An endogenous factor enhances ferulic acid decarboxylation catalyzed by phenolic acid decarboxylase from Candida guilliermondii

Materials

FA, CA, 4VG, and 6H2N were purchased from Wako Pure Chemical (Osaka, Japan). PCA was from MP Biomedicals (Solon, OH), and 4VP was from Sigma-Aldrich (Steinheim, Germany). All other chemicals used were of analytical grade.

Microorganisms and propagation

The source of PAD and its gene was C. guilliermondii (M. guilliermondii) ATCC 9058. The enzyme was induced aerobically by 6H2N (1 mM) in Yeast Nitrogen Base (YNB; Invitrogen, Carlsbad, CA) broth containing 0.5% glucose as described [Huang et al. 2011]. Briefly, the yeast was grown at 25°C for 1 d, with shaking, in 200-ml portions of the medium placed in 2-l flasks. E. coli DH5α (Takara Bio, Otsu, Japan) and E. coli BL21 (DE3) (Takara Bio) were used for plasmid preparation and sequencing and for expression and purification of recombinant CgPAD, respectively. The transformed E. coli cells were grown, with shaking, at 37°C in 50-ml portions of Luria-Bertani broth plus ampicillin (100 μg ml^-1) placed in 500-ml flasks to an A₆₀₀ of 0.5. After adding isopropyl β-D-galactosyl pyranoside (0.1 mM) to the culture, incubations were further continued at 18°C for 24 h. After cells were collected by centrifugation (12,000 × g for 10 min) at 4°C, cell pastes obtained from 600-ml culture were used as the starting materials for enzyme purification.

Purification of native and recombinant forms of CgPAD

Enzyme purification was done at a temperature not exceeding 4°C. The native CgPAD in C. guilliermondii was purified by successive column chromatographies on CM Toyopearl 650 M (Tosoh, Tokyo, Japan), DEAE Toyopearl 650 M (Tosoh), and Bio-Gel P-100 (Bio-Rad, Hercules, CA) columns, as described previously [Huang et al. 2011].

The wild-type and mutant recombinant enzymes highly expressed in E. coli cells were each purified by essentially the same procedure as that of the native enzyme [Huang et al. 2011]. The recombinant E. coli cells were washed twice with saline and then suspended in two volumes of the extraction buffer [20 mM sodium phosphate buffer (pH 7.0) plus 1 mM each of phenylmethanesulfonyl fluoride, MgCl₂, EDTA, and DTT]. The cells were disrupted six times for 50 s each with glass beads (0.5 mm in diameter) at 2,500 rpm in a homogenizer (Multi-Beads Shocker; Yasui Kikai, Osaka, Japan). After cell debris was removed by centrifugation (12,000 × g, 15 min), the supernatant obtained was applied directly to a column of DEAE Toyopearl 650 M (2.5 cm × 25.5 cm) previously equilibrated with 20 mM 2-morpholinoethanesulfic acid/NaOH (MES) buffer (pH 6.5). The column was initially washed with 200 ml of 50 mM NaCl in MES buffer (pH 6.5), and proteins were eluted with a 300-ml linear gradient of 50 mM to 0.5 M NaCl in the buffer. The active fractions were immediately concentrated and exchanged with 50 mM phosphate buffer (pH 7.0) by ultrafiltration (Amicon Ultra-15; Millipore, Billerica, MA) to a small volume. The concentrate was then put on a column of Bio-Gel P-100 (1.0 cm × 43 cm) equilibrated with 50 mM sodium phosphate buffer (pH 7.0) and eluted with the equilibration buffer. The active fractions were combined and concentrated by ultrafiltration, and the concentrate was stored at -20°C until use. Highly purified wild-type and mutant recombinant enzymes were obtained approximately 2- to 5-folds with yields of 40-70% within 2 d by the simple purification procedure as judged by SDS-acrylamide gel electrophoresis (see Additional file 2).

Assay of CgPAD activity

The enzyme assay method was essentially the same as described previously [Huang et al. 2011]. The initial velocity of decarboxylation activity was measured at 25°C with 4-hydroxycinnamic acid as substrate. The reaction mixture contained the suitably-diluted enzyme solution and a 5 mM substrate (neutralized with 1.0 N NaOH) in 0.1 M sodium phosphate buffer (pH 6.0) in a final volume of 1.0 ml. After the reactions were terminated by boiling for 10 min, the products formed were quantified by high-performance liquid chromatography (HPLC) using a packed column for reversed phase chromatography (Cosmosil 5C18-MS-II, 4.6 mm × 150 mm; Nacalai Tesque, Tokyo, Japan) with acetonitrile/0.05% phosphoric acid (7:3, v/v) as the mobile phase at a flow rate of 0.6 ml min^-1. One U of enzyme activity was defined as the amount of enzyme that released 1 μmol of 4VG or 4VP per min. Because the product, 4-vinylcatechol, from CA was not commercially available, the CA decarboxylation activity was expressed as formation of 4VG. Protein concentrations were measured using a BCA protein assay kit (Thermo Fisher Scientific, Rockville, MD) with bovine serum albumin as the standard.

