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First published online November 17, 2004; 10.1105/tpc.104.025288 © 2004 American Society of Plant Biologists Structural Basis for the Entrance into the Phenylpropanoid Metabolism Catalyzed by Phenylalanine Ammonia-LyaseInstitut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität, Freiburg im Breisgau, Germany 79104 1 To whom correspondence should be addressed. E-mail georg.schulz{at}ocbc.uni-freiburg.de; fax 49-761-203-6161.
Because of its key role in secondary phenylpropanoid metabolism, Phe ammonia-lyase is one of the most extensively studied plant enzymes. To provide a basis for detailed structure–function studies, the enzyme from parsley (Petroselinum crispum) was crystallized, and the structure was elucidated at 1.7-Å resolution. It contains the unusual electrophilic 4-methylidene-imidazole-5-one group, which is derived from a tripeptide segment in two autocatalytic dehydration reactions. The enzyme resembles His ammonia-lyase from the general His degradation pathway but contains 207 additional residues, mainly in an N-terminal extension rigidifying a domain interface and in an inserted  -helical domain restricting the access to the active center. Presumably, Phe ammonia-lyase developed from His ammonia-lyase when fungi and plants diverged from the other kingdoms. A pathway of the catalyzed reaction is proposed in agreement with established biochemical data. The inactivation of the enzyme by a nucleophile is described in detail.
Phe ammonia-lyase (PAL; EC 4.3.1.5) is at the gateway from the primary metabolism into the important secondary phenylpropanoid metabolism in plants (Hahlbrock and Scheel, 1989  ). PAL catalyzes the nonoxidative elimination of ammonia from L-Phe to give trans-cinnamate (Figure 1A). Trans-cinnamate is the precursor of numerous phenylpropanoid compounds that fulfill various essential functions as mechanical supports (lignins) (Whetten and Sederoff, 1995), as protectants against biotic and abiotic stress (antipathogenic phytoalexins, antioxidants, and UV-absorbing compounds) (Dixon and Paiva, 1995), as pigments like the anthocyanins (Holton and Cornish, 1995), and in signaling as, for instance, flavonoid nodulation factors (Weisshaar and Jenkins, 1998). Besides its important role in plant development, PAL is also a key enzyme in plant stress response. Its biosynthesis is stimulated on pathogenic attack, tissue wounding, UV irradiation, low temperature, or low levels of nitrogen, phosphate, or iron (Dixon and Paiva, 1995). The enzyme is accumulated in the vicinity of the affected tissue (Mauch-Mani and Slusarenko, 1996; Ehness et al., 1997). Because PAL is ubiquitously distributed in plants and absent in animals, it is a promising target for herbicides; various inhibitors are currently being developed (Laber et al., 1986; Appert et al., 2003).
The unusual nonoxidative deamination reaction of PAL requires an electrophilic group in the enzyme, which is not available among the 20 standard amino acid residues. Because PAL lacks a cofactor to provide such an electrophilic group, it was suggested that the electrophile is produced by a posttranslational dehydration of a Ser to form a dehydroalanine (Hanson and Havir, 1970 ). The Ser was later identified as Ser203 (Schuster and Retey, 1994). On elucidation of the crystal structure of the homologous enzyme His ammonia-lyase (HAL), however, the proposed dehydroalanine turned out to be part of a 4-methylidene-imidazole-5-one (MIO) group (Schwede et al., 1999), which is formed autocatalytically by cyclization and dehydration of an Ala-Ser-Gly tripeptide as shown in Figure 1B (Baedeker and Schulz, 2002a). In a sequence alignment between HAL and PAL, residues 202Ala-Ser-Gly of PAL were assigned to form the MIO (Schwede et al., 1999) in agreement with the identified Ser203. A closely related autocatalytic tripeptide cyclization was detected earlier with the green fluorescent protein (Ormö et al., 1996; Barondeau et al., 2003) which is, however, neither sequence- nor structure-related to HAL or PAL.
A medical application of PAL arose in context with the human genetic disease phenylketonuria that leads to severe mental retardation (Levy, 1999
Crystal Structure For protein production, we used a gene of PAL that had been adapted to Escherichia coli by numerous silent mutations (Baedeker and Schulz, 1999 ). The redesigned gene was cloned into vector pT7-7, transformed into E. coli strain BL21(DE3), and expressed in the cytosol. Narrow peaks were cut out during the chromatographic steps to obtain a pure and homogeneous enzyme. The crystals grew from an unbuffered solution within a couple of days, assuming feather-like shapes without a planar surface as depicted in the insert of Figure 2. However, these feathers were single crystals that diffracted to high resolution.
