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Originally published In Press as doi:10.1074/jbc.M411215200 on October 19, 2004

J. Biol. Chem., Vol. 279, Issue 52, 53947-53954, December 24, 2004
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Cloning, Heterologous Expression, and Characterization of a Phenylalanine Aminomutase Involved in Taxol Biosynthesis*

Kevin D. Walker{ddagger}§, Karin Klettke{ddagger}, Takumi Akiyama¶||, and Rodney Croteau¶

From the {ddagger}Department of Chemistry and Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824 and the Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164

Received for publication, September 30, 2004 , and in revised form, October 15, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Biosynthesis of the N-benzoyl phenylisoserinoyl side chain of the anticancer drug Taxol starts with the conversion of 2S-{alpha}-phenylalanine to 3R-{beta}-phenylalanine by phenylalanine aminomutase (PAM). A gene cloning approach was based on the assumption that PAM would resemble the well known plant enzyme phenylalanine ammonia lyase. A phenylalanine ammonia lyase-like sequence acquired from a Taxus cuspidata cDNA library was expressed functionally in Escherichia coli and confirmed as the target aminomutase that is virtually identical to the recombinant enzyme and clone from Taxus chinensis, acquired recently by a reverse genetics approach (Bristol-Myers Squibb (August 14, 2003) U. S. Patent WO 03/066871 A2). The full-length cDNA has an open reading frame of 2094 base pairs and encodes a protein of 698 residues with a calculated molecular mass of 76,530 Da. The recombinant mutase has a pH optimum of 8.5, a kcat value of 0.015 s–1, and a Km of 45 ± 8 µM for 2S-{alpha}-phenylalanine. The stereochemical mechanism of PAM involves the removal and interchange of the pro-3S hydrogen and the amino group, which rebonds at C-3 with retention of configuration. The recombinant enzyme appears to catalyze both the forward and reverse reactions with specificity for both 2S-{alpha}-phenylalanine and 3S- or 3R-{beta}-phenylalanine substrates, respectively, whereas the related phenylpropanoids 2S-aminocyclohexanepropanoic acid, 2R-{alpha}-phenylalanine, and 2S-{alpha}-tyrosine are not converted to their {beta}-isomers by the mutase.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
The final stages of Taxol biosynthesis (see Fig. 1A) in yew species involve the assembly and attachment to C-13 of the taxane core of the N-benzoyl phenylisoserinoyl side chain, which is an important pharmacophoric descriptor of this anticancer drug (1, 2). In the current practice of Taxol production, this side chain is attached by chemical semisynthesis to baccatin III, which is derived from 10-deacetylbaccatin III, a Taxus (yew) metabolite that is much more readily available than Taxol itself (3–5). In the biosynthetic pathway, five steps are involved in the construction of the side chain. The first step is considered to be the conversion of 2S-{alpha}-phenylalanine to 3R-{beta}-phenylalanine by an aminomutase that catalyzes an intramolecular migration of the amino group and a partial internal transfer of the pro-3S hydrogen (6, 7). This step is seemingly followed by the ligase-mediated activation to the corresponding CoA ester and then the transfer of {beta}-phenylalanoyl to the C-13 hydroxyl of baccatin III. The resulting intermediate (designated {beta}-phenylalanoyl baccatin III or N-debenzoyl-2'-deoxytaxol) then likely undergoes cytochrome P450-mediated hydroxylation at the side chain 2'-position to generate the isoserinoyl moiety and final N-benzoylation of this side chain (8) to complete the biosynthesis of Taxol. cDNAs encoding the two transferases involved in C-13 side chain assembly have been described previously (8, 9).