Sequencing of internal amino acid residues of CgPAD

Initially, peptides of native CgPAD were obtained by treatment with CNBr or Staphylococcus aureus V8 protease. The CNBr cleavage was done essentially by the method of [Steers et al. (1965)]. One mg of CgPAD was dissolved in 0.2 ml of 70% formic acid and cleaved with an excess of CNBr at room temperature for 24 h. After the remaining CNBr was removed by a rotary evaporator, the reaction mixture was filtered on a column of TSK gel G2000SWXL (Tosoh, 0.78 cm × 30 cm) in 30% acetic acid. The digestion of CgPAD with V8 protease was performed at 37°C for 6 h in 50 mM ammonium bicarbonate buffer (pH 7.8) plus 4 M urea and 2 mM EDTA. The peptide fragments obtained were fractionated by reverse-phase high-performance liquid chromatography on a C4 column (3.9 mm × 150 mm, Waters, Milford, MA). The amino acid sequences of peptides derived from CNBr or V8 protease digestion of native CgPAD were determined by automated sequential Edman degradation using a PPSQ-23A protein sequencing system (Shimadzu, Kyoto, Japan).

Cloning and sequencing of CgPAD gene

All primers used are presented in Additional file 3. By a reverse transcription polymerase chain reaction using appropriate degenerate primers, the internal cDNA fragments of CgPAD were sequenced. Total RNA was isolated from C. guilliermondii using an RNeasy kit (Qiagen, Valencia, CA). First-strand cDNA synthesis was performed with 5 μg of total RNA using a GeneRacer kit (Invitrogen, Carlsbad, CA) with oligo(dT) adaptor primer. The cDNA obtained was used as a template for PCR amplification with degenerate primers. The first PCR was performed with primers P1 (a forward primer designed from an internal LKNKHFQYTYDNG WKYEFHV) and P2 (a reverse primer designed from an internal AFSQGHWEHPEQAHGDK RED), and nested PCR was done with P1 and P3 (a reverse primer designed from an internal AFSQGHWEHPE QAHGDKRED) (the sequences used for primer design are underlined). The nested PCR product was then cloned into a pGEM-T vector (Promega, Madison, WI) and sequenced using the ABI Prism system (Model 310; Applied Biosystems, Foster City, CA).

To obtain the entire gene for CgPAD, both 5' and 3' rapid amplification of cDNA ends (RACE) were performed using a GeneRacer kit according to the manufacturer's instructions. The gene-specific primers P4 (first PCR) and P5 (nested PCR) were used for the 5'-RACE and the gene-specific primers P6 (first PCR) and P7 (nested PCR) for the 3'-RACE. Approximately 250 bp and 290 bp were amplified by 5'-RACE and 3'-RACE, respectively. Finally, a cDNA fragment containing the entire coding region of CgPAD cDNA was amplified using the forward primer P8 (designed from the 5'-RACE product) and reverse primer P9 (designed from the 3'-RACE product).

The nucleotide sequence of CgPAD was submitted to DDBJ under the accession number AB663499

Site-directed mutagenesis

The entire CgPAD gene was amplified by PCR and cloned into the Nde I/Hin dIII site of pET-22b (+) (Novagen, Darmstadt, Germany), yielding the construct designated pPAD22b. Amino acid replacements were performed using a QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA). PCR was performed using PfuUltra HF DNA polymerase (Promega) with pPAD22b as the template. The primer sets used were 5'-CATGGGGGGCCACTG GCTGGACGGCAC-3'/5'-GTGCCGTCCAGCCAG TGGCCCCCCATG-3' for the Met57→Leu (M57L) mutation, 5'-CATGGGGGGCCAACG GCTGGACGGCAC-3'/5'-GTGCCGTCCAGCCGT TGGCCCCCCATG-3' for the M57T mutation, and 5'-CATGGGGGGCCAGCG GCTGGACGGCAC-3'/5'-GTGCCGTCCAGCCGC TGGCCCCCCATG-3' for the M57A mutation (the underlined sequences indicate mutated codons). To express mutant proteins, the resulting plasmids harboring the respective mutated genes were each introduced into competent E. coli BL21 (DE3) cells.

Construction of model structure of CgPAD

The secondary structure of CgPAD was predicted by the method of [Kabsch and Sander (1983)]. The deduced amino acid sequence of the enzyme was aligned with that of the crystal structure of a PAD (PCA decarboxylase) from L. plantarum (LpPAD; PDB code 2GC9) [Rodríguez et al. 2010]. A model of the CgPAD structure built with method of homology modeling was constructed based on the structure of LpPAD [Sali et al. 1993]. All data sets were processed on a Windows XP personal computer using the Discovery Studio software package (Accelrys, San Diego, CA). Distance between intramolecular sulfur atom of methionine and side-chain carbonyl oxygen atoms of glutamic acid or amide nitrogen atom of arginine was calculated from the coordinate values. The figure was prepared using a DS Visualizer (Accelrys).