For phasing, we produced selenium-labeled protein using a Met-auxotrophic E. coli strain. In electrospray ionization mass spectrometry, the labeled PAL showed a mass increase of 976 D with respect to a parallel measurement of wild-type PAL. This difference is close to the expected value of 985 D for the incorporation of all 21 seleno-Met. Labeled PAL yielded the same crystals as the wild type. One labeled crystal was used in a multiwavelength anomalous diffraction (MAD) experiment reaching 2.2-Å resolution (Table 1). The MAD data revealed 34 clear selenium sites, which were then used for phasing, giving rise to an electron density map that was subsequently improved by solvent flattening and histogram matching. The quality of the resulting map sufficed to build a model of two PAL subunits in the crystallographic asymmetric unit, which was confirmed by all selenium positions being at Met. The model was then transferred to the wild-type crystals and refined to 1.7-Å resolution, yielding quality indices in a favorable range (Table 1).
The model comprises 692 of the 716 amino acid residues in each of the two asymmetric subunits (Figure 3A). These two subunits resemble each other closely as indicated by their B-factor distributions (Figure 2). A structural comparison resulted in an rmsd of 0.51 Å for the C atoms, with the largest deviations at packing contacts. Because of lacking electron density, residues 1 to 24 could not be modeled. Moreover, residues 104 to 128 and 334 to 347 had only weak density support in both asymmetric units, resulting in high B-factors (Figure 2). The tetrameric structure of PAL is clearly defined in the crystal, confirming gel permeation chromatography data in solution. The tetramer shows the common D2 symmetry (Figure 3B). One of the three molecular twofold axes is crystallographic, and the others deviate by 20° from the nearest translation vector of the orthorhombic unit cell. The four subunits are tightly interconnected. Each subunit has a surface of 30,330 Å2 and buries 29% of it in contacts of 4215 Å2, 3660 Å2, and 880 Å2 with the other subunits. Consequently, a single subunit is most likely unstable in solution. The interface residues are specified in Figure 2. The packing of PAL in the crystal is very dense, giving rise to a Vm of 2.18 Å3/D, which is at the lower end of the distribution for proteins of this size (Kantardjieff and Rupp, 2003). Despite dense packing, the involved contacts bury merely 7215 Å2 or 8% of each PAL tetramer surface of 86,940 Å2. The residues in packing contacts are marked in Figure 2.
Molecular Architecture PAL is a predominantly -helical protein with 52% of the residues in 23 -helices and only 5% of the residues in eight ß-strands. A chainfold superposition with HAL shows that each PAL subunit should be subdivided into four domains as indicated by arrowheads in Figure 2, by colors in Figure 3A, and in sequence detail in Figure 4. The mobile N-terminal 24-residue peptide occurs only in PAL and not in HAL. Such extensions are widespread; they may anchor the enzyme at other cell components. The MIO domain (residues 25 to 261) is also present in HAL, although HAL lacks helices 1 and 2, which fortify the interface to the core domain (residues 262 to 527 and 650 to 716). The core domain is also present in HAL, whereas the inserted shielding domain (residues 528 to 649) is a specialty of PAL. Further additions in PAL, mostly small insertions into external loops, are detailed in the structure-based sequence alignment of Figure 4. The size difference between PAL and HAL is demonstrated in Figure 5. The most prominent addition of PAL with respect to HAL is the shielding domain, two of which form an arch over the active center, restricting substrate access to a narrow channel.
The B-factor plot of Figure 2 reveals two highly mobile segments around positions 110 and 340. Both loops are located at the entrance of the active center. In particular, loop 110 is near the MIO of the same subunit, whereas loop 340 is near the MIO of the other subunit in the crystallographically asymmetric unit. Both are marked in Figure 3. The same highly mobile loops are known from HAL (Baedeker and Schulz, 2002b ), indicating that their mobility is required for catalysis. Figure 2 shows further that the whole shielding domain is very mobile, although it is tightly connected to the core domain through the exceptionally long 55-residue helix 17. Interestingly, the detected phosphorylation site of PAL from French beans (Phaseolus vulgaris) (Allwood et al., 1999) is located at the end of this helix as marked in Figures 3B and 4. This position has the highest mobility of the shielding domain (Figure 2). It seems quite possible that the phosphorylation shifts this domain, changing the access to the active center.
Prosthetic Group MIO
The prepared PAL was always kept in 5 mM DTT to avoid intermolecular disulfide bonds that may have hindered crystallization. Therefore, we concluded that the analyzed crystal contained a nucleophilic DTT bound to the electrophilic MIO. This conclusion is supported by the electron density distribution in front of MIO that was modeled as a covalently bound DTT showing the high densities of the sulfur atoms (Figure 6). It is further corroborated by the sp2 conformation of the 204-N atom that corresponds to a similarly modified HAL. It agreed also with the electrospray ionization mass spectrometry value for our wild-type PAL that was 163 D above the expected value and with the electrospray ionization mass spectrometry value for selenomethionine-labeled PAL that was 152 D above expectation. Both differences fit the 154-D mass of DTT very well.