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  		FIG. 1.
Outline of the late stages of Taxol biosynthesis and the semisynthesis of Taxol and the proposed MIO-dependent mechanism of PAM catalysis. A, sequence shows isomerization of 2S-{alpha}-phenylalanine to 3R-{beta}-phenylalanine, conversion of 3R- and 3S-{beta}-phenylalanine to 2S-{alpha}-phenylalanine, and {alpha},{beta}-elimination of ammonia from 2S-{alpha}-phenylalanine to trans-cinnamic acid catalyzed by the multifunctional Taxus phenylalanine ammonia lyase (a); thioesterification of 3R-{beta}-phenylalanine by a putative acyl group:coenzyme A ligase (b); transfer of {beta}-phenylalanoyl group from the CoA ester to baccatin III by the baccatin III 13-O-phenylpropanoyltransferase (c); conversion of 10-deacetylbaccatin III (10-DAB) to baccatin III by a 10{beta}-acetyltransferase (d); hydroxylation at C-2' of the taxoid side chain by a putative cytochrome P450 hydroxylase to N-debenzoyltaxol (g); and final N-benzoylation by a taxane N-benzoyltransferase (h). The semisynthesis of Taxol begins with conversion of 10-deacetylbaccatin III to baccatin III by semisynthesis (d) followed by 7-hydroxyl protection as the triethylsilyl (TES) derivative, coupling to a synthetic {beta}-lactam precursor of the N-benzoyl phenylisoserine side chain (e), and final deprotection (f). B, proposed mechanism of MIO-dependent catalysis by PAM is shown. Shown is the 1,4-addition of the aromatic ring to the MIO moiety (i) followed by elimination of the pro-3S benzylic hydrogen ion (j) and, last, either elimination of ammonia to form cinnamic acid or elimination then vicinal migration and final 1,4-Michael addition of ammonia to a cinnamate type intermediate to form 3R-{beta}-phenylalanine (k). B, putative general base residue within aminomutase active site; EE, 1-ethoxyethoxy; Bz, benzoyl; Enz, phenylalanine aminomutase enzyme; EnzB, enzyme base; Hs, pro-3s hydrogen of 2-{alpha}-phenylalanine; Ac, acetyl.

 
The relative abundance of {beta}-phenylalanine and side chaindeficient late pathway metabolites such as baccatin III and 10-deacetylbaccatin III in vivo (10) suggests that either the CoA ligase for {beta}-phenylalanine or the CoA ester-dependent {beta}-phenylalanoyltransferase (9), both of which function downstream of the aminomutase, may be rate limiting in side chain assembly and, thus, in Taxol biosynthesis. Because the phenylalanine aminomutase (PAM)1 catalyzes the first step of the side chain assembly process and shares its primary metabolite substrate, phenylalanine, with several competing, non-taxoid phenylpropanoid pathway enzymes in plants (11–13), it is therefore an important target for genetic engineering in yew or derived cell cultures to increase Taxol production yields. PAM is also of interest enzymologically because the reaction that is catalyzed is unusual, and, in addition to the adenosylcobalamin-dependent leucine 2,3-aminomutase from Andrographis paniculata and potato tubers (14–16), it is the only other aminomutase of plant origin described thus far.

Amino acid aminomutases are presently categorized mechanistically as 1) radical-based catalysts that use either cobalamin and pyridoxal 5'-phosphate (PLP) (17, 18) or S-adenosylmethionine, PLP, and iron-sulfur clusters (19) as cofactors, 2) ATP-dependent enzymes (20), or 3) enzymes that require no cofactors and rely solely on the electronegativity of an autocatalytically formed 4-methylideneimidazol-5-one (MIO) functional group within the active site (21) (see Fig. 1B). A recently cloned and functionally characterized tyrosine aminomutase (TAM) from Streptomyces globisporus (21) falls into the latter category and shows high sequence homology to a family of phenylalanine ammonia lyase (PAL) and histidine ammonia lyase (22) that contains a signature Ala-Ser-Gly motif, which rearranges to the MIO moiety (21). This recent information about the TAM mechanism and prosthetic group requirement coupled with the previous biochemical analysis of PAM from Taxus brevifolia cell free extracts (which also requires no external cofactors (6, 7)) suggests that the aminomutase from Taxus may share the same mechanism and overall peptide sequence homology with TAM and plant PALs. This assumption led to a homology-based strategy for cloning the corresponding gene. An expressed sequence tag project based on a cDNA library derived from Taxus cuspidata cells induced for taxoid biosynthesis with methyl jasmonate (10) revealed an abundant PAL-like sequence (23), and functional expression of the full-length form in Escherichia coli readily afforded the cDNA encoding PAM.

