Metabolism of Ferulic Acid to Vanillin
A BACTERIAL GENE OF THE ENOYL-SCoA HYDRATASE/ISOMERASE SUPERFAMILY ENCODES AN ENZYME FOR THE HYDRATION AND CLEAVAGE OF A HYDROXYCINNAMIC ACID SCoA THIOESTER*

Michael J. Gasson, Yoshie KitamuraDagger , W. Russell McLauchlan§, Arjan Narbad, Adrian J. Parr, E. Lindsay H. Parsons, John Payne, Michael J. C. Rhodes§, and Nicholas J. Waltonpar

From the Departments of Genetics and Microbiology and § Biochemistry, Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

A gene encoding a novel enoyl-SCoA hydratase/lyase enzyme for the hydration and nonoxidative cleavage of feruloyl-SCoA to vanillin and acetyl-SCoA was isolated and characterized from a strain of Pseudomonas fluorescens. Feruloyl-SCoA is the CoASH thioester of ferulic acid (4-hydroxy-3-methoxy-trans-cinnamic acid), an abundant constituent of plant cell walls and a degradation product of lignin. The gene was isolated by a combination of mutant complementation and biochemical approaches, and its function was demonstrated by heterologous expression in Escherichia coli under the control of a T7 RNA polymerase promoter. The gene product is a member of the enoyl-SCoA hydratase/isomerase superfamily.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Cinnamic acids are biologically important and abundant molecules; for example, ferulic acid (4-hydroxy-3-methoxy-trans-cinnamic acid) and 4-coumaric acid (4-hydroxy-trans-cinnamic acid) together represent up to 1.5% by weight of the cell walls of grasses (1). They function in the cross-linking of plant cell walls and are precursors of a variety of antimicrobial compounds, signaling molecules, and phytoalexins that play an important role in plant defense responses (2). They are precursors of lignin in wood (3) and, by contrast, of simple molecules such as vanillin (4-hydroxy-3-methoxybenzaldehyde) and salicylic acid (2-hydroxybenzoic acid). Because they are released during the breakdown of lignin and cell wall material, their catabolism is essential to the biodegradation of plant wastes. There is substantial interest in the potential of ferulic acid and related compounds as feedstocks, for example, for the biotechnological production of vanillin, one of the principal flavoring and aroma compounds in the world (4, 5).

The degradation of these molecules is not well understood. It was proposed (6) that the chain shortening of cinnamic acids might occur via a beta -oxidation process directly analogous to the well known beta -oxidation pathway of fatty acid oxidation. The proposed route for ferulic acid is shown in Fig. 1. Key steps in this scheme are activation to the CoASH thioester, hydration of the enoyl-SCoA to the beta -hydroxy derivative, beta -oxidation to the beta -keto thioester, and thioclastic cleavage to give the corresponding benzoyl-SCoA (vanilloyl-SCoA) together with acetyl-SCoA. The aldehyde, vanillin, would be formed by reduction of vanilloyl-SCoA.


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Fig. 1.   Conversion of ferulic acid to vanillin. The postulated beta -oxidative route is shown as a light line. The non-beta -oxidative route established in P. fluorescens biovar V, strain AN103, by the present work is shown as a heavy line.

Although it is known that an overall two-carbon cleavage of ferulic acid can occur to give vanillin or vanillic acid (4-hydroxy-3-methoxybenzoic acid) together with an acetate unit, the mechanism of such a route, whether in plants or microorganisms, has proved surprisingly elusive, perhaps on account of the assumed low abundance or instability of reaction intermediates (4). In Pseudomonas acidovorans, for example, the overall conversion was demonstrated many years ago (7), but no further investigation of the mechanism was reported subsequently.

In the work reported here, the close relationship of hydroxycinnamate catabolism to the beta -oxidation of fatty acids is fully confirmed and demonstrated. A gene encoding an enoyl-SCoA hydratase/lyase enzyme for the conversion of feruloyl-SCoA to vanillin and acetyl-SCoA was isolated from a Pseudomonas strain able to utilize ferulic acid as sole carbon source,1 and its function was confirmed by heterologous expression in Escherichia coli. The gene product was shown to be a member of the enoyl-SCoA hydratase/isomerase superfamily (9, 10) which includes bacterial, mitochondrial, peroxisomal, and glyoxysomal enzymes of the fatty acid oxidation pathway, together with bacterial dihydroxynaphthoate synthases of vitamin K biosynthesis and 4-chlorobenzoyl-SCoA dehalogenases of Pseudomonas sp. The gene encodes no NAD+ binding domain, and the enzyme does not exhibit beta -oxidation activity.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Chemicals and Strains-- Chemicals were obtained routinely from Sigma-Aldrich Chemical Co. Ltd., Gillingham, Dorset, UK or from BDH-Merck, Poole, Dorset, UK and were of analytical grade. The preparation of CoASH thioesters (feruloyl-SCoA, vanilloyl-SCoA, and 4-hydroxy-3-methoxyphenyl-beta -hydroxypropionyl coenzyme A thioester (HMPHP SCoA)2) is described below. Restriction enzymes were routinely obtained from Promega, Southampton, UK. The isolation of Pseudomonas fluorescens biovar V, strain AN103 by enrichment using ferulic acid as sole carbon source is described elsewhere.1

Preparation of Feruloyl-SCoA and Vanilloyl-SCoA-- These were produced from ferulic acid and vanillic acid, respectively, essentially as described by Semler et al. (11) for piperoyl-SCoA, except that in the case of feruloyl-SCoA, the N-succinimidyl derivative was not isolated before transesterification with CoASH. Final isolation was by preparative TLC using cellulose TLC plates (Avicel; 1,000 µm; Analtech, Newark, DE) developed in n-butanol/acetic acid/H2O (5:2:3, v/v/v) (13) and elution with 50% MeOH.

