Plant Physiol. (1 997) 1 1 3 : 175-1 79

Maize Phenylalanine Ammonia-Lyase Has Tyrosine Ammon ia- Lyase Act ivi ty

Jens Rosler, Florian Krekel, Nikolaus Amrhein*, and Jürg Schmid

lnstitute of Plant Sciences, Swiss Federal lnstitute of Technology, Universitatstrasse 2, CH-8092 Zurich, Switzerland

A full-length cDNA encoding phenylalanine ammonia-lyase (PAL) from Zea mays L. was isolated and the coding region was expressed in Escherichia coli as a C-terminal fusion to glutathione S-transferase. After purification by glutathione-Sepharose chroma- tography, the glutathione S-transferase moiety was cleaved off and the resulting PAL enzyme analyzed. In contrast to PAL from dicots, this maize PAL isozyme catalyzed the deamination of both i-phenylalanine (PAL activity) and i-tyrosine (tyrosine ammonia- lyase activity). These results provide unequivocal proof that PAL and tyrosine ammonia-lyase activities reside in the same polypep- tide. In spite of large differences in the Michaelis constant and turnover number of the two activities, their catalytic efficiencies are very similar. Also, both activities have the same p H and temperature optima. These results imply that maize can produce p-coumaric acid from both phenylalanine and tyrosine.

PAL (EC 4.3.1.5) catalyzes the elimination of ammonia from L-Phe to yield (E)-cinnamic acid (Fig. 1) (Koukol and Com, 1961), the first step in the phenylpropanoid pathway (for reviews, see Hahlbrock and Scheel, 1989; Dixon and Paiva, 1995; Whetten and Sederoff, 1995; Campbell and Sederoff, 1996). PAL activity has been found in some fungi and in a11 higher plants analyzed but not in animals. Be- cause of the central function of PAL at a branch point of metabolism, this enzyme is one of the best studied in plants; the same is true for its corresponding gene(s).

In a11 plants analyzed, PAL has been shown to occur as a tetramer, and multiple forms have often been isolated (Bolwell et al., 1985). PAL multigene families, such as those in tomato (Lycopersicon esculentum; Lee et al., 1992), parsley (Petroselinum crispum; Appert et al., 1994), or Arabidopsis thaliana (Wanner et al., 1995), explain at least in part these different forms. The occurrence of heteromers is likely but has as yet not been proven. On the other hand, apparent charge isoforms of a poplar PAL were recovered from insect cell cultures producing the protein from a baculovi- rus expression vector containing the respective cDNA (McKegney et al., 1996).

Results from severa1 studies (Neish, 1961; Young et al., 1966; Havir et al., 1971; Jangaard, 1974) indicated that the enzyme from monocots utilizes Tyr (TAL activity) in addi- tion to Phe, whereas the enzyme from dicots utilizes only Phe efficiently. The four homotetrameric parsley PAL

* Corresponding author; e-mail amrhein@ipw.agrl.ethz.ch; fax 41-1- 632-1 084.

175

isozymes, for example, were found to have K, values for Phe in the range of 15 to 25 PM and for Tyr the values were 2 to 8 mM (Appert et al., 1994). The turnover numbers determined for one of the parsley PAL isozymes (PAL-1) were about 22 s-l for Phe but only about 0.3 s-’ for Tyr (Appert et al., 1994). These results show that it is highly unlikely that Tyr is a substrate for these enzymes under physiological conditions. On the other hand, the K , values of purified PAL from maize have been reported to be 270 PM for Phe and 29 PM for Tyr (Havir et al., 1971), indicating that PAL from maize also has TAL activity. However, the uncertainty remains that a protein with TAL activity co- purifies with PAL activity. Referring to the heterologous expression of parsley PAL cDNAs (Schulz et al., 1989; Appert et al., 1994), Whetten and Sederoff (1995) stated: ”The same experiment could be performed with maize cDNA to test for PAL and TAL activities in the same polypeptide.” This is exactly the experiment we report here. Expression of a PAL-specific cDNA from maize in Escherichia coli provided the ultimate proof that PAL and TAL activities reside in one polypeptide.