During the course of this investigation, a patent was issued to Steele et al. (24) that described the purification, characterization, and microsequencing of native PAM obtained from induced Taxus chinensis cells and the isolation of the corresponding PAM cDNA by a reverse genetic approach. In this study, we describe the characterization of the PAM cDNA and recombinant enzyme from T. cuspidata and report on the substrate selectivity, mechanism, and stereochemistry of the reaction, and we compare the properties of the recombinant PAM from T. cuspidata with those of the native enzyme from T. chinensis and the tyrosine aminomutase from Streptomyces.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Enzymes, Substrates, and Reagents—Unless otherwise indicated, enzymes and vectors were obtained from Invitrogen. 2R- and 2S-{alpha}-phenylalanine, 2S-{alpha}-tyrosine, S-{alpha}-aminocyclohexanepropionic acid hydrate, and (–)-camphanic acid chloride were purchased from Aldrich, and 3R- and 3S-{beta}-phenylalanine were purchased from Peptech (Burlington, MA). Deuterium-labeled samples (2S,3S)-[ring-2,3-2H7]-(90%+ enantiomeric excess), (2SR)-[ring-2-2H6]-, (2SR,3RS)-[ring-3-2H6]-, and (2SR)-[ring-3,3-2H7]{alpha}-phenylalanines (each ≥98% deuterium-enriched) and R- and S-[ring-3-2H6]{beta}-phenylalanines (96% enantiomeric excess (each ≥98% deuterium enriched)) were gifts from Heinz Floss (University of Washington, Seattle, WA). A mixture of cis- and trans-cinnamic acid was synthesized from trans-cinnamic acid by an established photochemical procedure (25). All other reagents and chemicals were purchased from Aldrich or Sigma, unless noted otherwise, and used without further purification.

Acquisition of Full-length PAL-like Clone by 5'-RACE—Based on the assumption that the phenylalanine aminomutase would share sequence similarity with tyrosine aminomutase, which exhibits significant homology with ammonia lyases but not with cofactor-dependent aminomutases (21), an annotated expressed sequence tag library (23) prepared from mRNA isolated from T. cuspidata cells elicited with methyl jasmonate for increased Taxol production (10, 26) was screened for PAL-like sequences. The library contained a 5'-truncated cDNA sequence (designated PAL1981) that was related closely (50–70% similarity) to functional PAL sequences (GenBankTM accession nos. X15473 [GenBank] , AY443341 [GenBank] , and M84466 [GenBank] ) and tyrosine aminomutase (GenBankTM accession no. AY048670 [GenBank] ) (see Fig. 2). The 5'-truncated PAL1981 cDNA in the original pBluescript vector was sequenced fully, and the full-length version was obtained by 5'-RACE (version 2.0, Invitrogen). Thus, from the sequence of the partial cDNA, three gene-specific reverse primers were designed and synthesized for nested PCR: 5'-CAA GAG TTT CCC CAG CCC-3' (GSP1), 5'-GGC GAC TGC GAT GCG CAC GTA GTC-3' (GSP2), and 5'-AAT TCG CAC CAT GGA GAG CCC-3' (GSP3). The 5'-RACE abridged anchor forward primer (AAP) 5'-GGC CAC GCG TCG ACT AGT ACG GGI IGG GII GGG IIG-3' was supplied by the manufacturer (Invitrogen).



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  		FIG. 2.
Alignment of PAM with related ammonia lyases and aminomutases. The deduced amino acid sequence of PAM from T. cuspidata (cusp) (PAM_cusp, GenBank accession no. AY582743 [GenBank] ) was aligned with those of phenylalanine aminomutase acquired from T. chinensis (chin) (PAM_chin (24)), TAM from S. globisporus (GenBank accession no. AY048670 [GenBank] ), seven PALs of plant origin (PAL_1 from Nicotiana tabacum (GenBankTM accession no. M84466 [GenBank] ), PAL_2 from Petroselinum crispum (GenBankTM accession no. X15473 [GenBank] ), and PAL_3 from Quercus suber (GenBankTM accession no. AY443341 [GenBank] )), and one histidine ammonia lyase from Pseudomonas putida (GenBankTM accession no. P21310 [GenBank] ). Residues boxed in black indicate positional identity for at least five compared sequences, and those boxed in gray are physiochemically or structurally conserved. *, the conserved ASG motif that forms the catalytic MIO moiety; #, the putative carboxylate binding residues Tyr-80 and Arg-325; @, the reported amino group binding residue Asn-231; ^, residues presumed to be involved in deprotonation of the substrate.