Preparation of HMPHP SCoA-- HMPHP SCoA was prepared starting from a Reformatsky condensation of vanillin with ethyl bromoacetate (12) followed by purification of the resulting ethyl 4-hydroxy-3-methoxyphenyl-beta -hydroxypropionate (ethyl HMPHP) by HPLC, hydrolysis to the free acid, N-succinimidylation and, finally, exchange of the N-succinimidyl group with CoASH and isolation of the CoASH thioester by preparative TLC.

Vanillin (3 g) was mixed with 1.9 ml of ethyl bromoacetate and 2 g of dry zinc dust in 60 ml of dry 1,4-dioxane. The reaction mixture was heated to boiling using a heating mantle and refluxed gently for about 1 h. After being allowed to cool, the mixture was acidified with 60 ml of 10% H2SO4 and extracted with 4 × 120 ml of Et2O. The combined Et2O phases were dried with anhydrous Na2SO4, and unreacted vanillin was removed by washing with 3 × 100 ml of saturated K2S2O5. The ether phase was then rotary evaporated under vacuum at about 30 °C to remove the ether, leaving a liquid residue (about 10 ml). This was then applied to a preparative C-18 reverse phase HPLC column (Dynamax 60A, 8 µm, 250 × 41 mm; Rainin, Woburn, MA) and eluted at 12 ml min/min with a gradient of MeOH/H2O, containing 1 mM trifluoroacetic acid. Solvent A comprised 40% MeOH and 1 mM trifluoroacetic acid. Solvent B comprised 100% MeOH and 1 mM trifluoroacetic acid. At time t = 0 min, the solvent was 20% B, rising linearly to 40% B at 28 min and 100% B at 35 min. Fractions were monitored by absorbance at 280 nm, and material eluting between 37 and 45 min was collected. The solvent was removed under vacuum at about 35 °C, and the remaining material brought to -20 °C overnight. The precipitate that formed was then filtered off rapidly and freeze-dried to give 300 mg of white substance. This was identified as ethyl HMPHP by mass spectrometry ([M-] = 239).

To generate the N-succinimidyl ester, 30 mg of ethyl HMPHP was first hydrolyzed for 40 min at room temperature in 0.5 ml of M KOH and the pH adjusted to about 3-4 with 0.6 ml of 0.5 M oxalic acid. The solution was extracted successively with Et2O (5 × about 10 ml) and the organic phases pooled and evaporated to dryness. N-Hydroxysuccinimide (0.1 mmol; 11.5 mg) was then added in 1.2 ml of dry 1,4-dioxane. This was followed gradually, with stirring, by 0.1 mmol (20.7 mg) of dicyclohexylcarbodiimide in 0.6 ml of dry 1,4-dioxane. Subsequent steps were essentially those described above for trans-feruloyl-SCoA, based on the procedure of Semler et al. (11). Identification was confirmed by mass spectrometry ([M-] = 960) and by hydrolysis to the free acid (as with ethyl HMPHP).

Growth of P. fluorescens Biovar V, Strain AN103-- Cells were grown routinely at 25 °C on minimal medium, with shaking, using vanillic acid (10 mM) or ferulic acid (10 mM) as the sole carbon source; 50 ml of medium was used in a 250-ml Erlenmeyer flask. Nutrient medium (LB-Mod) was prepared as indicated by Narbad and Gasson.1 Growth was measured routinely by monitoring absorbance at 600 nm.

Preparation and Incubation of Cell-free extracts; Assay of Ferulate:CoASH Ligase-- Cell-free extracts of logarithmic phase cultures (6-10 h after inoculation) were prepared by sonication. Cells from about 200 ml of medium were pelleted by centrifugation and resuspended in 5-10 ml of extraction buffer (routinely 40 mM KPi, pH 7, containing 10 mM dithiothreitol). They were then sonicated (MSE Soniprep 150; Fisons Instruments, Crawley, Sussex, UK) at 4 °C (5 × 20 s; 22 amplitude microns on full power) and centrifuged (20,000 × g for 20 min at 4 °C). For storage, extracts were frozen at -70 °C. In some instances, extracts were buffer changed using a PD-10 column (Pharmacia, Milton Keynes, UK) before use. Protein contents of extracts varied between 0.25 and 1.8 mg/ml.

Extracts were incubated routinely at 30 °C and pH 7.5 in a reaction mixture (1 ml) containing 90 mM Tris-HCl buffer and 2.5 mM MgCl2, together with (as appropriate) 0.5 mM ferulic acid, 0.2 mM CoASH (lithium salt), and 2.5 mM ATP. This complete reaction mixture constituted an assay for ferulate:SCoA ligase, where the initial increase in absorbance at 345 nm was monitored against a blank reaction mixture from which CoASH was omitted. A value for epsilon 345 of 1.9 × 104 M-1 cm-1 was used (13). Incubations with HMPHP SCoA (see below for preparation) were performed similarly, but with the omission of ferulic acid, CoASH, and ATP.

Assay of Vanillin:NAD+ Oxidoreductase-- Vanillin:NAD+ oxidoreductase was assayed at 30 °C and pH 7.0 by monitoring the initial decrease in absorbance at 340 nm against a blank reaction mixture from which NAD+ had been omitted. Because of the similarity in the extinction coefficients of vanillin and NADH, regeneration of NADH to NAD+ was achieved by providing lactate dehydrogenase and pyruvate in the reaction mixture. Reaction mixtures contained, in a 1-ml volume, 75 mM KPi buffer, pH 7.0, 0.125 mM vanillin, 1.2 mM sodium pyruvate, 1.1 unit of lactate dehydrogenase (rabbit muscle; Boehringer, Lewes, Sussex, UK), and 0.5 mM NAD+.

