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Originally published In Press as doi:10.1074/jbc.M501988200 on May 2, 2005

J. Biol. Chem., Vol. 280, Issue 28, 26435-26447, July 15, 2005
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A Two-component Hydroxylase Involved in the Assimilation of 3-Hydroxyphenyl Acetate in Pseudomonas putida*

Elsa Arias-Barrau{ddagger}§, Ángel Sandoval{ddagger}, Germán Naharro||, Elías R. Olivera{ddagger}§, and José M. Luengo{ddagger}**

From the {ddagger}Departamento de Bioquímica y Biología Molecular, Facultad de Veterinaria, Universidad de León, 24007 León and the ||Departamento de Patología Animal (Sanidad Animal), Facultad de Veterinaria, Universidad de León, 24007 León, Spain

Received for publication, February 22, 2005 , and in revised form, April 21, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
The complete catabolic pathway involved in the assimilation of 3-hydroxyphenylacetic acid (3-OH-PhAc) in Pseudomonas putida U has been established. This pathway is integrated by the following: (i) a specific route (upper pathway), which catalyzes the conversion of 3-OH-PhAc into 2,5-dihydroxyphenylacetic acid (2,5-diOH-PhAc) (homogentisic acid, Hmg), and (ii) a central route (convergent route), which catalyzes the transformation of the Hmg generated from 3-OH-PhAc, L-Phe, and L-Tyr into fumarate and acetoacetate (HmgABC). Thus, in a first step the degradation of 3-OH-PhAc requires the uptake of 3-OH-PhAc by means of an active transport system that involves the participation of a permease (MhaC) together with phosphoenolpyruvate as the energy source. Once incorporated, 3-OH-PhAc is hydroxylated to 2,5-diOH-PhAc through an enzymatic reaction catalyzed by a novel two-component flavoprotein aromatic hydroxylase (MhaAB). The large component (MhaA, 62,719 Da) is a flavoprotein, and the small component (MhaB, 6,348 Da) is a coupling protein that is essential for the hydroxylation of 3-OH-PhAc to 2,5-diOH-PhAc. Sequence analyses and molecular biology studies revealed that homogentisic acid synthase (MhaAB) is different from the aromatic hydroxylases reported to date, accounting for its specific involvement in the catabolism of 3-OH-PhAc. Additionally, an ABC transport system (HmgDEFGHI) involved in the uptake of homogentisic acid and two regulatory elements (mhaSR and hmgR) have been identified. Furthermore, the cloning and the expression of some of the catabolic genes in different microbes presented them with the ability to synthesize Hmg (mhaAB) or allowed them to grow in chemically defined media containing 3-OH-PhAc as the sole carbon source (mhaAB and hmgABC).


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Bacteria belonging to the genus Pseudomonas are able to assimilate a very large number of organic compounds that may be dangerous or toxic for many other living organisms (animals, plants, and microbes) (1-5). Most of such molecules are unnatural compounds (xenobiotics) that are mainly produced by the pyrolysis of organic molecules generated in industrial activities (6, 7). These may accumulate in the biosphere, and because of their very stable molecular structures, they require long periods of time (up to decades) to become mineralized (5, 7-9). Most of these compounds are degraded through unusual catabolic pathways that imply the participation of uncommon enzymes (encoded by genes with anarchical subcellular locations) that generate rare catabolites as intermediates and that seem to be controlled by some rather specific regulators (10-15).

The aerobic degradation of aromatic compounds usually involves the participation of hydroxylating enzymes, which catalyze the introduction of two hydroxyl groups into the ring in order to facilitate an intra- or extradiol cleavage mediated by molecular oxygen (16-18). The enzymes that catalyze the incorporation of O2 into substrates (oxygenases) are a fairly heterogeneous group of enzymes, involved in the degradation of many different compounds that have important cellular functions (drug resistance, solvent elimination and detoxification, ecological restoration of natural habitats, etc.) (18-36).

Although many benzene-derived aromatic compounds (see above) must be hydroxylated as an initial catabolic event, we have observed that there are some important exceptions. Thus, the degradation of phenylacetic acid (PhAc)1 in Pseudomonas putida U is carried out through a new catabolic pathway that involves the activation of this acid to phenylacetyl-CoA prior to hydroxylation (2, 5). Nevertheless, hydroxylated derivatives of phenylacetic acid (3-OH-PhAc, 4-OH-PhAc, or dihydroxylated compounds -3,4-diOH-PhAc and 2,5-diOH-PhAc-) are not degraded by any of these pathways, suggesting that they would require other catabolic routes involving the participation of hydroxylases, as indicated above (37-45). Indeed, the degradative pathway of 4-OH-PhAc seems to be fairly similar to the one that catalyzes the assimilation of this compound in Escherichia coli through the homoprotocatechuate pathway (3,4-diOH-PhAc) (46). However, in E. coli, 4-OH-PhAc and 3-OH-PhAc are degraded through the same pathway (42-44), but in P. putida the assimilation of 3-OH-PhAc requires a different route, which has only been partially clarified. In previous work (32) we showed that 3-OH-PhAc is oxidized to Hmg through an unknown mechanism and that this intermediate is further catabolized through the homogentisate pathway, a central route involved in the degradation of L-Phe and L-Tyr.

Here we approached the study of the enzymatic steps involved in the conversion of 3-OH-PhAc into Hmg in P. putida U, reporting for the first time that the 3-OH-PhAc upper catabolic pathway requires the participation of the following: (i) a permease; (ii) a novel two-component hydroxylating enzyme (3-hydroxyphenylacetate 6-hydroxylase, homogentisate synthase); (iii) an ABC transport system; and (iv) several regulatory elements.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Materials—Molecular biology products were supplied by Amersham Biosciences. Commercial vectors used for cloning or subcloning genomic fragments were from Stratagene and Promega. Aromatic compounds were obtained from Merck, Lancaster Synthesis, Ltd. (France), or Sigma. Biochemical and reagents were supplied by different commercial firms. All other products employed were of analytical quality or HPLC grade.