 
Briefly, the product of first strand synthesis, using 5 µg of mRNA and 2 pmol of primer GSP1, was purified by gel permeation (spin column, Edge Biosystems) and employed for 3'-homopolymer-C tailing and second strand amplification with primers AAP and GSP1 and Taq polymerase (94 °C for 1 min and then 35 cycles at 94 °C for 45-s denaturation, 50 °C for 45-s annealing, and 72 °C for 2-min extension, with a final extension at 72 °C for 10 min). The PCR product of appropriate size was gel-purified, extracted (gel extraction kit, Qiagen, Valencia, CA), and employed in second and third round PCR (same conditions), but reverse primers GSP2 and GSP3 were substituted, respectively, for GSP1, and then the PCR products were gel-purified as before. The final double-stranded cDNA product of the appropriate size (≥1000 bp) was excised and purified as before and ligated into PCR 2.1-TOPO to transform E. coli TOP 10' cells for kanamycin selection and PCR-based insert screening using M13 forward and reverse primers as described by the manufacturer (TOPO TA cloning kit) (Invitrogen).

Positive transformants were grown for plasmid preparation (miniprep kit) (Qiagen), sequencing, and analysis (version 10.0, GCG Wisconsin package, Madison, WI) to compare the newly acquired sequence with phenylalanine ammonia lyase sequences and the original partial PAL1981 gene sequence. Following verification, forward primer 1981FOR (5'-ATG GGG TTT GCC GTG GAA TCG CG-3') and reverse primer 1981REV (5'-TCA CGC AGA TTT GTT CCA AAC ACT TTG GAA CGA CTC G-3') were prepared to isolate the full-length gene from the original Taxus canadensis transcript pool. Reverse transcription was carried out as for the 5'-RACE procedure, except that primer 1981REV was used in place of GSP1. The full-length cDNA was obtained by PCR amplification of the single strand antisense product using the 1981FOR and 1981REV primers and Pfu polymerase (PCR conditions were essentially the same as before, except the extension time at 72 °C was increased to 4.5 min). The resulting amplicon (~2000 bp) was gel-purified and excised as before.

Heterologous Expression—For directional ligation of the full-length PAL1981 clone into pET32b(+), which was digested previously with NdeI and BamHI, sticky-end primers consisting of pair 1 (1981NDE1F (5'-TGG GGT TTG CCG TGG AAT CGC GTT C-3') and 1981BAM1R (5'-GAT CCT CAC GCA GAT TTG TTC CAA ACA CTT TGG-3') and pair 2 (1981NDE2F (5'-TAT GGG GTT TGC CGT GGA ATC GCG TTC-3') and 1981BAM2R (5'-CTC ACG CAG ATT TGT TCC AAA CAC TTT GG-3') were used to install 5'-NdeI and 3'-BamHI overhangs by a PCR method described previously (27). Additionally, primers 1981XHO1R (5'-TCG AGC GCA GAT TTG TTC CAA ACA CTT TGG-3') and 1981XHO2R (5'-GCC CAG ATT TGT TCC AAA CAC TTT GG-3') were used in place of 1981BAM1R and 1981BAM2R, respectively, to delete the stop codon and incorporate a 3'-XhoI overhang and to permit ligation into appropriately digested pET32b(+) for the expression of PAL1981 as an N-terminal His6-tagged fusion. pET vector containing the PAL1981 gene insert (designated pET1981 or pET1981His for expression as the His6 fusion) was used to transform E. coli BL21(DE3) cells for ampicillin selection to provide BL21(DE3)/pET1981 or pET1981His. Positive transformants were grown for plasmid preparation, determination of proper insert orientation, and sequence verification (with T7 promoter and T7 terminator primers).