Protein Assay-- Protein was assayed by the method of Bradford (14) using commercially prepared reagent (Bio-Rad Laboratories, Hemel Hempstead, Hertfordshire, UK).

Analysis of Metabolites-- Metabolites, including the components of cell-free reaction mixtures, were routinely analyzed and quantitated by HPLC using a Lichrosorb RP-18 column (20 cm × 4.6 mm; Capital HPLC, Broxburn, West Lothian, UK) with a multiphasic gradient. Solvent A was 20 mM NaOAc, adjusted to pH 6, and solvent B was MeOH. The flow rate was 1.2 ml/min. The proportion of solvent B rose linearly from 0% at 0 min to 10% at 15 min and then to 50% at 40 min and 75% at 45 min, finally decreasing to 0% at 50 min. Detection was with a Spectra Focus detector (ThermoSeparation Products, Stone, Staffordshire, UK) which permitted UV analysis of each eluting component. Typical approximate retention times were: CoASH, 3 min; vanillic acid, 7 min; ferulic acid, 19 min; acetyl-SCoA, 22 min; HMPHP SCoA, 29 min; vanilloyl-SCoA, 31 min; vanillin, 31.5 min; feruloyl-SCoA, 34 min. Quantitation was based on absorbance measurements at 260 nm, using reference compounds as standards; aromatic CoASH thioesters (both as substrates and products) were also quantitated independently by alkaline hydrolysis to the free acids, which were then measured by HPLC.

Mass Spectrometry-- Mass spectra (+-ve and --ve ion) were recorded on a 9/50 mass spectrometer (Kratos Instruments, Manchester, UK) using xenon fast atom bombardment at a potential of 6-8 kV with glycerol as matrix. Liquid chromatography/mass spectrometry of cell-free reaction mixtures was performed on a Platform I instrument (Micromass, Manchester, UK) using positive electrospray ionization and liquid chromatography conditions adapted from the HPLC conditions described above; data were processed using Micromass MassLynx software.

Isolation of Mutants in Ferulate Utilization-- A suspension of vanillate-grown cells (1 ml; 4 × 109 cells/ml in 0.1 M KPi) was incubated with 0.08 ml of ethyl methane sulfonate in a total of 3 ml of 0.1 M KH2PO4 at 37 °C for 45 min. The cells were then precipitated by centrifugation at 4 °C, washed, resuspended, and inoculated into 50 ml of LB-Mod medium. After overnight incubation at 25 °C, the mutagenized cells were enriched for mutants in ferulate utilization by treatment with carbenicillin in minimal medium in the presence of ferulic acid. The cells were harvested by centrifugation at 4 °C and then washed and resuspended in 20 ml of minimal medium. A sample (1 ml) was inoculated into minimal medium (15 ml) and incubated at 25 °C for 1 h; then ferulic acid (10 mM final concentration) and carbenicillin (2 mg/ml final concentration) were added. (A control flask was prepared containing ferulic acid, but not carbenicillin.) The flask was incubated overnight at 25 °C for 16 h, monitoring A580 to confirm the effectiveness of the antibiotic. Penicillinase (10 units) was then added to destroy the carbenicillin, incubating overnight at 25 °C. The cells were harvested by centrifugation at 4 °C, washed twice, and resuspended in 5 ml of minimal medium; 1 ml of these resuspended cells was then inoculated into 50 ml of medium containing 10 mM vanillic acid and incubated at 25 °C for about 24 h. These enriched cells were finally screened by replica plating for mutants unable to use ferulic acid. The enriched stock was diluted, plated onto LB-Mod medium, incubated at 25 °C for 2 days, and then replica plated onto minimal medium containing 10 mM vanillic acid or 10 mM ferulic acid. The plates were incubated at 25 °C for 2-3 days and screened for colonies able to grow on vanillate but unable to grow on ferulate.

Preparation of Genomic Library of P. fluorescens, Biovar V, Strain AN103-- A genomic library was prepared in the broad host range cosmid cloning vector, pLAFR3 (15). Genomic DNA was isolated from strain AN103 and partially digested with Sau3AI at 37 °C for 7-10 min. The DNA was then size fractionated on a NaCl gradient (1.25-5 M). The fraction containing DNA of 20-40 kb was selected and 0.5 µg ligated into the dephosphorylated BamHI site of pLAFR3. Half of the ligation mix was packaged into bacteriophage lambda  particles using a Gigapack II XL kit (Stratagene, Cambridge, UK). The packaged cosmids were transfected into E. coli strain 803 (16). Approximately 10,000 primary transfectants were obtained. The lawn of cells obtained was washed from the selection plates, and glycerol-containing stocks were prepared for storage at -70 °C.

Complementation of Mutants of P. fluorescens, Biovar V, Strain AN103 Defective in Ferulate Utilization-- The genomic library in cosmid pLAFR3 was introduced into mutant strains by conjugation using the helper plasmid, pRK2013 (17). Mutant strains were inoculated into minimal medium containing 10 mM vanillic acid and incubated at 25 °C for 2 days. The E. coli strain carrying the helper plasmid (E. coli 803pRK2013) was inoculated into LB-Mod medium (10 ml) and incubated at 37 °C for 6 h. A sample of the AN103 genomic library was similarly inoculated and incubated. Equal populations of the three organisms were then combined. The mixture of cells was centrifuged, resuspended in a minimal volume of the supernatant solution, and spread over a sterile gridded cellulose nitrate membrane filter (47-mm diameter, Whatman, Maidstone, Kent, UK) on a moist LB-Mod agar plate. The suspension was allowed to air dry onto the filter for a few minutes and then incubated overnight at 25 °C. The bacteria were washed from the filter, and aliquots (0.1 ml) were applied to selection plates of minimal medium with 10 mM vanillic acid and 5 µg/ml of tetracycline. These were incubated at 25 °C for 2 days and the colonies obtained (>1,000/plate) were replica plated to similar plates containing ferulic acid in place of vanillic acid. Colonies (two to three/plate) able to grow on these plates containing ferulic acid were selected and inoculated into fresh minimal medium containing 5 µg/ml tetracycline.