Microorganisms and Vectors—The P. putida U (Coleccion Española de Cultivos Tipo, CECT4848) was from our collection (2, 34-35, 46). This strain was originally obtained from R. A. Cooper (Department of Biochemistry, University of Leicester, UK). Pseudomonas fluorescens A2-2 (47) was kindly supplied by PharmaMar S. A. (Colmenar Viejo, Madrid, Spain). E. coli HB101 containing the plasmid pGS9 (48), which includes the transposon Tn5, was kindly supplied by Dr. J. L. Ramos (Estación Experimental del Zaidín, CSIC, Granada, Spain). E. coli DH5{alpha}' (Invitrogen) and E. coli DH10B were used for plasmid propagation (49). E. coli strain NM538 was employed for the genomic amplifications of genomic libraries. E. coli (pRK600) was used as a helper strain in the triparental filter mating (50). E. coli MC4100 and plasmid pRS551 were specifically used for the identification of promoters (51). E. coli W ATCC11105 (52) and E. coli W14 (a 33-kb deletion mutant derived from E. coli W that have lost the PhAc and the 2-phenylethylamine catabolic pathways) were provided by Dr. J. L. García (Consejo Superior de Investigaciones Científicas, Madrid, Spain).

The commercial plasmid pGEM-T Easy (Promega) were used for subcloning genomic fragments, and the pK18::mob (53) and the pJQ200KS (54) were employed to induce specific gene disruption. pBBR1MCS-3 (Tcr), a broad host range cloning and expression vector (55), was used to analyze the expression of different genes in P. putida U. DNA manipulations and sequence analyses were performed as indicated elsewhere (56, 57). All strains, mutants, and vectors used in this work are summarized in Table I.


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  		TABLE I
Strains, mutants, and vectors used in this work

 
The sequences reported in this paper have been deposited in the GenBankTM data base (accession numbers AY929299 [GenBank] , AY929300 [GenBank] , and AY937229 [GenBank] ).

Culture Media and Growth Conditions—P. putida U and P. fluorescens were maintained on trypticase soy agar (Difco), and growth slants (12 h at 30 °C) were used to inoculate liquid media. 500-ml Erlenmeyer flasks containing 100 ml of a chemically defined medium (MM, see Ref. 2) were inoculated with 2 ml each of a bacterial suspension (A540 = 0.5). Incubations were carried out in a rotary shaker (250 rpm) at 30 °C for the time required in each set of experiments. The carbon sources used for culture were phenylacetic acid (10 mM), 3-OH-PhAc (10 mM), 4-OH-PhAc (10 mM), L-tyrosine (5 mM), succinic acid (42 mM), or combinations thereof. In some experiments different antibiotics were also supplied.

E. coli strains were maintained on Luria-Bertani (LB) agar plates and cultured in the required medium at 37 °C overnight or for the time indicated in each experiment (34, 52, 58). In all the experiments in which solid media were employed, 25 g liter-1 Difco agar was added.

Isolation of Mutants Handicapped in the Degradation of 3-OH-PhAc—The isolation of mutants unable to catabolize 3-OH-PhAc was carried out by mutagenesis with the transposon Tn5, following the mating procedure (48, 50). Bacteria grown on the mating filters were resuspended in 3 ml of LB medium and seeded on LB plates containing rifampicin (20 µgml-1) and kanamycin (25 µgml-1) and incubated 48 h at 30 °C. Colonies were seeded by replica plating in two different media (containing either 0.5 g liter-1 fructose or 10 mM 3-OH-PhAc as the sole carbon source). Mutants were those strains able to grow on plates containing fructose but unable to grow on those containing 3-OH-PhAc as the sole carbon source. Following this procedure, several mutants were isolated (see "Results").

For gene disruption through single homologous recombination, an internal fragment (usually 99-300 bp) of the gene to be disrupted was cloned in the polylinker of pK18::mob or pJQ200KS (two mobilizable plasmids that do not replicate in Pseudomonas) (53-54), and the resulting construct was introduced into P. putida U by triparental filter mating (50). Exconjugants harboring the disrupted gene were isolated on LB medium containing rifampicin and kanamycin after 48 h of incubation at 30 °C. Deletion of a specific gene (or a set of genes) was accomplished by using the methodology described by others (54, 59), which involves a double-recombination event and the selection of the required mutant by expression of the lethal sacB gene. All mutants were analyzed by PCR to define the insertional position of the disrupting element or to confirm the position and extent of the deletion.

Throughout the text, deletions are indicated as {Delta} followed by the name of the gene that has been eliminated. Genetic disruptions, caused by the insertion of the transposon Tn5 or of the plasmids pK18::mob and pJQ200KS in a particular gene, are summarized as {Delta}gene::Tn5 {Delta}gene::pK18::mob and {Delta}gene::pJQ200KS, respectively.

HPLC Equipment and Chromatographic Procedure—The intermediates accumulated by the different mutants were analyzed by HPLC. Samples taken at different times from the culture broths of the mutants studied were centrifuged (31,000 x g, 20 min) to eliminate the bacteria and were filtered through a Millipore filter (0.45-µm pore size). Aliquots of 50 µl were taken and analyzed by using a high performance liquid chromatograph (Spectra Physics SP8800) equipped with a variable wavelength UV-visible detector (SP8450), a computing integrator (SP4290), and a microparticulate (10 µm particle size, 1 µm pore size) reverse-phase column (Nucleosil C18, 250 x 4.6 mm inner diameter; Phenomenex Laboratories). The mobile phase was 0.05 M K2HPO4 (pH 4) and acetonitrile (99:1 by volume)). The flow rate was 2.5 ml·min-1, and the eluate was monitored at 254 nm. Under these conditions the retention times for L-Tyr, homogentisic acid (2,5-diOH-PhAc), 3,4-diOH-PhAc, 4-OH-PhAc, and 3-OH-PhAc were 3, 7, 11, 20, and 23 min, respectively.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Isolation and Identification of P. putida U Mutants Unable to Catabolize 3-OH-PhAc—The aerobic degradation of 3-OH-PhAc in E. coli and in other microorganisms (3, 4, 42, 46, 60-61) involves the hydroxylation of this aromatic compound to 3,4-diOH-PhAc and later the degradation of this intermediate through a specific pathway that allows its transformation to central metabolites (succinate and pyruvate) (46, 61). However, P. putida U, a strain able to grow in minimal media containing 3-OH-PhAc as the sole carbon source (Fig. 1), assimilates this compound through a different catabolic pathway that, in a first step, catalyzes the introduction of a second hydroxyl group in ortho position of the aromatic ring to generate 2,5-diOH-PhAc (so-called homogentisic acid) (62-63). This compound is then catabolized through the homogentisate pathway as has been reported for the catabolism of L-phenylalanine and L-tyrosine (Fig. 2). Very recently, we have described the genetic organization, enzymatic activities, and molecular regulation of the route involved in the degradation of L-Tyr to homogentisic acid (upper pathway) as well as that required for the catabolism of Hmg to fumarate and acetoacetate (central pathway) (32). However, although this pathway has been characterized in detail, nothing is known about the enzymatic step(s) involved in the uptake or in the conversion of 3-OH-PhAc into Hmg. Accordingly, here we approached the identification of the gene(s) and protein(s) required for the uptake of 3-OH-PhAc and for the synthesis of Hmg from 3-OH-PhAc (mha, meta-hydroxyphenylacetic acid catabolic genes). The enzyme or enzymatic system responsible for the hydroxylation of 3-OH-PhAc to 2,5-diOH-PhAc will henceforth be designated homogentisate acid synthase (HmgS).

