cleavage of the meta-ring fission product on the phenylpropionic acid catabolic pathway. The crystal structure of E. coli MhpC has revealed a number of active-site amino acid residues that may participate in catalysis. Site-directed mutants of His263, Ser110, His114, and Ser40 have been analysed using steady-state and stopped-flow kinetics. Mutants H263A, S110A and S110G show 10(4)-fold reduced catalytic efficiency, but still retain catalytic activity for C-C cleavage. Two distinct steps are observed by stopped-flow UV/Vis spectrophotometry, corresponding to ketonisation and C-C cleavage: H263A exhibits very slow ketonisation and C-C cleavage, whereas S110A and S110G exhibit fast ketonisation, an intermediate phase, and slow C-C cleavage. H114A shows only twofold-reduced catalytic efficiency, ruling out a catalytic role, but shows a fivefold-reduced K(M) for the natural substrate, and an ability to process an aryl-containing substrate, implying a role for His114 in positioning of the substrate. S40A shows only twofold-reduced catalytic efficiency, but shows a very fast (500 s(-1)) interconversion of dienol (317 nm) to dienolate (394 nm) forms of the substrate, indicating that the enzyme accepts the dienol form of the substrate. These data imply that His263 is responsible for both ketonisation of the substrate and for deprotonation of water for C-C cleavage, a novel catalytic role in a serine hydrolase. Ser110 has an important but non-essential role in catalysis, which appears not to be to act as a nucleophile. A catalytic mechanism is proposed involving stabilisation of reactive intermediates and activation of a nucleophilic water molecule by Ser110. << Less

  J. Mol. Biol. 346:241-251(2005) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.