SDS-PAGE of Cell-free Extracts and Electroelution of Protein Bands-- SDS-PAGE, with Coomassie staining, was carried out essentially as described by Schägger and von Jagow (18), using an Atto AE6450 gel electrophoresis apparatus (supplied by Genetic Research Instrumentation, Dunmow, Essex, UK). Electroelution of protein bands from fixed, stained gels was performed using a Bio-Rad model 422 electroeluter according to the manufacturer's instructions. Eluted protein was then deposited by centrifugation onto a Pro-Spin membrane (Applied Biosystems, Foster City, CA) used as directed by the manufacturer.

NH2-terminal Sequencing-- Protein NH2-terminal sequencing was undertaken under contract by Alta Bioscience, Department of Biochemistry, University of Birmingham, Birmingham, UK.

Isolation, Digestion, and Probing of Cosmid DNA-- Cosmid DNA was isolated using Qiagen minicolumns (Qiagen, Crawley, West Sussex, UK) according to the manufacturer's instructions and was digested with restriction endonucleases EcoRI and PstI. Fragments were separated by agarose gel electrophoresis and Southern blotted to a Hybond-N filter (Amersham International, Aylesbury, Buckinghamshire, UK) as described elsewhere (19). Probe DNA was linearized, denatured, and labeled with digoxygenin as described by the kit manufacturer (Boehringer).

Polymerase Chain Reaction (PCR)-- Oligonucleotide primers were prepared using an ABI 394 Synthesizer (Perkin-Elmer, Warrington, UK). For PCR, Perkin-Elmer UltmaTM DNA polymerase was used with a "hot start" according to the manufacturer's directions; a Hybaid OmnigeneTM instrument (Teddington, Middlesex, UK) was used, with the following thermal cycling conditions: for amplification using the degenerate primers based on the NH2-terminal sequence of the 31-kDa protein (see Fig. 5), there was one cycle of a 2-min denaturation at 95 °C followed by 25 cycles, each consisting of a 2-min denaturation at 95 °C, 2-min annealing at 50 °C, and 2-min extension at 72 °C, and finally, one cycle of a 7-min extension at 72 °C. For the amplification of open reading frame (ORF) A, there was one cycle of a 2-min denaturation at 96 °C followed by five cycles, each consisting of a 2-min denaturation at 96 °C, 2-min annealing at 47 °C, and 2-min extension at 72 °C, then 20 cycles, each consisting of a 2-min denaturation at 96 °C, 2-min annealing at 60 °C, and 2-min extension at 72 °C, and finally, one cycle of a 5-min extension at 72 °C. The following primers were used for the amplification of ORF A before cloning in the E. coli expression vector, pSP72 (Promega): NH2-terminal, 5'-TATATAGAATTCAAAACCCAGAACAAGA-3', incorporating a synthetic EcoRI site; COOH-terminal, 5'-ATATATGGATCCTTCAGCGTTTATACG-3', incorporating a synthetic BamHI site. For amplification of ORF A before cloning in the vector pRK415 (20), the NH2-terminal primer used was 5'-TATATAAGCTTGGCCGGTGATTGC-3', incorporating a synthetic HindIII site; this was homologous to a sequence CGACCTTGGCCGGTGATTGCTACGGCCAATATCGCTCGGC running from -1 to -40 bp upstream of the start of the sequence shown in Fig. 5.

DNA Sequencing-- Cosmid restriction fragments for sequencing were subcloned into the E. coli vector pUC19 (Pharmacia), using strain XLI (Blue) (Stratagene). Automated fluorescent sequencing by the Sanger dideoxy termination method (21) was carried out by means of an Applied Biosystems DNA Sequencer (model 373; Perkin-Elmer) together with the manufacturer's Taq DyeDeoxy Terminator Cycle sequencing kit, used according to the manufacturer's instructions. A primer walking strategy was employed.

Sequence Homologies-- Homologies of nucleotide sequences and their translation products with sequences available in the data libraries were determined using the Wisconsin Package, Version 8, September 1994, Genetics Computer Group, Madison WI and EGCG Package, Peter Rice, The Sanger Center, Hinxton, Cambridge, UK.

Nucleotide Sequence Accession and Strain Deposit-- The nucleotide sequence data reported in this paper have been submitted to the EMBL, GenBankTM, and DDBJ nucleotide sequence data bases under accession number Y13067. P. fluorescens biovar V, strain AN103, has been deposited with the National Collection of Industrial and Marine Bacteria, Aberdeen, UK under accession no. NCIMB 40783 as required under the Budapest Treaty governing deposits of organisms in connection with patent applications (8, 22).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Conversion of Ferulate in Cell-free Extracts of P. fluorescens Biovar V, Strain AN103-- It was shown previously1 that the metabolism of ferulate by extracts of cells of strain AN103 grown in the presence of ferulate required CoASH, ATP, and Mg2+ ions. The reaction products were feruloyl-SCoA and equimolar quantities of vanillin and acetyl-SCoA (Fig. 2A). Because there was no requirement for NAD+ and because vanilloyl-SCoA (0.5 mM) was found not to be converted to vanillin by these extracts even in the presence of NADH (0.5 mM), the cleavage reaction appeared to proceed without beta -oxidation (see Fig. 1).