The DTT derivative was finally confirmed by enzyme kinetics. Wild-type PAL showed a specific activity of 1.9 units/mg with a Km of 68 µM, which is in the commonly observed range (Baedeker and Schulz, 1999 Therefore, we suggest that the inactivated PAL of the crystal contains an oxidized DTT-MIO adduct, which is a MIO (Figure 1B) with a sulfur-substituted 203-Cß atom. Obviously, the sulfur substitution at 203-Cß changes the electronic structure of MIO to an sp2 conformation at the 204-N atom, which by itself, should also prefer sp2 because it forms an amide with 203-O. Presumably, the sp3 conformation of 204-N observed in wild-type HAL and expected in wild-type PAL is probably enforced by the polypeptide as it increases the electrophilicity of the 203-Cß atom and thus promotes the enzymatic reaction.
Reaction Pathway
The suggested reaction pathway is shown in Figure 8. First, an electron pair of the phenyl ring attacks the 203-Cß atom of MIO (state I). On this attack MIO becomes aromatic and the positively charged  -complex of the substrate phenyl group is stabilized by the produced 203-O anion (state II). Moreover, the positive charge is stabilized by an interaction with the  -electrons of Phe400 (Figure 7B). The conversion of MIO to the aromatic state changes the conformation of 204-N from sp3 to sp2, causing a small peptide displacement, which was observed in HAL where it was easily accommodated. The electron-deficient phenyl ring of the substrate renders the two hydrogens at the Cß atoms acidic, and the HS proton is abstracted by Tyr351' from another subunit in agreement with isotope labeling experiments (Hermes et al., 1985). Tyr351' is in turn hydrogen bonded via Wat1088 and Glu484 to the bulk solvent so that the proton is easily released. The following rearrangement of the Phe-MIO adduct in state III eliminates the amino group and regenerates MIO. The resulting ammonia is occluded by the other product trans-cinnamate and will be released into the solvent after the cinnamate has diffused away, as was suggested by kinetic data (Hermes et al., 1985).
The active center residues of PAL were also confirmed by mutational studies guided by a homology model based on HAL (Röther et al., 2002 ). Most intriguingly, the mutation Tyr110 Phe resulted in a complete loss of activity, although this Tyr should not be that important for the reaction as it is expected to contact merely the substrate carboxylate group. However, because the introduced Phe110 is in a highly mobile loop (Figure 2), it may reach the active center to bind like the substrate and inhibit the enzyme.
The PAL and HAL Superfamily The PAL sequences are from 46 plant and five fungi species as well as from one bacterium, whereas the HAL sequences are spread over all species. The bacterial PAL is special and will be discussed below. Using the reported PAL from parsley (Petroselinum crispum) as a reference, the most distant PAL is from a fungus and has 29% identical amino acid residues, whereas the closest HAL is from Pseudomonas syringe and shows only 25% identities. Because all HAL sequences are related to each other and have >32% identities in binary comparisons, the superfamily splits clearly into a PAL and a HAL family. The 97 and 65 residues strictly conserved within the PAL and HAL families, respectively, are marked in Figure 4. Among them, 31 are conserved in the superfamily. The homology within the plant PAL species is above 60% identical residues. Several plants contain multiple isoenzymes. Parsley has the three isoenzymes PAL1 (reported here), PAL2, and PAL3, whereas Arabidopsis thaliana, for instance, has the four isoenzymes PAL1 to PAL4. Sequence comparisons among these seven isoenzymes showed that they cluster by species so that those within one species are more closely related to each other than to isoenzymes of the other species. Accordingly, the diversification occurred in each species separately.
PAL is known to accept Tyr as a poor substrate (Langer et al., 2001
Sequence alignments with the TAL from R. capsulatus indicated that it was developed from HAL rather than from PAL because it lacks the N-terminal extension and the shielding domain characteristic for the plant and fungi PAL (Figure 3A). Recently, a bacterial PAL was detected in Streptomyces maritimus, which uses the produced trans-cinnamic acid as a precursor for the antibiotic enterocin (Xiang and Moore, 2002
Taken together, HAL is the basic enzyme of the superfamily because it participates in a central metabolic pathway and is therefore ancient and distributed over numerous species. PAL and TAL were derived from HAL to fulfill special tasks. Applying merely amino acid exchanges, the bacterial PAL and TAL were developed directly from HAL and used for the syntheses of an antibiotic and a chromophore, respectively. The plant and fungi PAL was derived from HAL by inserting
The structure of PAL shows that this key plant enzyme is most likely an offspring of HAL from the central metabolic His degradation pathway. In contrast with HAL, the active center of the plant and fungi PAL is well shielded by a separate domain. The shielding domain restricts the access to the active center so that the risk of inactivation by nucleophiles in conjunction with dioxygen is minimized. This may help PAL to function, for instance, in stressed plant tissue. It should be noted that PAL forms its electrophilic prosthetic group autocatalytically from its own polypeptide, rendering it independent of any cofactor and thus facilitating its upregulation. In a medical application, the known surface of PAL can now be used for designing a recombinant nonimmunogenic enzyme for the treatment of the human genetic disease phenylketonuria.