MATERIALS A N D METHODS

The basic molecular techniques were adopted from Aus- ubel et al. (1987) and Sambrook et al. (1989).

lsolation of Maize PAL-Specific cDNA Clones

RNA was extracted by a method based on phenol extrac- tion of roots of 4-d-old dark-grown maize (Zea mays L. cv Corso) seedlings homogenized in liquid nitrogen. Poly(A)+ RNA was purified by two passages over oligo(dT)- cellulose and used as a template for the construction of a cDNA library. A commercial cloning kit (ZAP-cDNA Syn- thesis Kit, Stratagene) was used according to the manufac- turer’s instructions, with the exception that the cDNA was size-selected ( 2 4 kb) on an agarose gel. The resultirig library (9 X 105 recombinant phages) was amplified once and 106 plaque forming units were screened in duplicate with a labeled maize PAL-specific cDNA fragment (gift from C. Baysdorfer) as a probe. Positive clones were

Abbreviations: GST, glutathione S-transferase; k,,, turnover number; PAL, Phe ammonia-lyase; TAL, Tyr ammonia-lyase; Zm- PAL1, PAL isozyme 1 from Z. mays (corresponding cDNA in italics).

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176 Rosler et al. Plant Physiol. Vol. 11 3 , 1997

J

6 OH

L-tyrosine

G

L-phenylalanine

phenylalanine armnonla-lyase NH37 dmH

(E)-cinnamic acid

Q 6H

p-coumadc acid

Figure 1. Scheme of the two alternative pathways of p-coumaric acid formation, either by deamination of Phe and subsequent p-hydroxylation of (0-cinnamic acid or by deamination of Tyr.

plaque-purified and the phage DNAs converted into plas- mids by in vivo excision.

Expression Construct

Maize PAL was expressed in Escherichia coli as the C-terminal fusion to GST (Smith and Johnson, 1988) in such a way that recombinant PAL protein without any addi- tional amino acids could be released by cleavage with the protease factor Xa (Boehringer Mannheim). The cDNA se- quence spanning coding region, the 3' untranslated region, and part of the polylinker of the vector pSK (Stratagene) that contained a KpnI site was amplified by the PCR with a 5' primer corresponding to the first 17 nucleotides of the coding region and a 3' primer corresponding to 17 nucle- otides of the polylinker. The generated PCR fragment was phosphorylated by T4 polynucleotide kinase and cleaved with the restriction endonuclease KpnI, and the resulting fragment was ligated into the vector pGEX-G (Gorlach and Schmid, 1996), which had first been treated with the two restriction endonucleases StuI and KpnI and dephosphory- lated. To exclude alterations in the sequence due to errors of the Tuq polymerase, the sequence from position 364 shown in Figure 2 (a BamHI site) to the KpnI site in the polylinker was exchanged by the corresponding fragment

from the original cDNA, and the portion that remained from the PCR fragment was verified by sequencing.

Protein Purification

E. coli cells (strain DH5; Hanahan, 1985) containing the expression construct were grown in Luria broth at 37°C to an optical density (Aeoo) of 1.0, and isopropyl-P-D- thiogalactoside was then added to a final concentration of 80 PM. After 3 h, cells were harvested by centrifugation. The purification of the fusion protein was as described by Appert et al. (1994). The fusion protein was digested with protease factor Xa (50 pg / 100 Fg fusion protein) for 48 h at 4°C. For the determination of the turnover numbers, ZmPALl was chromatographically separated from both the GST moiety and factor Xa on a Mono-Q HR 5 / 5 column

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Maize Phe Ammonia-Lyase 177

(Pharmacia) with a linear gradient of O to 0.4 M NaCl in 50 mM Tris-HC1, pH 7.8, 1 mM DTT. The ZmPAL1-containing fractions were desalted by Centricon-10 columns (Amicon, Beverly, MA) and taken up in 50 mM Tris-HC1, pH 8.0.