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Fig. 2.   Formation of (panel A) feruloyl-SCoA (bullet ), vanillin (open circle ), and acetyl-SCoA (×) from ferulate and of (panel B) vanillin (open circle ), acetyl-SCoA (×), and feruloyl-SCoA (bullet ) from HMPHP SCoA (black-square) by a cell-free extract of P. fluorescens biovar V, strain AN103. Cells were grown on 10 mM ferulate as the carbon source; extract was passed through a PD-10 column (Pharmacia) before use and incubated (7 µg of protein/reaction mixture) in the presence of (panel A) 0.5 mM ferulate, 0.2 mM CoASH, 2.5 mM MgCl2, and 2.5 mM ATP or (panel B) 0.4 mM HMPHP SCoA and 2.5 mM MgCl2, for the time periods indicated, as described under "Experimental Procedures."

Extracts also converted the hydrated derivative of feruloyl-SCoA, HMPHP SCoA. This too was converted to vanillin and acetyl-SCoA in equimolar quantities without a requirement for NAD+ (Fig. 2B); and in addition it was dehydrated by the extract to feruloyl-SCoA, demonstrating the reverse of the enoyl-SCoA hydratase reaction (see Fig. 1). (The feruloyl-SCoA so formed was assumed to be the trans-isomer, but cis- and trans-isomers were not distinguished by the HPLC analysis used.) The reaction was relatively rapid, and greater than 90% of the substrate was converted to products.

Vanillin formation was therefore independent of added NAD+; however, as indicated previously,1 when NAD+ was provided in the reaction mixture, much of the vanillin was oxidized to vanillate by the activity of vanillin:NAD+ oxidoreductase present in the extracts.

Isolation and Complementation of Mutants in Ferulate Utilization-- A classical mutation and complementation strategy was used to isolate the gene(s) responsible for ferulate cleavage. Mutants of P. fluorescens AN103 which were unable to utilize ferulate as sole carbon source were isolated, and their defect was then complemented by the introduction of cosmid clones from a genomic library of the same strain. Chemical mutagenesis of P. fluorescens AN103 using ethyl methane sulfonate was followed with enrichment and screening for mutants unable to grow on ferulate. These mutants all retained the ability to grow on vanillate and fell into two biochemical classes distinguished by the activities of ferulate:CoASH ligase and vanillin:NAD+ oxidoreductase. As shown in Table I, class I mutants (typified by van 1) had severely reduced activities of both ferulate:CoASH ligase and vanillin:NAD+ oxidoreductase, whereas class II mutants (typified by van 10) had wild-type levels of both enzymes. Despite the presence of these enzyme activities, cell-free extracts of the class II mutants were impaired in the ability to form vanillate from ferulate in the presence of ATP, Mg2+, CoASH, and NAD+. This suggested that the class II mutants were defective in a structural gene for vanillin formation from feruloyl-SCoA. The absence of two distinct enzyme activities in the class I mutants suggested that they may be defective in a common gene control process.

                              
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Table I
Metabolism of ferulate in cell-free extracts of P. fluorescens biovar V, strain AN103, and of mutant strains van 1 and van 10 
Data were obtained using extracts from cells grown in medium containing 10 mM ferulate plus 10 mM vanillate. Preliminary experiments showed that the induction of ferulate:CoASH ligase in strain AN103 in response to 10 mM ferulate in the growth medium was unaffected by the simultaneous presence of 10 mM vanillate. Extracts were prepared and assayed for ferulate:CoASH ligase and vanillin:NAD+ oxidoreductase as described under "Experimental Procedures." Extracts (about 300 µg of protein) were also incubated for 4 h in the presence of CoASH, Mg2+, ATP, and NAD+ (see "Experimental Procedures,") and the production of vanillate determined.

A class II mutant was used for complementation experiments designed to isolate a wild-type copy of the mutant gene. A gene library of P. fluorescens AN103 DNA was constructed using the broad host range cosmid cloning vector pLAFR3 in E. coli. Cosmid clones were introduced from the E. coli library into a class II mutant derivative of P. fluorescens AN103 by triparental mating using an E. coli strain carrying the helper plasmid pRK2013. Two cosmids, pFI793 and pFI794, were identified as complementing the class II mutants.

Identification of a 31-kDa Polypeptide Induced by Ferulate-- The protein profiles of P. fluorescens AN103 following growth on ferulate and vanillate were compared using SDS-PAGE with Coomassie Blue staining. As shown in Fig. 3, the ferulate-grown cells produced a strongly enhanced or additional band corresponding to a polypeptide of 31 kDa. It was likely that this polypeptide was an enzyme involved in the conversion of ferulate to vanillin. Accordingly, the 31-kDa band was excised from the fixed and stained polyacrylamide gel, electroeluted, and subjected to NH2-terminal sequencing. The following sequence was determined: Ser-Thr-Tyr-Glu-Gly-Arg-Trp-Lys-Thr-ValLys-Val-Glu-Ile-Gln-Asp-Gly-Ile-Ala-Phe.


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Fig. 3.   Electrophoretic comparison of crude extracts of P. fluorescens biovar V, strain AN103, from cells grown on 10 mM ferulate (F) and 10 mM vanillate (V), respectively, as sole carbon source. SDS-PAGE followed by Coomassie staining for protein was carried out as described under "Experimental Procedures." Marker proteins were phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), hen egg white ovalbumin (45.5 kDa), bovine carbonic anhydrase (31.0 kDa), soybean trypsin inhibitor (21.5 kDa), and hen egg white lysozyme (14.4 kDa).