The characterization of the genes involved in the catabolic pathway required for the specific degradation of 3-OH-PhAc (Mha upper pathway) was performed by mutagenesis with the transposon Tn5 (48, 50). Following this procedure, several mutants (seven) were isolated. It was a priori expected that all of them would belong to one of the two following groups: (i) mutants unable to catabolize L-Tyr and 3-OH-PhAc (group I), and (ii) mutants unable to catabolize 3-OH-PhAc to 2,5-PhAc (Hmg) (group II). Analysis of the different exconjugants revealed that all them belonged to the first group (mutants in which the transposon was inserted in some of the genes encoding the enzymes of the Hmg pathway, the central pathway) (see Fig. 2). However, none of these mutants was specifically affected in the degradation of 3-OH-PhAc (group II). To identify this specific gene (or genes), we used a different strategy. We had observed previously that all the P. putida U mutants lacking a functional gene encoding homogentisate dioxygenase (hmgA), accumulated Hmg in the broth when they were cultured in any media containing 3-OH-PhAc, L-Tyr, peptides, or proteins containing this amino acid (32). This accumulation was easily visualized because, when oxidized to quinoid derivatives, Hmg became a dark-brown pigment that stained the culture broths (Fig. 3). Because we were interested in the isolation of mutants affected in the transformation of 3-OH-PhAc into Hmg, we designed a P. putida U strain in which the hmgABC cluster had been deleted (see "Materials" and Table I). This strain accumulated the Hmg pigment derivative when cultured either in the presence of 3-OH-PhAc or in media containing L-Tyr or L-Tyr-precursors (Fig. 3a). For this reason, a mutant handicapped in the enzymatic step(s) responsible for the production of Hmg from 3-OH-PhAc would not be detected when cultured in media containing L-Tyr or its precursors, because the red-brown pigment would be generated from L-Tyr. To improve the selection method, the hpd gene (encoding the 4-hydroxyphenylpyruvate dioxygenase) (32) was also eliminated. This double deleted mutant, P. putida U {Delta}hmgABC{Delta}hpd (Table I), only accumulated pigment in the presence of 3-OH-PhAc, because the pathway responsible for the transformation of L-Tyr into homogentisic acid has been blocked (see Fig. 3b). Mutagenesis with transposon Tn5 revealed that all the mutants in which the transposon had been inserted in any gene that was unrelated to the hydroxylation of 3-OH-PhAc to Hmg accumulated pigment when cultured in different media (LB or others) supplemented with 3-OH-PhAc. Despite this, three of them lost the capacity to produce homogentisic acid (and its pigment derivative) (Fig. 3b). Genetic analyses revealed that in those mutants the transposon had been inserted in a DNA fragment in which three ORFs were identified (Fig. 4).



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  		FIG. 1.
Bacterial growth of P. putida U ( and {circ}), P. putida KT2440 ({blacksquare} and {square}), and P. fluorescens A2-2 ({blacktriangleup} and {triangleup}) cultured in MM + 3-OH-PhAc (, {blacksquare}, and {blacktriangleup}) or in MM + L-Tyr ({circ}, {square}, and {triangleup}) (a), and P. putida U {Delta}mhaA::Tn5 ( and {circ}) and P. putida U {Delta}mhaB::Tn5 ({blacksquare} and {square}) cultured in MM +3-OH-PhAc ( and {blacksquare}) or in MM + L-Tyr ({circ},{square}) (b). The concentrations of the carbon sources were 10 mM for 3-OH-PhAc and 5 mM for L-Tyr.

 
Identification of the Genes and Enzymes Belonging to the Peripheral Pathway Involved in the Catabolism of 3-OH-PhAc—Sequence analysis of the DNA adjacent to the transposon Tn5 in the mutants of P. putida U unable to produce Hmg from 3-OHPhAc (P. putida U {Delta}hmgABC{Delta}hpd{Delta}mhaA::Tn5 and P. putida U {Delta}hmgABC{Delta}hpd{Delta}mhaB::Tn5, see Table I) revealed that in both cases Tn5 had been inserted into a discrete piece of DNA containing three ORFs that seemed to be involved in the uptake and in the hydroxylation of aromatic compounds (see below). The precise insertion of the transposon occurred in two genes (ORF1 and ORF2, mhaA and mhaB, meta-hydroxyphenylacetic acid genes A and B) encoding two proteins MhaA and MhaB. The small one (MhaB, 6,348 Da) showed certain homology (33% identity) with a hypothetical protein of Burkholderia fungorum LB400. Upstream from mhaB, a different gene (ORF1) was identified (Fig. 4). Sequence analyses revealed that this gene (henceforth designated mhaA) encodes a protein (MhaA, 62,719 Da) showing high homology (46% identity) with the one encoding the 2-polyprenyl-6-methoxyphenol hydroxylase from B. fungorum LB400. Further studies revealed that MhaA contains a FAD binding domain (from amino acids 36 to 167, VVVVGA...... LAQHDD) (64-67), a monooxygenase consensus sequence (from amino acids 184 to 388, ADWV-VA.... RFMAPP) (68), a pyridine nucleotide-disulfide oxidoreductase motif (from amino acids 36 to 62, VVVVGA.... IACVVL) (68), and ribityl moiety of the FAD-binding motif (from amino acids 314 to 348, RVIFAG.... LVWKLA), showing that this protein is a FAD-dependent oxidoreductase (69). Moreover, MhaA showed a certain homology with the Coq6p gene product (UbiH), a protein involved in the biosynthesis of coenzyme Q in Saccharomyces cerevisiae that catalyzes the introduction of a second hydroxyl group on the aromatic ring of a biosynthetic intermediate of coenzyme Q (ubiquinone) (68). Furthermore, sequence alignment with other proteins included in the data base revealed that MhaA is related to UbiH and UbiF of E. coli (70-72) as well as to the Homo sapiens, Mus musculus, Drosophila melanogaster, and Caenorhabditis elegans homologues (68). It is also worth noting that UbiH and MhaA catalyze the introduction of a second OH group on aromatic rings and that, in both cases, the hydroxyl group is introduced in para position, respecting the existing one.