Enzyme Expression, Purification, Activity, and Crystallization Because of inefficient codon usage, heterologous expression of the 2.2-kb gene of isoenzyme-1 of the PAL from parsley (Petroselinum crispum) in Escherichia coli failed to yield enough homogenous protein for a structure analysis (Schuster and Retey, 1995 ). Therefore, we used a gene especially designed for E. coli (Baedeker and Schulz, 1999). For expression, we took vector pT7-7-PAL in E. coli BL21(DE3) together with the Hsp60 chaperone-containing vector pREP4-groESL. The cells were grown at 37°C to OD600 0.8 using 2YT medium supplemented with 100 µg/mL of ampicillin (PAL plasmid) and 25 µg/mL of kanamycin (groESL plasmid). The temperature was then lowered to 20°C, and the expression was induced by adding 0.1 mM isopropylthio-ß-D-thiogalactoside to run for 20 h. The cells were harvested, resuspended in 35 mL of buffer A (50 mM KH2PO4, pH 7.5, and 5 mM DTT) with 1 mM tosylfluorid (phenylmethylsulfonyl fluoride) and 500 units of Benzonase (Merck, Darmstadt, Germany), and sonicated for 10 min. After centrifugation (60 min at 48,000g), the supernatant was dialyzed overnight against buffer A and then applied to an anion-exchange column (Source 30Q; Pharmacia, Uppsala, Sweden), which was subsequently eluted using buffer A with a gradient of 0 to 2 M NaCl. PAL was identified by SDS-PAGE, separated, concentrated, and applied to a gel permeation column (Superdex 200-26/60; Pharmacia) equilibrated in buffer A with 600 mM NaCl and 3% (w/v) maltose. PAL-containing fractions were concentrated and dialyzed overnight against deionized water with 5 mM DTT. A typical yield was 15 to 20 mg PAL per liter of culture.
The activity of PAL was determined by monitoring the reaction product trans-cinnamate at 290 nm (
Selenomethionine (SeMet)-labeled PAL was produced by transforming the expression vector pT7-7-PAL into the Met-auxotrophic E. coli strain B834(DE3) without cotransforming pREP4-groESL. Cells were cultured in LeMaster medium (Hendrickson et al., 1990 The protein was crystallized using the hanging drop vapor diffusion method at 20°C. After screening, suitable crystals were obtained from a 1:1 mixture of a 8-mg/mL protein solution with a reservoir solution containing 6 to 11% (w/v) polyethylene glycol 6000 and 30 to 60 mM MgCl2. Fewer and larger crystals grew if the reservoir solution was diluted with water to a volume of 120 to 160% after the drop had been placed. Wild-type and SeMet-labeled PAL crystallized under identical conditions and yielded similar crystals. The crystals appeared within 2 d and grew to final dimensions of 500 x 150 x 100 µm3. For data collection, the crystals were transferred in three steps to a solution containing 30% (w/v) glycerol, 18% (w/v) polyethylene glycol 6000, and 40 mM MgCl2 before they were mounted on a cryoloop and flash-frozen to 100 K in a nitrogen gas stream.
X-Ray Data Collection and Analysis The atomic coordinates and structure factors are deposited in the Protein Data Bank under accession code 1W27. The accession codes for the sequences of Figure 4 are Y07654 (PAL) and M35140 (HAL). The accession code for the coordinates of HAL is 1B8F.
We thank the team of the Swiss Light Source (Villigen, Switzerland) for their help in data collection. The project was supported by the Deutsche Forschungsgemeinschaft under SFB-388.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Georg E. Schulz (georg.schulz{at}ocbc.uni-freiburg.de). Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.104.025288. Received June 18, 2004; accepted September 20, 2004.
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90 complete PAL sequences, 
290 = 10,000 M–1cm–1). The assay contained 0.1 M Tris-HCl, pH 8.8, and various concentrations of L-Phe and DTT in 1 mL of water and was run at 30°C. The reaction was started by adding 9.3 µg of PAL dissolved in 5 µL of water. The L-Phe and DTT concentrations were 0.01 to 10 mM and 0.01 to 2.0 mM, respectively.