Standard Assays for PAL and TAL Activities

PAL activity was assayed by following (E)-cinnamic acid formation at 280 nm in a spectrophotometer at 40°C in buffer (0.2 M sodium borate-NaOH, either pH 8.7 or 7.7) containing 61 mM L-Phe. TAL activity was assayed by monitoring p-coumaric acid formation at 310 nm in a spec- trophotometer at 40°C in the same buffers containing 1.9 mM L-Tyr.

ldentification of Reaction Products

PAL and TAL reactions (using 10 mL of assay mixture each) were allowed to proceed overnight and the reaction products were extracted into ethylacetate after acidification of the reaction mixtures. After the sample dried over an- hydrous CaCl,, the solvent was evaporated and the resid- ual material was subjected to methylation with diazometh- ane in ether. GC-MS analysis of the methylated products was carried out on a gas chromatograph (Hewlett-Packard HP5890; 30 m X 0.25 mm X 0.25 pm DB-5ms column, J&W Scientific, Folsom, CA) coupled to a mass spectrometer (model TSQ7000, Finnigan, Bremen, Germany) run at 70 eV and 800 pA (mass range 45400 in 0.5 s).

Analyses of Data

Sequence analyses were done with the Wisconsin Se- quence Analysis Package (version 8, Genetics Computer Group, Madison, WI). The kinetic data were analyzed us- ing the program Enzfitter (version 1.05, Elsevier Biosoft, Cambridge, UK).

RESULTS

ldentification of Maize PAL-Specific cDNAs

A size-selected maize cDNA library (106 plaque forming units) in the A expression vector Uni-ZAP XR was screened with a 600-bp maize PAL-specific cDNA fragment. Eigh- teen positive A clones were identified, plaque-purified, and converted to plasmids by in vivo excision. To identify full-length clones, the 5' ends of these cDNAs were deter- mined. Of the 18 cDNAs, 17 had identical sequences (named ZmPALl) as far as they overlapped and, of these, four were full-length clones. The remaining cDNA (not a full-length clone) also encoded PAL (ZmPALZ), which in- dicated that at least two distinct PAL genes exist per hap- loid maize genome. The complete sequence of the full- length clone with the longest 5' untranslated region was determined (Fig. 2). The GenBank accession no. for Zm- PALl is L77912. The cDNA without the poly(A) tail is 2487 bp long and contains an open reading frame that has the capacity to code for a polypeptide of 703 amino acids with a calculated molecular mass of 74,926 D. The deduced amino acid sequence shows sequence identity with the

PALl sequences of rice (Oryza sativa), tomato, parsley, and Arabidopsis thaliana: 89, 69, 62, and 68%, respectively. The dendrogram (Fig. 3) clearly indicates that the two PAL sequences known until now from monocots belong to one group and a11 of those from dicots belong to a second group. The only exception is PAL3 from A. thaliana (Wan- ner et al., 1995), which is distinctly different from a11 of the other plant sequences, as well as from the fungus se- quences.

Expression of a CST-ZmPAL1 Fusion Protein in E. coh and Purification of ZmPALl

To obtain large quantities of the maize PAL isozyme ZmPALl, the protein was expressed as a fusion protein with GST. It was known from previous studies of PAL from parsley (Appert et al., 1994) that the enzyme can be ex- pressed in E . coli as such a fusion protein and that a single purification step by affinity chromatography would yield the pure fusion protein. In addition, it was known that the PAL moiety could efficiently be released by cleavage of the fusion protein with protease factor Xa. Therefore, a con- struct in the E. coli expression vector pGEX-G (Gorlach and Schmid, 1996), which directed the expression of a GST- ZmPALl fusion protein (Fig. 4A), was made in such a way that after treatment with protease factor Xa the recombi- nant ZmPALl isozyme was released (Fig. 48) and could be purified to homogeneity by an additional step (Fig. 4C).