For the overexpression of clone pET1981 or pET1981His, 1 liter of Luria-Bertani medium (in a 3-liter flask) was inoculated with 1 ml of an E. coli culture, grown overnight, consisting of BL21(DE3)/pET1981 or pET1981His. The inoculum was grown in the medium for ~3 h to A600 = 0.5 at 37 °C with ampicillin selection. Expression was then induced with 1 mM isopropyl-D-thiogalactoside, and the transformed bacteria were grown for 18 h at 15 °C.

Purification of Recombinant Aminomutase—The following procedures were carried out at 4 °C. After expression, the bacteria were harvested by centrifugation at 5000 x g (10 min), resuspended in 50 ml of sonication buffer (50 mM Tris-HCl, pH 7.5), and lysed by brief sonication (five 20-s bursts at 50% power (Misonix sonicator, Farmingdale, NY)) followed by removal of cellular debris by centrifugation at 45,100 x g (2 h). The supernatant was either dialyzed (using Spectra/Por 10,000-Da cut-off tubing (Spectrum, Rancho Domingez, CA)) against 25 mM sodium phosphate buffer (pH 8.5), concentrated (with a Stir Cell concentrator using a YM10, 10,000-Da cut-off membrane (Amicon)) to 3–5 ml without further purification, and used directly for assays, or the clarified supernatant containing overexpressed His6-tagged protein from pET1981His was purified by affinity chromatography using a His-Select nickel affinity gel (Qiagen) according to the batch procedure described by the manufacturer. The aminomutase was eluted from the affinity gel with 50 mM histidine, and then the eluant was dialyzed and concentrated as before. The protein purity was assessed by SDS-PAGE according to Laemmli (28), and protein concentrations (~2.5 µg/µl) were determined by the Bradford method or by Coomassie Blue staining (29).

Enzyme Assays—Typical assay mixtures contained 500 µM 2S-{alpha}-phenylalanine and 25–50 µg of enzyme and were supplemented with 25 mM sodium phosphate buffer (pH 8.5) to bring the volume to 500 µl. The assay mixtures were incubated for 1 h at 28 °C, the reaction was terminated by the addition of 1 N NaOH (to pH >10), and the mixtures were then treated with 2.5 equivalents of acetic anhydride (undiluted) to N-acetylate the {alpha}- and {beta}-phenylalanines. For stereochemical analysis of the {beta}-phenylalanine product of the mutase reaction, chiral auxiliary (–)-camphanic acid chloride at 10 mg/ml in tetrahydrofuran was used in place of acetic anhydride as the acyl group donor. After derivatization, the mixture was acidified to pH 2 with HCl and extracted twice with 1 ml of ethyl acetate. The organic fraction was treated with excess diazomethane to make the amino acid methyl esters. The organic solvent was removed in vacuo, and the residue was resuspended in 100 µl of ethyl acetate. A 1-µl aliquot of this material was loaded onto an HP 5HS GC column (0.25-mm inner diameter x 30 m, 0.25-µm film thickness) (Agilent, Palo Alto, CA) mounted in a GC (model 6890N, Agilent) coupled to a mass selective detector (model 5973 inert®, Agilent). The GC conditions were as follows. Column temperature was programmed from 70 °C (3 min hold) to 320 °C at 10 °C/min and then a 3-min hold at 320 °C, splitless injection was selected, and helium was used as the carrier gas (1.2 ml/min). The mass detector was operated in the scan mode (50–380 atomic mass units) with 70 eV ionization potential. To calculate the isotopic abundance of the product isolated from mutase assays in which the substrates were deuterium-labeled, the ratios of diagnostic MS fragment ions (P – 1:P:P + 1) of the product were assessed by comparison with the natural abundance distribution of fragment ions of the authentic standard.