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  		FIG. 2.
Schematic representation of the different catabolic pathways involved in the degradation of L-Tyr, 3-OH-PhAc, and 4-OH-PhAc in P. putida U (solid arrow), P. putida KT2440 (dotted arrow), and E. coli W (dashed arrow). 1, tyrosine aminotransferase; 2, 4-OH-phenylpyruvate dioxygenase; 3, homogentisate dioxygenase; 4, maleylacetoacetate isomerase; 5, fumarylacetoacetate hydrolase; 6, homogentisate synthase; 7 and 8, 4-OH-phenylacetate 3-monooxygenase; 9, homoprotocatechuate 2,3-dioxygenase; 10, 5-carboxymethyl-2-hydroxymuconic-semialdehyde dehydrogenase; 11, 5-carboxymethyl-2-hydroxymuconate isomerase; 12 and 13, 5-oxopent-3-ene-1,2,5-tricarboxylate decarboxylase; 14, 2-oxohept-3-ene-1,7-dioate hydratase; 15, 2,4-dihydroxy-hept-2-ene-1,7-dioate aldolase; 16, succinic semialdehyde dehydrogenase.

 



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  		FIG. 3.
a, cultures of P. putida U (A) and their mutants P. putida U {Delta}hmgA::Tn5 (B) and P. putida U {Delta}hpd (C) on LB solid medium; b, cultures of P. putida U {Delta}hpd{Delta}hmgABC (A) and P. putida U {Delta}hpd{Delta}hmgABC{Delta}mhaB::Tn5 (B) in LB + 3-OH-PhAc (10 mM).

 
The participation of heterodimeric enzymes in the degradation of aromatic compounds is not a rare event. Thus, the hydroxylases involved in the catabolism of 4-OH-PhAc (a close structural analogue of 3-OH-PhAc) in several microorganisms are dimeric enzymes also constituted by two different subunits ({alpha}{beta} enzymatic complex) (3, 4, 14, 25, 30, 40-45).

All the above observations, together with the fact that mutants in which the transposon Tn5 had been inserted in the genes mhaA or in the mhaB (Fig. 4) were unable to catabolize 3-OH-PhAc (Fig. 1), suggest that MhaB (or its analogues) could constitute the short component (coupling protein) of a {alpha}{beta}-hydroxylating enzymatic complex (3, 25, 42) in which MhaA would be the FAD-dependent hydroxylase required to introduce a hydroxyl group on the aromatic ring (ortho position) of 3-OH-PhAc. Thus, it could be argued that in P. putida U MhaAB would be a functional dimeric enzyme showing homogentisate synthase activity.

The study of the genetic organization of mhaAB and its homologues in the bacterial chromosome of P. putida U and B. fungorum has revealed that it is very similar in both bacteria, suggesting that they could constitute a single operon able to ensure their co-ordinate translation (68, 70-72). Downstream from ORF2 there is a third ORF (ORF3), the gene mhaC (see Fig. 4) encoding a protein related to permeases that shows high homology with the permease involved in the uptake of 4-hydroxyphenylacetic acid in other microbes (HpaX from E. coli W, Yersinia pestis, and Pseudomonas aeruginosa), suggesting that this protein could be involved in the cellular incorporation of 3-OH-PhAc.

In sum, genetic studies and comparative analyses revealed that two of the three ORFs (ORF1 and ORF2, mhaAB) described above encode the two components of a novel flavoprotein (FAD-associated) aromatic hydroxylase, constituted by a small subunit (MhaB, 6,348 Da) and a large subunit (MhaA, 62,719 Da), which seems to be responsible for the specific conversion of 3-OH-PhAc into Hmg (homogentisic acid synthase). Additionally, the third ORF (mhaC) encodes a permease that seems to be involved in the uptake of 3-OH-PhAc in P. putida U. Further analyses of this piece of DNA revealed that upstream and downstream from the genes (mhaABC) (Fig. 4) there is no essential genetic information for the degradation of 3-OH-PhAc.

Functional Analyses of the Enzymes Belonging to the 3-OH-PhAc-specific Pathway (Mha Upper Pathway)—To analyze the function of the enzymes involved in the transformation of 3-OH-PhAc into Hmg, several genetic engineering experiments aimed at determining the biochemical role played by all these proteins were designed. Thus, the disruption or the deletion of some of the above genes in P. putida U (see below) led to the loss of the ability to catabolize 3-OH-PhAc in this strain, suggesting that all these proteins would be essential for the catabolism of this aromatic acid.