Enzymatic Properties of ZmPALl

The GST-ZmPAL1 fusion protein was found to have both PAL and TAL activities. To analyze the enzyme in detail, the properties of the purified recombinant ZmPALl were analyzed (Table I). In previous experiments (Havir et al.,

R. rubra PALl

R. toruloides PALl

O. sativa PAU

2. mays PALl

A. thaliana PALl

F! crispum PALl LI+ I L. esculentum PALl L A. thaliana PAL3

Figure 3. Comparison of selected PAL sequences. The dendrogram shows a comparison of the sequence of maize ZmPALl with those of rice PALl (accession no. X16099), parsley PALl (accession no. X16772/X15473), tomato PALl (accession no. P3551 I), Arabidopsis PALl and PAL3 (accession nos. L33677 and L33679, respectively), Rhodotorula rubra PALl (accession no. X13094), and Rhodospo- ridium toruloides PALl (accession no. P11544). The dendrogram was created with the program PileUp (Genetics Computer Group, version 8).

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178 Rosier et al. Plant Physiol. Vol. 113, 1997

factor Xa

Met... GST ... HeGlu Gly Arg Meti... ZmPALI ... Lys703

BM 1 2 3

M

kDa

94- — »* —67- — ~™

43- - I

30- _ .i •=.

'. GST-ZmPALl! ZmPALI

. factor Xa: GST

kDa-200-118-78

-47

*» -31,̂ -25

Figure 4. Purification of ZmPALI expressed in E. coli. A, Scheme ofthe GST-ZmPALl fusion protein. Digestion of the fusion protein withprotease factor Xa, which has the indicated recognition sequenceIle-Glu-Gly-Arg and cleaves C-terminal of Arg, yields recombinantZmPALI, which has an amino acid sequence identical to the de-duced amino acid sequence shown in Figure 2. B, Aliquots offractions from each purification step were analyzed, along with sizemarkers (M) by SDS-PAGE (10% acrylamide). Lane 1, Crude extract;lane 2, affinity-purified by glutathione-Sepharose; lane 3, GST-ZmPAL! fusion protein after treatment with protease factor Xa. C,Aliquots of the purified GST-ZmPAL fusion protein (lane 1) andZmPALI (lane 2), along with size markers (M) analyzed by SDS-PAGE (10% acrylamide).

1971), the Km values for Phe and Tyr had been measured insodium borate buffer at pH 8.7 and 7.7, respectively, andwe therefore adopted these conditions (Table I). At bothpH values, the Km values for Phe were about 15 timeshigher than those for Tyr, and the ratios of the Km values atpH 7.7 versus 8.7 were 2 to 2.5 in both cases (Table I).Turnover numbers for the TAL activity were about one-tenth of those for the PAL activity (Table I).

Since no difference was found between GST-ZmPALland ZmPALI with respect to their kinetic properties (datanot shown), and because of the high costs of the prepara-tion of ZmPALI, further characterization of the enzymewas done with GST-ZmPALl. The pH optima for both thePAL and TAL activities were uniformly in the range of pH8.0 to 8.5. Likewise, their temperature optima (in the rangeof 55-60°C) did not differ significantly (data not shown).

GC-MS analysis of the methylated reaction products re-vealed the exclusive presence of (E)-cinnamic acid methylester and p-methoxy-(E)-cinnamic acid methyl ester in theethylacetate extracts; their mass spectra were identical withthose of the authentic reference compounds. (E)-cinnamicacid methyl ester: m/e 162(M+, 64); 161(36); 144(3); 132(10);131(100); 117(5); 104(8); 103(66); 102(15); 91(3); 77(33); 63(3);51(16). p-methoxy-(E)-cinnamic acid methyl ester: m/e192(M+, 74); 191(3); 162(11); 161(100); 147(3); 134(15);133(33); 132(7); 118(13); 103(7); 90(9); 89(13); 79(5); 77(8);63(5); 51(3).