Enzyme Characterization, Stereochemical Analysis, and Substrate Specificity—Assays were performed after evaluating the optimal protein concentration (1.5 µM) and reaction time between 45 min and 1 h to ensure linear reaction rates at the lowest substrate concentration. Kinetic parameters were obtained with KALEIDAGRAPH version 3.08 (Synergy Software, Reading, PA) by fitting all initial rate data to the Michaelis-Menten equation using nonlinear regression (R2 = 0.97). For determination of the pH optimum, single time point assays containing ~50 µg of enzyme, purified by nickel affinity column chromatography according to the manufacturer's protocol (Sigma) and dialyzed as described, was diluted to 500 µl with 25 mM sodium phosphate (pH 5.0 –9.0) or 25 mM CAPSO (pH 9.0 –10.5) buffer at increments of 0.5 pH units.

To assess the conferred configuration at C-3 after amino group transfer from C-2 of 2S-{alpha}-phenylalanine and to track the fates of the hydrogens, (2S,3S)-[ring-2,3-2H7]-, (2SR)-[ring-2-2H6]-, (2SR,3RS)-[ring-3-2H6]-, and (2SR)-[ring-3,3-2H7]{alpha}-phenylalanines were employed as substrates at 500 µM in the standard assay. The cofactor dependence of the reactions was assessed by incubating the enzyme under standard conditions with 500 µM substrate and either ATP, S-adenosylmethionine and PLP, or PLP alone, each at 1 mM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Cloning and Sequence Analysis of PAM—Aminomutases are a subgroup of isomerases that typically catalyze the vicinal interchange of an amino group and hydrogen. These mutases cleave and form bonds during catalysis either via a radical-dependent homolytic mechanism or via an ion-dependent heterolytic mechanism. The former reaction requires pyridoxal phosphate and either cobalamin (30, 31) or S-adenosylmethionine (32, 33) as cofactors, whereas the latter reaction employs either exogenous pyridoxal phosphate (34) or ATP (20, 35) or initiates mutase activity by the action of an internal electronegative MIO moiety formed autocatalytically from a conserved active site motif (Asp-Ser-Gly) and requires no external cofactors (21) (Fig. 1B). The only MIO-dependent aminomutase described to date is the TAM, which shows significant homology (56% similarity) to several members of the well known ammonia lyase family but not to the cofactor-dependent aminomutases (21). PAM activity in cell-free extracts of Taxus requires no exogenous cofactors (6), suggesting a mechanism that is fundamentally different from that of cofactor-dependent aminomutases and more likely related to that of TAM.

The evaluation of sequences in an expressed sequence tag library (23) derived from mRNA isolated from T. cuspidata cells (induced for taxoid biosynthesis with methyl jasmonate (10)) revealed an abundant but 5'-truncated PAL-like sequence designated PAL1981. The 5'-terminus of the partial PAL1981 was used to design primers for 5'-RACE to obtain the full-length cDNA (GenBankTM accession no. AY582743 [GenBank] ). Clone PAL1981 (shown subsequently to encode PAM, see below) has an open reading frame of 2094 base pairs, encodes a protein of 698 amino acids with a calculated molecular mass of 76,530 Da, and shares significant sequence homology (50–70% similarity) with several PAL and the TAM sequences deposited in the GenBankTM data base (Fig. 2).

Expression, Purification, Functional Confirmation, and Stereochemical Analysis of the Recombinant PAM—PAL1981 was overexpressed as an N-terminal His6 fusion protein in E. coli BL21(DE3) and yielded approximately 10% of the target protein as inclusion bodies (as determined by SDS-PAGE) with approximately 20 mg of target protein/liter in operationally soluble form (migrating on SDS-PAGE with the expected size of ~80 kDa). The recombinant protein was purified to near homogeneity by His-Select nickel affinity chromatography and shown to possess PAM activity by cofactor-independent conversion of {alpha}-phenylalanine to {beta}-phenylalanine, as determined by GC-MS analysis of the derived N-acetyl methyl ester. The calculated Km value (Michaelis-Menten plotting and non-linear regression curve fitting (R2 = 0.97) (KALEIDAGRAPH version 3.08) for the recombinant enzyme was ~45 ± 8 µM for 2S-{alpha}-phenylalanine, with a kcat value of 0.015 s–1 and a pH optimum at approximately pH 8.5, which is similar to the pH optima reported for TAM (21) and aromatic ammonia lyases (36). PAM seems to be highly specific for 2S-{alpha}-phenylalanine as a substrate; neither 2R-{alpha}-phenylalanine, S-{alpha}-tyrosine, nor S-{alpha}-aminocyclohexanepropanoic acid hydrate was detectably isomerized to the corresponding {beta}-isomer by this recombinant enzyme. Furthermore, no significant change in activity was detectable when either pyridoxal phosphate, S-adenosylmethionine, or ATP, each at 1 mM, was added to PAM assays under standard conditions and compared with the activity observed in assays lacking these cofactors. Although the present data suggest that PLP is not required for catalysis, ongoing structurefunction analysis and mutagenesis of the phenylalanine aminomutase will ultimately provide additional information to investigate thoroughly whether PLP is cryptically present as a tightly bound prosthetic aldimine group situated at an active site lysine.