Nucleotide and deduced amino acid sequences of CgPAD

We initially cloned and sequenced the CgPAD gene. The entire CgPAD gene was 504 nucleotides in length, and an open reading frame encoded 168 amino acid residues (Figure 1). The calculated molecular mass was 19,828 Da http://web.expasy.org/compute_pi/, a value very close to the 20 kDa determined for the native enzyme by SDS-polyacrylamide gel electrophoresis [Huang et al. 2011].

The deduced amino acid sequence of CgPAD was aligned with those of functional PADs reported to date from different bacteria [Thompson et al. 1997]; http://www.genome.jp/tools/clustalw). As shown in Figure 2, CgPAD exhibited very low similarity of sequence to functional PADs reported to date from L. plantarum WCFS1 ([Rodríguez et al. 2010] and L. plantarum LPCHL2 [Cavin et al. 1997a] with 39%, Enterobacter sp. Px6-4 [Gu et al. 2011a, 2011b] with 34%, Klebsiella oxytoca[Uchiyama et al. 2008] with 27%, and B. subtilis 168 [Cavin et al. 1998] with 26% identity, and B. pumilus PS213 [Zago et al.1995], P. pentosaceus ATCC 25745 [Barthelmebs et al. 2000], and Lactobacillus brevis ATCC 367 (RM84) [Landete et al. 2010] each with 25% identity. Nevertheless, four residues (Tyr18, Tyr20, Arg48, and Glu71) involved in the catalysis of LpPAD [Rodríguez et al. 2010] were well conserved in CgPAD as Tyr30, Tyr32, Arg60 [but Asn23 in the Enterobacter enzyme [Gu et al. 2011b]], and Glu82 and of PADs from other bacteria as these residues at the corresponding positions.

Construction of model structure and creation of mutant proteins of CgPAD

The absence and/or replacement of methionine residues adjacent to catalytic residues or in the proximate area of active-site pockets has been reported to confer resistance to oxidation, as based on the catalytic activity of enzymes [Estell et al. 1985, Hagihara et al. 2001, 2003, Nonaka et al. 2003]. Further, we demonstrated that the replacement or oxidation of such the Met residues altered the conversion rates of substrates by some enzymes [Hagihara et al. 2001, 2003, Nonaka et al. 2003, 2004, Saeki et al. 2007]. Then, we first postulated that the fluctuation of the ratio of decarboxylation toward FA to PCA might result from oxidation of heat-labile CgPAD, because the deduced amino acid sequence contained two oxidizable Met residues at positions 57 and 103 (Figure 1). It was expected that the replacement of either Met57 or Met 103 with the non-oxidizable amino acids would increase the ratio of decarboxylation activity of CgPAD toward FA to PCA.

According to this scenario, we constructed a model of CgPAD using the crystal structure of LpPAD (PDB code 2GC9) as the template. In the result, the Met residues at positions 57 and 103 in the modeled CgPAD appeared to be located in the active-site pocket (see Additional file 4). Especially, the Met57 residue is located at the entrance of the pocket and in the immediate vicinity of possible catalytic residues Arg60 and Glu82. However, the Met103 residue is spatially more distant from the two catalytic residues and located deeper in the active-site pocket (see Additional file 5).

Acceleration of FA decarboxylation activity of CgPAD by thiol compounds

For measurement of enzyme activities in the cell-free extracts, we disrupted the cells in the extraction buffer supplemented with 1 mM DTT. Then, we examined the effect of this thiol on the activity of purified native CgPAD. As the result, the FA decarboxylation activity was found to be enhanced by DTT at 0.2-1 mM (Figure 3), while the PCA decarboxylation activity was not affected by DTT at the concentrations examined.

Activation of FA decarboxylation activity by ultrafiltrate of cell-free extract

Finally, we supposed that C. guilliermondii inherently possessed a physiological thiol activator, which might have been removed during the enzyme purification. Then, we prepared an ultrafiltrate of the cell-free extract (Mr 10,000-cut-off) of induced C. guilliermondii cells and incubated it with native and recombinant CgPADs at 25°C for 20 min before enzyme assays. As the results shown in Figure 4, the ultrafiltrate was found to remarkably increase the FA decarboxylation activities of both enzymes (approximately up to 5-folds) with an increase in its volume, while it exhibited little effect on their PCA and CA decarboxylation activities. The ultrafiltrate of the cell-free extract of recombinant E. coli did not exhibit such an activation effect.

Partial purification of true activator in the ultrafiltrate

The ultrafiltrate was subjected to gel-filtration chromatography on Bio-Gel P-2. As shown in Figure 5, a possible true activator associated with the FA decarboxylation activity was detected in fractions corresponding to a Mr larger than 1,400. The fractions reacted positively with the 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) and ninhydrin reagents. In the presence of 50 μl of the filtrate, the values of K_m, k_cat, and k_cat/K_m for FA were 5.67 mM, 278 s^-1, and 49.0 s^-1 mM^-1, whereas those of the control (without filtrate) were 5.31 mM, 89.7 s^-1, and 16.9 s^-1 mM^-1, respectively.