Fig. 5 shows that when the mutant P. putida U {Delta}hmgABC{Delta}hpd{Delta}mhaB::Tn5 was cultured in a chemically defined medium (MM, see Ref. 32) containing 3-OH-PhAc (10 mM) and succinic acid (42 mM) or in LB + 3-OH-PhAc (10 mM), neither homogentisic acid or its pigment derivatives were produced. These results indicate that either the mhaB gene is essential for the hydroxylation of 3-OH-PhAc to Hmg or that disruption of this gene indirectly affects the expression of mhaC (the gene encoding the permease), if all them (mhaABC) constitute a single operon, and therefore that in the absence of uptake of 3-OH-PhAc Hmg cannot be synthesized. To clarify this point and to define further the function of these proteins, both genes (mhaA and mhaB) were amplified (independently or in tandem), cloned in the appropriate vectors (pGEM-T Easy, pBBR1MCS-3), and expressed in different microorganisms unable to synthesize homogentisic acid from 3-OH-PhAc (P. putida U {Delta}hmgABC{Delta}hpd{Delta}mhaB::Tn5, P. fluorescens A2-2, P. putida KT2440 or E. coli W). We observed that transformation of P. putida U {Delta}hmgABC{Delta}hpd{Delta}mhaB::Tn5 with a genetic construction in which the gene mhaB had been cloned into the plasmid pBBR1MCS-3 (Tcr), which replicates autonomously in P. putida (55), restored the ability of this mutant to produce homogentisic acid (see Fig. 5 and Table II). Moreover, PCR analyses revealed that in all the transformants the location of transposon Tn5 was not modified (data not shown). Thus, by taking into account that the original mutation (mhaB knockout) remained unaltered, it may be assumed that disruption of the mhaB alone blocks the synthesis of Hmg and that this metabolic alteration is not due, as we speculated above, to a hypothetical lack of expression of the permease (mhaC gene product).


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  		TABLE II
Functional analysis of different recombinant aromatic hydroxylases when expressed in several bacterial strains

The abbreviations used are as follows: G, growth; NG, no growth; hmg, homogentisate; hmp, homoprotocatechuate. S and L correspond to the small and to the large subunits of the hydroxylases, respectively. The number 3 or 4 indicates the position of the hydroxyl group present in the molecule of substrate to be hydroxylated; 3L3S, homogentisate synthase; 4L4S, 4-OH-PhAc hydroxylase. 3L4S corresponds to a recombinant hydroxylase containing the large subunit of homogentisate synthase and the small subunit of 4-OH-PhAc hydroxylase. 4L3S is a recombinant hydroxylase containing the large subunit of 4-OH-PhAc hydroxylase and the small subunit of homogentisate synthase. Colors indicate the carbon source(s) present in the media used for bacterial growth (white, 10 mM 3-OH-PhAc; light gray, 10 mM 4-OH-PhAc; black, 10 mM 3-OH-PhAc + 42 mM succinic acid; and dark gray, 10 mM 4-OH-PhAc + 42 mM succinic acid).

 
To establish further the function of the mhaAB genes, each of them was disrupted in P. putida U by homologous recombination using the plasmid pK18::mob. Thus, internal fragments of mhaA or mhaB were amplified by PCR (from position 1263 to 1569 in mhaA and from position -9 to 49 in mhaB), cloned into this vector, and used to transform P. putida U by triparental mating (see "Materials"). All the recombinant strains were analyzed, and we observed that none of the mutants in which mhaA or mhaB had been disrupted was able to catabolize 3-OH-PhAc, despite being able to grow well in chemically defined medium containing L-Tyr (Fig. 1), PhAc, 4-OH-PhAc, or different aromatic compounds as the sole carbon source. These results reinforce the above data suggesting that mhaAB expression is essential for the catabolism of 3-OH-PhAc in P. putida U (Table II), and they indicate that MhaAB is not a general enzymatic complex involved in the hydroxylation of aromatics but, instead, a specific one required for the catabolism of 3-OH-PhAc in this bacterium.



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  		FIG. 4.
Genetic organization of the cluster (genes mhaABC) involved in the hydroxylation of 3-OH-PhAc to homogentisic acid (upper pathway). Arrows indicate the insertion of the transposon Tn5 into mhaA (two mutants) and mhaB.

 



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  		FIG. 5.
Cultures of different mutants of P. putida U (A, P putida U {Delta}hpd{Delta}hmgABC; B, P. putida U {Delta}hpd{Delta}hmgABC- {Delta}mhaB::Tn5; and C, P. putida U {Delta}hpd{Delta}hmgABC{Delta}mhaB::Tn5 transformed with the plasmid pBBR1MCS-3 (Tcr) carrying the gene mhaB from P. putida U) in LB + 3-OH-PhAc (10 mM).

 
To analyze the exact biochemical function of these two genes, mhaA, mhaB, or both (mhaAB) were cloned into pBBR1MCS-3 (Tcr) (see Table I), and these constructions were used to transform P. putida KT2440, E. coli W, and P. fluorescens A2-2 (see "Materials"), three microorganisms unable to generate homogentisic acid from 3-OH-PhAc. The independent expression of mhaA or mhaB in these bacteria did not lead to the production of Hmg, but when both genes were cloned together, the recombinant microbes synthesized Hmg as long as 3-OH-PhAc was added to the culture medium. In the recombinant strain E. coli W harboring the pBBR1MCS-3mhaAB, the presence of Hmg was rapidly visualized (accumulation of red pigment in the medium) (Fig. 6). However, the recombinant strains P. putida KT2440 and P. fluorescens A2-2 transformed with the same genetic construction did not accumulate pigment because they have a functional L-Tyr catabolic pathway that allows the degradation of the Hmg generated from 3-OH-PhAc (Fig. 2). To study the expression of the mhaAB genes in P. fluorescens A2-2 in an easy way, the genes encoding the homogentisate pathway were deleted in this strain, and the P. fluorescens A2-2 {Delta}hmgABC (Table I) mutant was transformed with the plasmid pBBR1MCS-3mhaAB. HPLC analyses of the culture broths of the recombinant strains when cultured in chemically defined media (32), containing 3-OH-PhAc (10 mM) as a precursor of intermediates and succinic acid (42 mM) as the carbon source, revealed that when mhaAB were expressed together, Hmg is produced, whereas it was not detected when mhaA or mhaB were expressed independently (Table II).

In sum, all these results allow us to conclude that MhaA and MhaB are two subunits of an enzymatic system involved in the specific hydroxylation of 3-OH-PhAc to 2,5-diOH-PhAc (Hmg). Thus, in P. putida U these two proteins constitute a new enzyme with 3-hydroxyphenylacetate 6-monooxygenase activity that we refer to as homogentisic acid synthase (HmgS).