To assign the absolute configuration of the enzymatically formed {beta}-phenylalanine product, recombinant PAM was incubated with 2S-{alpha}-phenylalanine under standard assay conditions, and the resulting product mixture was treated with (–)-camphanic acid chloride and diazomethane to convert the amino acids to the corresponding (–)-camphanamide methyl esters for subsequent analysis by GC-MS. The sample contained an analyte with an identical retention time (21.35 min) and corresponding fragment ions, by GC-MS analysis, as that of authentic N-[(–)-camphanoyl]R-{beta}-phenylalanine methyl ester; no detectable analyte was observed at 21.50 min corresponding to the derivatized S-{beta}-diastereoisomer. Therefore, the configuration of the {beta}-phenylalanine was established as the R-enantiomer.

Deuterium-labeled {alpha}-phenylalanines were employed to assess the steric course of the enzymatic reaction. (2S,3S)-[ring- 2,3-2H7]-, (2SR,3RS)-[ring-3-2H6]-, (2SR)-[ring-2-2H6]-, and (2SR)-[ring-3,3-2H7]{alpha}-phenylalanine were incubated individually with recombinant PAM, and the resulting product mixture was treated with acetic anhydride and diazomethane to convert the resulting amino acids to the corresponding acetamide methyl esters for GC-MS analysis. The structural assignment of two diagnostic fragment ions (m/z 178 (P – CH3CO) and m/z 148 (P – CH2COOCH3), designated P178 and P148, respectively) derived from the parent ion (P = m/z 221, very weak) of an authentic unlabeled standard allowed for quantitative analysis of isotopic enrichment and distribution (Fig. 3). Each labeled {alpha}-phenylalanine substrate contained five deuteriums on the aromatic ring as well as containing deuterium on the propanoate side chain to trace the fates of the hydrogens on the ring and at the {alpha}- and {beta}-carbons. Incubation with (2S,3S)-[ring-2,3-2H7]{alpha}-phenylalanine yielded product that on MS fragmentation of the parent ion generated diagnostic ions P153 (P148 + 5), carrying five deuteriums, and P184 (P178 + 6 (100% relative abundance)), and P185 (P178 + 7 (~50% relative abundance)), carrying six and seven deuteriums ions, respectively (Fig. 3). The data indicated that in addition to carrying five deuteriums on the aromatic ring, the propanoyl side chain of the {beta}-isomer product contained either one or two deuteriums at C-2, and thus the deuterium present originally at C-3 of the substrate was removed and underwent at least partial intramolecular migration (~50%) to C-2 during catalysis (Fig. 3). In contrast, the incubation of PAM with (2SR,3RS)-[ring-3-2H6]{alpha}-phenylalanine2 yielded the {beta}-isomer possessing six deuterium atoms in both ions P184 and P154 (P148 + 6), thus indicating that the deuterium remained at C-3 of the propanoate side chain in the course of the reaction (Fig. 3). Incubations with (2SR)-[ring-2-2H6]- and (2SR)-[ring-3,3-2H7]{alpha}-phenylalanines2 were also conducted to verify the stereochemical analysis. The (2SR)-[ring-2-2H6]-isotopomer gave product for which the MS fragment ions contained P184 (P178 + 6) and P153 (P148 + 5) as expected, indicating that the {beta}-amino isomer carried at C-2 the deuterium that was originally at this carbon of the substrate. As the substrate, (2SR)-[ring-3,3-2H7]{alpha}-phenylalanine gave product for which the MS fragment ions contained P184 (P178 + 6 (100%)), P185 (P178 + 7 (~50%)), and P154, indicating a distribution of deuterium that was identical to that found in the product derived from the (2S,3S)-[ring-2,3-2H7]-isotopomer (Fig. 3) and supporting the proposed intramolecular migration mechanism. These results suggest that the mutase reaction proceeds with removal of the pro-3S hydrogen from the substrate followed by migration of the amino group from C-2 to C-3 with retention of the configuration. The reaction is completed by the addition at C-2 of a hydrogen for which the former pro-3S hydrogen of the substrate serves as a partial source.