DISCUSSION

The ZmPALI Isozyme Has TAL Activity

The results presented here provide definitive evidencethat the analyzed PAL isozyme of maize (ZmPALI) also

has TAL activity. It is interesting to note that, whereas theKm values for Tyr are lower by an order of magnitudecompared with Phe, the turnover numbers for Phe areabout 10 times higher than those for Tyr, which results incomparable catalytic efficiencies (/ccat/Xm) for the two ac-tivities (Table I). The pH optima for both activities were inthe range of 8.0 to 8.5, which is not in agreement with theresults of Havir et al. (1971), who obtained pH optima forPhe at 8.7 and for Tyr at 7.7. However, this difference maybe explained by the fact that these authors most likelyanalyzed a mixture of different PAL isozymes, whereasonly one isozyme, ZmPALI, was analyzed in the currentstudy.

Potential Physiological Implications

As indicated by several earlier reports (Neish, 1961;Young et al., 1966; Havir et al., 1971; Jangaard, 1974), PAL,not only from maize but from monocot plants in general,has TAL activity. This implies that these plants can, inprinciple, produce p-coumaric acid from both Phe and Tyr(Fig. 1), which raises the question of the physiologicalsignificance and relative contribution to phenylpropanoidsynthesis of these two pathways. Without complementingin vivo studies, our data do not permit any conclusionconcerning the quantitative importance of PAL and TALactivities in maize, but with variable ratios of concentra-tions of the two aromatic amino acids, the relative contri-bution of the two pathways to p-coumaric acid productionmay change. There are several factors that may potentiallyregulate the concentration of these two aromatic aminoacids in the cytosol, such as a differential expression andactivities of arogenate dehydratase and arogenate dehy-drogenase or differential contributions of plastidic and cy-tosolic pathways from chorismate to Phe and Tyr (Eber-hard et al., 1996a, 1996b), as well as differential transport ofthe two amino acids across the plastid membranes. Also,the consumption of Phe and Tyr in protein synthesis aswell as by other enzymes would have an impact on theirconcentrations. Finally, the concentrations will be affectedby differential sequestration of the two amino acids in thevacuole. Therefore, only in vivo experiments will reveal towhat extent either of these two routes contributes to thebiosynthesis of p-coumaric acid and its derivatives. Previ-

Table I. Enzymatic properties of recombinant ZmPAL 1Km values were determined spectrophotometrically by following

CfJ-cinnamic acid formation at 280 nm and 40°C in a buffer containing0.2 M sodium borate-NaOH at pH 8.7 or 7.7, or by monitoring thep-coumaric acid formation at 310 nm and 40°C in the same buffers.The Km values are the mean values ± SE of 12 independent measure-ments and the turnover numbers of 6 independent measurements.

Enzymatic Properties

Km L-Phe (,XM)Km L-Tyr (/IM)fc c a lPAL(s- ' )fcca,TAL (s^1)kcJKm PALkcJKm TAL

pH 7.7

658 ± 4741 ± 2

11.9 ± 3.40.94 ± 0.19

0.0180.023

pH 8.7

270 ± 2019 ± 1

10.6 ± 3.20.92 ± 0.22

0.0390.048

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Maize Phe Ammonia-Lyase 179

ous experiments with the PAL and TAL, inhibitor ~ - a - amino-oxy-p-phenylpropionic acid revealed a n increase of both Phe and Tyr i n wheat a n d barley leaves in response to inhibitor treatment (Hollander e t al., 1979), indicating that both aromatic amino acids are utilized in the deamina- tion reaction. On the other hand, L-a-amino-oxy-p-phe- nylpropionic acid is also known to be a potent inhibitor of Tyr decarboxylase (Chapple e t al., 1986) and it therefore remains to be seen to what extent inhibition of either enzyme contributes t o the accumulation of Tyr i n vivo.

ACKNOWLEDCMENTS

We would like to thank Drs. C. Baysdorfer (California State University, Hayward) for the maize PAL probe (CSUH00156), E. Weiler and P. Hennig (Ruhr University, Bochum, Germany) for the GC-MS analysis of the reaction products, and D. Rubli (ETH, Zurich, Switzerland) for photographic assistance.

Received July 16, 1996; accepted October 15, 1996. Copyright Clearance Center: 0032-0889/97/ 113/0175/05.

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