Design of Genetically Engineered Hydroxylases (Hybrid Recombinant Hydroxylases)—As indicated above, the two components (MhaA and MhaB) of the homogentisate synthase of P. putida U have certain similarities with the subunits of other aromatic hydroxylases. Taking into account that the combination of subunits corresponding to different hydroxylases could allow the design of new enzymatic systems with different substrate specificities, using genetic engineering we addressed the constructions of several hydroxylases by combining the genes encoding the small subunits (S) and those encoding the large ones (L). To perform these experiments, we selected the genes mhaA and mhaB, involved in the synthesis of Hmg in P. putida U, and hpaB and hpaC from E. coli W, which are required for the hydroxylation of 4-OH-PhAc to 3,4-diOH-PhAc (4-hydroxyphenylacetate 3-monooxygenase) (14, 42-44). The two subunits from P. putida U were designated 3S and 3L because they correspond to the small and to the large subunits involved in the hydroxylation of 3-OH-PhAc, whereas those from E. coli were abbreviated as 4S (HpaC) and 4L (HpaB), respectively. Thus, in all the engineered hydroxylases the number 3 or 4 indicates the position of the hydroxyl group present in the molecule of the substrate to be hydroxylated, whereas the letter S or L refers to the size of the subunit integrating each dimeric protein ({alpha}{beta}-hydroxylases). After these experiments, the following genetic constructions were obtained: 3L3S (homogentisate synthase); 3L4S, 4L4S (4-OH-PhAc hydroxylase); and 4L3S. Additionally, other genetic constructions containing the gene encoding each single subunit (3S, 3L, 4S, or 4L) were obtained (see Table I). To determine the functionality of the recombinant hydroxylases, the above genetic constructions were cloned in different microorganisms (see Table II).



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  		FIG. 6.
Cultures of E. coli W (A) and of three recombinant strains (B-D) containing the plasmid pBBR1MCS-3 (Tcr) carrying the genes mhaAB from P. putida U (B) in MM + 3-OH-PhAc (10 mM).

 
The expression of 3L3S in P. putida KT2440 and in P. fluorescens A2-2, two bacterial strains able to catabolize L-Tyr through the homogentisate pathway but unable to assimilate 3-OH-PhAc, enabled them to grow in chemically defined media containing 3-OH-PhAc as the sole carbon source (Table II). However, when 3L or 3S were expressed independently, none of these bacteria were able to catabolize 3-OH-PhAc (Table II). Furthermore, when constructions designed to express 3L3S, 3S, or 3L were used to transform P. fluorescens A2-2 {Delta}hmgABC (Table I), we observed that the recombinant strain expressing 3L3S (cultured in MM containing 3-OH-PhAc as source of intermediates and succinic acid to support the bacterial growth, see "Materials") released homogentisic acid into the medium; and when oxidized, the former turned into a red-brown pigment. However, neither Hmg nor pigment was detected if the strain only expressed one protein (3L or 3S) (see Table II).

To analyze the function of the two subunits (4L and 4S) corresponding to the 4-OH-PhAc 3-monooxygenase of E. coli W, their encoding genes were cloned and expressed in different microorganisms (see Table II). We observed that the transformation of P. putida A2 (a mutant in which the transposon Tn5 had been inserted in the gene encoding the large subunit of 4-OH-PhAc 3-monooxygenase and which was therefore unable to grow in MM containing 4-OH-PhAc as the sole carbon source, see Table I), with a construction containing the genes encoding 4L4S or 4L alone, restored the ability to grow in that medium in both cases, indicating that the enzyme from E. coli is also functional in P. putida U. Moreover, when these genes were cloned in P. putida U {Delta}hmgABC, a mutant that cannot grow in MM + 3-OH-PhAc because of deletion of the homogentisate pathway, the recombinant strain (P. putida U {Delta}hmgABC pBBR1MCS-3hpaBC, see Table I) was able to grow in this medium and also to accumulate Hmg in the culture (see Table II). In this bacterium, part of the 3-OH-PhAc added to the medium was oxidized to Hmg, which cannot be further catabolized, whereas the rest of the 3-OH-PhAc was transformed into 3,4-diOH-PhAc and used as carbon source through the 4-OH-PhAc catabolic pathway. However, in the wild-type (P. putida U) the catabolism of 3-OH-PhAc via 3,4-diOH-PhAc cannot occur, because in this bacterium there is no enzyme that hydroxylates 3-OH-PhAc to 3,4-diOH-PhAc. By contrast, in E. coli W (which does not have the homogentisate route and which is unable to synthesize Hmg from 3-OH-PhAc) the enzyme that hydroxylates 4-OH-PhAc also recognizes 3-OH-PhAc, generating the same product (3,4-diOH-PhAc) with both substrates.

Transformation of P. putida KT2440 and P. fluorescens A2-2 {Delta}hmgABC with a genetic construction containing the gene encoding 4L (pBBR1MCS-3hpaB) revealed that the expression of this enzyme in P. putida KT2440 and in P. fluorescens A2-2 {Delta}hmgABC, when cultured in MM containing succinic acid + 3-OH-PhAc, led to the synthesis of 3,4-diOH-PhAc, which was accumulated in the culture broth (see Table II). In contrast, in the control strains (P. putida KT2440 and in P. fluorescens A2-2 {Delta}hmgABC transformed with the plasmid pBBR1MCS-3) synthesis of this compound did not take place. Taking into account that only 4L was expressed and that the synthesis of 3,4-diOH-PhAc requires both 4L and 4S, it must be assumed that an enzyme that takes over the function of the 4S subunit must exist in these bacteria.

When genetically engineered hydroxylases (hybrid hydroxylases, 3L4S or 4L3S) were obtained, we observed that 3L4S was unable to oxidize 3-OH-PhAc to homogentisic acid (2,5-diOH-PhAc) and that it did not catalyze the hydroxylation of 4-OH-PhAc to 3,4-diOH-PhAc either (see Table II). Additionally, we observed that 4L3S was unable to synthesize Hmg, although in some strains (those having an unknown enzyme with an analogous function to 4L), this protein did catalyze the transformation of 4-OH-PhAc into 3,4-diOH-PhAc (see Table II).

In sum, all these results allow us to conclude that E. coli does not contain a hydroxylase able to synthesize Hmg from 3-OH-PhAc and neither is there a pathway that transforms Hmg into general metabolites. However, in this bacterium the same hydroxylase converted 3- and 4-OH-PhAc into 3,4-diOH-PhAc. Unlike E. coli, in P. putida U there are two different hydroxylases. One of them hydroxylates 4-OH-PhAc to 3,4-diOH-PhAc but does not recognize 3-OH-PhAc. The other one hydroxylates 3-OH-PhAc to Hmg (2,5-diOH-PhAc) but does not use 4-OH-PhAc as a substrate.