Identification and Cloning of the Gene for a Ferulate-induced Polypeptide-- The NH2-terminal sequence of the ferulate-induced 31-kDa polypeptide was used to design degenerate oligonucleotide primers (Fig. 4) which were shown to PCR amplify a 60-bp sequence of DNA from the equivalent P. fluorescens AN103 gene. The amplified fragment was used as a probe to locate this gene on EcoRI/PstI digests of the two cosmids pFI793 and pFI794. The fragments identified were 6 kb and 4.3 kb, respectively, and the latter smaller fragment was targeted for subcloning and sequence determination.


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Fig. 4.   Degenerate oligonucleotide primers designed from the NH2-terminal amino acid sequence of the ferulate-induced 31-kDa polypeptide.

Open Reading Frame of the 31-Kb Polypeptide-- The sequence of part of the 4.3-kb fragment of cosmid pFI794 is shown in Fig. 5. An open reading frame ORF A (831 bp), corresponding to the ferulate-induced polypeptide and encoding the NH2 terminus determined previously, commenced at position 126 and was terminated by a stop codon at position 954; it encoded a polypeptide of 276 amino acids, with a molecular mass of 31.010 kDa, in good agreement with the SDS-PAGE data (see Fig. 3). A native ribosome binding site (GAGA) could be identified at -9 bp and inverted repeats at -37 bp, -64 bp, and -83 bp.


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Fig. 5.   Nucleotide sequence of part of the 4.3-kb fragment of cosmid pFI794, showing ORF A. The enoyl-SCoA hydratase signature sequence (es), inverted repeat regions (ir), and ribosome binding site (r) are shown. ORF 2 indicates the start of the open reading frame ORF2 reported by Priefert et al. (23).

A second open reading frame (ORF B) was found to begin at position 1058 and was terminated by a stop codon beginning at position 2504 (data not shown). This open reading frame of 1,449 bp encoded a derived polypeptide sequence of 482 amino acids and was preceded by a native ribosome binding site (GAGG) at -8 bp.

Expression of ORF A in E. coli and Demonstration of the Encoded Enzyme Activity-- To determine the function of ORF A and to assay its gene product, ORF A was amplified by PCR, cloned into an E. coli expression vector, and then transformed into E. coli. From the sequence of ORF A, PCR primers (see "Experimental Procedures") were designed to amplify the gene such that it was flanked by restriction endonuclease sites EcoRI and BamHI. The amplified gene retained its native ribosome binding site, being initiated at base -29 and ending 6 bp downstream of the stop codon. The amplified fragment was cloned into the equivalent sites of the E. coli expression vector pSP72 (under the control of the bacteriophage T7 RNA polymerase promoter) to produce pFI1039, which was transformed into E. coli JM109(DE3) (Promega). Cells of the E. coli strain containing the vector pSP72 alone, designated JP1, and of the strain containing pFI1039, designated JP2, were each grown in the presence and absence of the inducer, isopropyl-beta -D-thiogalactoside, and then extracted and assayed for activity with feruloyl-SCoA and HMPHP SCoA, determining the reaction products by HPLC. For comparison, a crude extract of strain AN103 was assayed under the same conditions.

As indicated previously in Fig. 2, extracts of strain AN103 grown in the presence of ferulate were shown to convert both feruloyl-SCoA and HMPHP SCoA to vanillin and acetyl-SCoA in equimolar proportions. HMPHP SCoA was in addition dehydrated to feruloyl-SCoA. Table II shows that the same reaction products in strikingly similar ratios were also produced by extracts of E. coli strain JP2 provided with these substrates. Activity in E. coli strain JP2 was found to be independent of the inducer, isopropyl-beta -D-thiogalactoside, and indeed the specific activity in the extract made after induction was slightly lower than in the extract prepared from uninduced bacteria. (It is possible that increased protein expression occurred upon induction but resulted in the production of incorrectly folded or inactive enzyme; this could not be assessed by SDS-PAGE because of coelectrophoresis of the 31-kDa beta -lactamase polypeptide responsible for ampicillin resistance.) No activity could be demonstrated in extracts of the unmanipulated E. coli strain JP1 (data not shown), demonstrating unequivocally that both the enoyl-SCoA hydratase activity and the subsequent lyase activity were encoded by ORF A. 

                              
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Table II
Expression of feruloyl-SCoA hydratase/lyase in E. coli
Extracts were prepared and activities determined with feruloyl-SCoA and HMPHP SCoA as substrates (0.28 mM and 0.40 mM, respectively), using HPLC to determine the reaction products, as described under "Experimental Procedures." Reaction mixtures contained about 10 µg of extract protein. For comparison, the activity with each substrate was determined for an extract of P. fluorescens biovar V, strain AN103 (13 µg of extract protein).

Ability of ORF A to Complement Pseudomonas Mutant van 10-- As shown in Table I, class II mutant strains were deduced to be defective in the conversion of feruloyl-SCoA to vanillin, because although they showed wild-type activities of ferulate:CoASH ligase and vanillin:NAD+ oxidoreductase, the overall conversion of ferulate to vanillate in the presence of the required cosubstrates (CoASH, ATP, Mg2+, and NAD+) was severely impaired. It was therefore predictable that ORF A would be capable of complementing these strains, restoring their ability to catabolize ferulate and to utilize this compound as a sole carbon source. To test this hypothesis, ORF A plus its putative promoter sequence, TTGACAT (bp 59-65 in Fig. 5), was first PCR amplified and cloned into the tetracycline-resistant vector pRK415 (20) to give pFI2046. E. coli clones containing this plasmid were then triparentally mated with the class II mutant, van 10, essentially as described above in relation to complementation by the cosmid clones pFI793 and pFI794. The complemented mutant recovered the ability to grow on ferulic acid as anticipated, and furthermore extracts of the complemented mutant showed wild-type ability to produce vanillate and acetyl-SCoA from ferulate in the presence of CoASH, ATP, Mg2+, and NAD+ (data not shown).