Studies with hybrid proteins (3L4S and 4L3S) revealed that none of them was functional, indicating that although the native enzymes (3L3S and 4L4S) function in E. coli and in Pseudomonas, the subunits corresponding to the homogentisate synthase from P. putida U and to the 4-OH-PhAc 3-monooxygenase from E. coli W are not exchangeable.

Identification and Functional Analyses of Other Genes and Proteins Involved in the Degradation of 3-OH-PhAc—Further Tn5 mutational studies allowed us to identify other genes needed for the assimilation of 3-OH-PhAc in P. putida U. We isolated a mutant in which the transposon was inserted into an ATPase component (HmgG) of an ABC transport system (Hmg-DEFGHI) integrated by a periplasmic binding protein (HmgD), two ATPases (HmgGH), two permeases (HmgEF), and a regulator (HmgI) showing certain homology with other ABC systems and that is essential for the degradation of 3-OH-PhAc. Biochemical studies revealed that this mutant, although unable to degrade 3-OH-PhAc, grew well in other media containing close structural aromatic analogues (PhAc, 4-OH-PhAc, 2-phenylethanol, and 2-phenylethylamine) as the sole carbon source. It was observed that although this mutant was unable to grow in a chemically defined medium containing 3-OH-PhAc as the sole carbon source, in the presence of L-Tyr (5 mM) the growth of this strain, although undergoing a certain delay, was even able to reach one-half of the absorbance measured in the wild-type strain cultured in the same medium and conditions (Fig. 1 and Fig. 7). These results suggest that the mutation performed in this strain either affects the rate of transport of 3-OH-PhAc and L-Tyr (being more dramatic in the first case) or that it modifies the rate of transport (efflux or secondary influx) of an intermediate common to 3-OH-PhAc and L-Tyr. Taking into account that the only common intermediate (convergent catabolite) is homogentisic acid, it could be argued that this ABC system could be involved in the transport of Hmg.

It is interesting to note that although in P. putida U Hmg is produced intracellularly (at least the homogentisate synthesized from L-Tyr), part of this compound was excreted to the medium. Thus, HPLC analyses of the culture broth of P. putida U (wild type) grown in MM + L-Tyr as the sole carbon source revealed that a certain amount of Hmg (equivalent to 30% of the L-Tyr added) was released to the broth, even though it disappeared some hours later (between 10 and 15 h). It may therefore be assumed that, once synthesized, part of the Hmg (probably the part due to the diphase between the rate of synthesis and the degradation of Hmg in that bacterium) is released to the broth and later (once the intracellular pool has decreased) taken up again and catabolized.

A similar effect was observed when P. putida U was cultured in MM + 4-OH-PhAc. HPLC analysis of the broth revealed that some 3,4-diOH-PhAc was also released (a quantity equivalent to the 40% of the 4-OH-PhAc supplied), suggesting that the intracellular accumulation of diOH-PhAc derivatives in this microbe must be toxic or dangerous and, therefore, that they should be eliminated rapidly (before undergoing oxidation to quinones or polymerization). This hypothesis is supported by the observation that the microbial degradation of these dihydroxy derivatives (3,4-diOH-PhAc and 2,5-diOH-PhAc) always required proteins encoded in genes that are always organized in two different operons: one encoding the proteins required for the hydroxylation of either 3-OH-PhAc or 4-OH-PhAc to 2,5-diOH-PhAc and 3,4-diOH-PhAc, respectively, and a second one containing all the genes required for the conversion of 3,4-diOH-PhAc or 2,5-diOH-PhAc into general catabolites (14, 61). Therefore, because Hmg is released to the broth, there would probably exist an efflux system that would allow the rapid elimination of this compound, and in order to avoid unnecessary loss of intermediate, a system (the same or another) that would permit the recovery of Hmg must also exist.

To confirm the participation of the ABC transport system in the uptake of 3-OH-PhAc, L-Tyr either in the efflux or in the re-uptake of homogentisic acid, the following experiments were performed. The operon involved in the catabolism of Hmg (hmgABC) was deleted in the mutant P. putida U {Delta}hmgG::Tn5 (see Table I). Thus, if the ABC transport system is not involved in the uptake of 3-OH-PhAc or L-Tyr, this strain (P. putida U {Delta}hmgABC{Delta}hmgG::Tn5) should accumulate Hmg when cultured in MM containing 3-OH-PhAc or L-Tyr (as Hmg precursors) and a different carbon source (PhAc, 4-OH-PhAc, succinic acid, or octanoic acid) for supporting bacterial growth. HPLC analyses of the culture broths revealed that the mutant P. putida U {Delta}hmgABC{Delta}hmgG::Tn5 accumulated Hmg in the media whenever 3-OH-PhAc or L-Tyr was added as carbon source. These results indicate that the ABC transport system is not involved in the uptake of these compounds and that it is not responsible for the efflux of Hmg either. If this were not so, the Hmg generated from L-Tyr would not have been accumulated extracellularly. Moreover, when P. putida U {Delta}hmgG::Tn5 (containing a functional Hmg catabolic route) was cultured in MM containing 3-OH-PhAc as the sole carbon source, it did not grow, whereas in the presence of 3-OH-PhAc and an additional carbon source (succinic acid), this mutant accumulated Hmg as well as its pigment derivatives in the medium, suggesting that in this mutant the permease responsible for 3-OH-PhAc uptake and the hydroxylase involved in the synthesis of Hmg (HmgS) would be independent of the mutated ABC transport system. Furthermore, the fact that this mutant was unable to grow in MM supplied with 3-OH-PhAc implies that its transformation into Hmg does not occur intracellularly (as in the case of the Hmg synthesized from L-Tyr), because if this were the case this mutant should be able to grow in MM containing 3-OH-PhAc as the sole carbon source. Additionally, the absence of growth observed when P. putida U {Delta}hmgG::Tn5 was cultured in MM + 3-OH-PhAc reinforces our hypothesis that a functional ABC system (HmgDEFGHI) would be required for the efficient catabolism of the Hmg generated from 3-OH-PhAc.



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  		FIG. 7.
Bacterial growth of a P. putida U mutant affected in the ABC transport system (P. putida U {Delta}hmgG::Tn5) when cultured in MM + 10 mM 3-OH-PhAc ({square}) or in MM + 5 mM L-Tyr ({circ}) (a); and P. putida U (wild type) cultured in MM + 10 mM Hmg ({blacktriangleup}); MM + 10 mM Hmg + 10 mM 3OH-PhAc ({blacksquare}), and MM + 10 mM Hmg + 5 mM L-Tyr () (b).

 
When the P. putida U {Delta}hmgG::Tn5 mutant was cultured in MM + L-Tyr, it grew to one-half of the absorbance attained by the wild-type (P. putida U, see Fig. 7), and it accumulated Hmg in the broth; this did not occur in the wild-type grown under the same conditions. These results indicate that some of the Hmg generated from L-Tyr is catabolized through the Hmg pathway, whereas another part (30%, see above) is released to the medium, and hence the ABC transport system must be required to ensure the re-uptake of the excreted Hmg. Thus, the ABC transport system encoded by the genes hmgDEFGHI seems to be needed for the uptake of Hmg from the medium.

By taking into account that P. putida U was unable to grow in MM containing Hmg and that this bacterium only catabolized this compound when either 3-OH-PhAc or L-Tyr was supplied to the medium (Fig. 7), the following must be assumed: (i) the degradation of Hmg requires the induction of the specific catabolic pathway (HmgABC) as well as the ABC transport system involved in the uptake of this compound (HmgDEFGHI); and (ii) this induction is not caused by extracellular Hmg.

Additional studies revealed the existence of other mutants that were unable to assimilate 3-OH-PhAc but that were also affected (to a lesser extent) in the degradation of other aromatic compounds (PhAc, 4-OH-PhAc, and L-Tyr). Genetic analyses revealed that in these mutants the transposon had been inserted in the genes encoding a two-component regulatory system (mhaSR) integrated by a sensor (MhaS) and a regulator (MhaR) similar to the CbrA-CbrB system that controls the utilization of multiple carbon and nitrogen sources in P. aeruginosa (73). Disruption of either of these two genes in P. putida U (see Table I) handicapped this strain in the catabolism of 3-OH-PhAc and also slowed down the rate of assimilation of its close structural analogues (PhAc, 4-OH-PhAc, and L-Tyr). These results suggest that the two-component regulatory system altered in this kind of mutant would correspond to a general system responsible for controlling the different catabolic pathways involved in the assimilation of several carbon sources, including aromatic molecules. Identification of this system could be important because knowledge of its functional meaning could allow us to pinpoint the basic molecular mechanisms involved in signal transduction in this bacterium. Furthermore, by taking into account that the alteration of this system affected the degradation of 3-OH-PhAc, PhAc, 4-OH-PhAc, and L-Tyr to different extents, its study could perhaps shed some light on the establishment of the regulatory mechanisms that control the hierarchical utilization of different aromatics as carbon sources, as well as the flux of intermediates through the different pathways involved in the degradation of such compounds.

Finally, a different mutant unable to catabolize 3-OH-PhAc was identified. In this strain, insertion of transposon Tn5 took place at the phgdh gene encoding the enzyme phosphoglycerate 3-dehydrogenase (Phgdh) (P. putida U {Delta}phgdh::Tn5, see Table I). Because this enzyme catalyzes the synthesis of phosphoenolpyruvate, and because this compound is used as high energy phosphate intermediate by important active transport systems (i.e. phosphoenolpyruvate:sugar phosphotransferase system) involved in the uptake of different compounds (sugars and hexitols) (74-75), it could be argued that the catabolism of 3-OH-PhAc would require the formation, during its transport, of a phosphoderivative, phosphoenolpyruvate being the phosphate donor.

The existence of different hydroxylases, such as those described here, involved in the synthesis of close aromatic structural analogues could be of special interest for understanding the basic mechanisms involved in the evolution of the catabolic pathways required for the assimilation of such compounds. It could be speculated that the existence of different degradative routes involved in the assimilation of close structural analogues in the same bacterium (P. putida U model) could have forced the acquisition of different genes to encode specific enzymes that would catalyze the first step(s) required for the transformation of a substrate into a product that can be easily and specifically catabolized through a pre-existing pathway (i.e. 3-OH-PhAc through the Hmg route and 4-OH-PhAc through the 3,4-diOH-PhAc pathway). Success in this evolutionary attempt would have led to a more complex metabolic situation (metabolic differentiation) that would have later required the acquisition of new genes (encoding regulators) responsible for establishing the hierarchical organization that controls the metabolic fluxes through these pathways.

However, in the absence (or due to the lost) of a concrete route (i.e. the Hmg pathway, which does not exist in the E. coli model), some bacteria must have modified the substrate specificity of a pre-existing enzyme in order to broaden the number of structural analogues that can be assimilated through the same pathway (i.e. 3-OH-PhAc and 4-OH-PhAc are transformed by the same enzyme to 3,4-diOH-PhAc and degraded through a single pathway). Both strategies seem to be plausible, and by taking into account the models studied (P. putida U and E. coli W), both have been successfully used throughout evolution.

In sum, the identification of the genes and enzymes involved in the transformation of 3-OH-PhAc into homogentisic acid, which has allowed this upper catabolic pathway to be elucidated, is not only important from an academic point of view (the description of a new enzyme and a novel pathway) but also for its important biotechnological and ecological implications.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY929299 [GenBank] , AY929300 [GenBank] , and AY937229 [GenBank] .

* This work was supported in part by the Comisión Interministerial de Ciencia y Tecnología, Madrid, Spain, Grant BIO2003-05309-C04-01, and by a Grant of Excma Diputación de León (2005). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Recipient of a fellowship from the Comisión Interministerial de Ciencia y Tecnología. Back

Recipient of a fellowship from the University of León. Back

** To whom correspondence should be addressed. Tel.: 34-987-291228; Fax: 34-987-291226; E-mail: dbbjlr{at}unileon.es.

1 The abbreviations used are: PhAc, phenylacetic acid; Hmg, homogentisic acid; 3-OH-PhAc, 3-hydroxyphenylacetic acid; 3,4-diOH-PhAc, 3,4-dihydroxyphenylacetic acid; 2,5-diOH-PhAc, 2,5-dihydroxyphenylacetic acid; HPLC, high pressure liquid chromatography; ORF, open reading frame. Back



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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