DNA Sequence Analysis-- ORF A was found to show homology to an open reading frame of unknown function, ORF2, of another Pseudomonas species, designated HR199 (23). The deduced translation product of ORF2 was 90% identical with the corresponding translated sequence of ORF A but was 32 residues shorter at the NH2 terminus. ORF B, commencing downstream of ORF A at position 1058, was homologous with vdh, a gene downstream of ORF2 and encoding vanillin:NAD+ oxidoreductase (23). The deduced amino acid sequences of vdh and ORF B showed 81% identity (not shown), and ORF B has been confirmed directly by heterologous expression to encode vanillin:NAD+ oxidoreductase.3

The alignment between the deduced amino acid sequence of ORF A and the translated sequences of five other genes encoding enzymes known to catalyze related reactions of CoASH thioesters is depicted in Fig. 6. These genes are: the fadB gene of E. coli, encoding the large alpha -subunit of the fatty acid oxidation complex (24); the faoA gene of Pseudomonas fragi, encoding the corresponding protein of this organism (25); the menB gene of E. coli, encoding dihydroxynaphthoate synthase (26); the gene of Pseudomonas sp. CBS-3 which encodes 4-chlorobenzoyl-SCoA dehalogenase (27); and mvab, from Pseudomonas mevalonii, which encodes 3-hydroxymethylglutaryl SCoA lyase (28). Residues conserved in four or more of these sequences are shown together with conservative substitutions at the same positions. Approximately 25-30% sequence identity was found between ORF A and each of these sequences, with the exception of the translated sequence of mvab, which was only about 19% identical.


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Fig. 6.   Sequence alignment of the translated sequences of ORF A and members of the enoyl-SCoA hydratase/isomerase superfamily. Fadb_Ecoli, E. coli fadB gene (to residue 285) (24); Faob_Psefr, P. fragi fao gene (to residue 286) (25); Orfa, ORF A; Menb_Ecoli, E. coli menB gene (26); 4-Cba-Scoa-Dehal, Pseudomonas sp. CBS-3 gene for 4-chlorobenzoate dehalogenase (27); Pmmvab, P. mevalonii mvab gene (28). Residues conserved in four or more sequences are shown in reverse type; conservative substitutions are shaded. The translated sequence of ORF2 (23) differs principally from that of ORF A in being 32 residues shorter at the NH2 terminus.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The results presented here characterize the pathway for cleavage of a hydroxycinnamic acid. It is shown that the hydration and cleavage reactions of the CoASH thioester of ferulic acid are catalyzed by an enzyme encoded by a single gene of 828 bp. The products of the reaction are vanillin and acetyl-SCoA. The catalysis of both reactions by a single enzyme was shown by heterologous expression of the gene in E. coli, with demonstration of the formation of vanillin and acetyl-SCoA when feruloyl-SCoA was supplied to cell-free extracts. The conclusions were further supported by the demonstration of the formation of three reaction products, feruloyl-SCoA, vanillin, and acetyl-SCoA, when the putative hydrated reaction intermediate, HMPHP SCoA, was supplied. This gene and its encoded enzyme offer important new possibilities for the biotechnological production of vanillin (4, 5, 8, 22).

Recently Priefert et al. (23) described an ORF of unknown function in Pseudomonas sp. HR199 which exhibits strong homology with ORF A. This gene, ORF2, is shorter than the P. fluorescens AN103 ORF A, lacking 32 codons at the 5'-end as well as the putative ribosome binding site and promoter. It remains to be seen whether the difference in length and the small number of nonconservative substitutions, most notably at ORF A residues 166 (Gln to Glu), 184 (Glu to Lys), 190 (Glu to Gln), and 250 (Thr to Pro), influence or modify enzyme activity. Pseudomonas sp. HR199 was isolated from soil on the basis of its ability to metabolize eugenol (29). The route proposed in outline for the metabolism of eugenol to vanillate envisages coniferyl alcohol, coniferyl aldehyde, ferulate, and vanillin as successive intermediates (29-31). Although ferulate is almost certainly an intermediate in this pathway, the reaction sequence from eugenol to vanillin requires further clarification, and the enzyme activities responsible have not been identified.

It is clear that the proteins encoded by ORF A and ORF2 are additional members of the superfamily that includes enoyl-SCoA hydratases of fatty acid beta -oxidation, together with a number of other proteins that catalyze, or are assumed to catalyze, related reactions of CoASH thioesters (9, 10). A compilation of 33 available sequences belonging to this superfamily, across a strongly conserved alpha -helical region, has recently been made (10). The equivalent region of the derived amino acid sequence of ORF A stretches from Gly114 to Met172; within this region, the derived sequences of ORF A and ORF2 are almost identical, differing in only one residue, at Gln166. It is known from site-directed mutagenesis studies that a conserved Glu residue functions as a catalytic base in the reactions catalyzed by mitochondrial enoyl-SCoA hydratase and mitochondrial Delta 3-cis-Delta 2-trans-enoyl-SCoA isomerase (9, 32, 33) and by the enoyl-SCoA hydratase activity of the multifunctional fatty acid oxidation large alpha -subunit of E. coli (34). This residue appears conserved in all members of the superfamily with enoyl-SCoA hydratase or Delta 3-cis-Delta 2-trans-enoyl-SCoA isomerase activity, and it is present in the derived amino acid sequences of ORF A and ORF2, at residue 143 and 111, respectively. A second Glu residue functions as a general acid-base catalyst for the activation of the nucleophilic water required in the enoyl-SCoA hydratase reaction and appears conserved in the sequences of proteins that possess enoyl-SCoA hydratase activity, but it is absent from the sequences of those that do not, including the mitochondrial Delta 3-cis-Delta 2-trans-enoyl-SCoA isomerases (10, 35). This residue is not conserved in the translated sequences of ORF A and ORF2, where it is replaced by a Ser residue, at positions 123 and 91, respectively. In this respect, therefore, this pair of sequences appears unique among enoyl-SCoA hydratases. In most other respects, however, the highly conserved residues characteristic of the superfamily are present in the translated sequences of ORF A and ORF2; these include an Asp residue at position 129, a Gly at 140, a Gly at 147, a Pro at 150, a Gly at 151, and a Gly at 163 (numbering of ORF A). Interestingly, however, residue 160 is Asp, rather than the Lys or Arg found in almost all other members of the superfamily, and residue 169 is a Tyr, whereas in most other sequences this is Glu or Asp and very rarely a nonpolar residue (10).

Multifunctional fatty acid oxidation proteins of the enoyl-SCoA hydratase superfamily, including the mitochondrial (36, 37), peroxisomal (38, 39) and plant glyoxysomal (40, 41) multifunctional proteins, the large alpha -subunit encoded by the fadB gene of E. coli (24, 42), and the corresponding protein encoded by the fao gene of P. fragi (25), all possess L-3-hydroxyacyl dehydrogenase activity. This is responsible for the beta -oxidation reaction to the corresponding 3-oxo acid. This activity is associated not with the NH2-terminal region, where enoyl-SCoA hydratase activity resides, but with a separate domain, which includes an NAD+ binding site. This domain and the sequence motif associated with NAD+ binding are not present in the shorter sequences of ORF A (276 residues) and ORF2, and hence this provides additional proof that the pathway in P. fluorescens biovar V, strain AN103 is non-beta -oxidative (see Fig. 1). Further evidence is provided by the close proximity of ORF A to ORF B, encoding vanillin:NAD+ oxidoreductase and by the fact that ORF A and ORF B are cotranscribed.

The enzyme encoded by ORF A possesses both feruloyl-SCoA hydratase and HMPHP SCoA lyase activities. This appears to be the first enzyme of the superfamily with a lyase activity. The closest formal analogy is with 3-hydroxy-3-methylglutaryl SCoA lyase, an enzyme that catalyzes a reverse Claisen ester condensation to acetoacetate and acetyl-SCoA (43). The gene, mvab, encoding this enzyme in P. mevalonii has been isolated (28). As shown in Fig. 6, this shows very little homology to ORF A and members of the enoyl-SCoA hydratase/isomerase superfamily and appears not to belong to this gene family. It is therefore not possible to use this sequence to provide indications of the residues of ORF A which might be associated particularly with the lyase activity.

The proteins encoded by fadB (44) and fao (25) possess a 3-hydroxyacyl epimerase activity. It remains to be investigated whether the enoyl-SCoA hydratase/lyase enzyme encoded by ORF A might also possess such an activity. This would explain the almost complete utilization of HMPHP SCoA (Fig. 2B), which was initially surprising since it was assumed that this intermediate would be a mixture of L- and D-isomers. The isomeric composition of this substrate has not been determined, however, and further work is clearly required to investigate this question.

It is probable that homologous genes and enzymes may be found elsewhere in the microbial and plant kingdoms. In plants, both CoASH-dependent and CoASH-independent mechanisms have been proposed for the conversion of 4-coumarate to 4-hydroxybenzoate (45-50); in particular, in extracts of cell cultures of Lithospermum erythrorhizon, both a non-CoASH-dependent mechanism via 4-hydroxybenzaldehyde (48) and a CoASH-dependent, beta -oxidative mechanism via 4-hydroxybenzoyl-SCoA (50) have been reported recently. The bacterial mechanism described here is of a third type, CoASH-dependent yet non-beta -oxidative. It will therefore be particularly interesting to discover whether this mechanism can also be demonstrated in plants.

    ACKNOWLEDGEMENTS

We thank John Eagles and Fred Mellon for performing the mass spectrometric analyses, Hugh Griffin for designing degenerate oligonucleotide primers, and Paul Needs (Institute of Food Research) and Jack Woolley (De Montfort University, Leicester, UK) for helpful discussions.

    FOOTNOTES

* This work was supported in part by the Biotechnology and Biological Sciences Research Council Competitive Strategic Grant to the Institute of Food Research. The names of authors appear in alphabetical sequence.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) Y13067.

Dagger Supported by a sabbatical leave grant from the Ministry of Science, Education and Culture of Japan. Permanent address: School of Pharmaceutical Sciences, Nagasaki University, Nagasaki 852, Japan.

Present address: Celsis Limited, Cambridge Science Park, Cambridge CB4 4FX, United Kingdom

par To whom correspondence should be addressed. Fax: 44-1603-507-723; E-mail: nicholas.walton{at}BBSRC.ac.uk.

1 Narbad, A., and Gasson, M. J. (1998) Microbiology, in press.

2 The abbreviations used are: HMPHP SCoA, 4-hydroxy-3-methoxyphenyl-beta -hydroxypropionyl coenzyme A thioester; HPLC, high performance liquid chromatography; kb, kilobase(s); PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; bp, base pair(s).

3 J. Payne, unpublished observations.

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Abstract
Introduction
Procedures
Results
Discussion
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