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  		FIG. 3.
Mass spectral fragment ions of N-acetyl-{beta}-phenylalanine methyl esters. Diagnostic fragment ions of N-acetyl-{beta}-phenylalanine methyl ester detected by electron impact mass spectrometric analysis result from the cleavage of the N-acetyl group to form the {beta}-amino propanoyl methyl ester ion at m/z 178 (P178, base peak) (route A) and from cleavage between the {alpha}- and {beta}-carbons to form an N-acetyl benzenemethanimine ion at m/z 148 (P148) (route B). A most useful and abundant diagnostic ion from N-acetyl- or N-[(–)-camphanoyl]{alpha}-phenylalanine methyl esters results from the {alpha},{beta}-elimination of a proton (without distinction for H1 or H2) and the amide to form a cinnamate methyl ester ion m/z 162 (P162). Also shown are the four deuterium-labeled {alpha}-phenylalanine substrates used (2S,3S)-[ring-2,3-2H7]-, (2SR,3RS)-[ring-3-2H6]-, (2SR)-[ring-2-2H6]-, and (2SR)-[ring-3,3-2H7]{alpha}-phenylalanines and designated, respectively, as (2S,3S), (2SR,3RS), (2SR-ring-2-D6, and (2SR)-ring-3,3-D7, along with) the diagnostic isotopomer fragment ions derived from the corresponding {beta}-phenylalanine products (see "Experimental Procedures" for further detail).

 

Interestingly, trans-cinnamic acid was produced by PAM at a rate of ~10% relative to {beta}-phenylalanine, with {alpha}-phenylalanine as substrate at maximal velocity. A similar observation was made with tyrosine aminomutase, which converts {alpha}-tyrosine to 4-hydroxycinnamic acid in increasing concentration over time (21).

The reverse mutase reaction was examined by employing R- and S-[ring-3-2H6]{beta}-phenylalanines as substrates under standard assay conditions. After a 1-h incubation and treatment of the product mixture with (–)-camphanic acid chloride and diazomethane to convert the amino acids to the corresponding camphanamide methyl ester derivatives, analysis by GC-MS revealed that both {beta}-enantiomers were converted exclusively to 2S-{alpha}-phenylalanine. Surprisingly, the S-{beta}-enantiomer (with stereochemistry opposite to that of the PAM forward reaction product) was converted by the mutase to 2S-{alpha}-phenylalanine much faster (Vrel = 100) than the naturally occurring R-{beta}-enantiomer (Vrel = 5). GC-MS analysis of authentic N-camphanoyl 2S-{alpha}-phenylalanine methyl ester revealed a weak parent ion (P = m/z 359) and a diagnostic fragment ion at m/z 162 (P–(camphanoyl–NH) minus either hydrogen at C-3, designated P162) (Fig. 3). Similar analysis of the deuterium-labeled {alpha}-phenylalanine product isolated from the reverse catalysis reaction assays revealed fragment ions P167 (P162 + 5) and P168 (P162 + 6) in equal abundance, thus demonstrating that the product carries deuterium and hydrogen in a 1:1 ratio at C-3 and indicating that the hydrogen present originally at C-3 of the {beta}-isomer does not migrate during the course of the reverse reaction.

Inhibition—The MIO prosthetic group formed posttranslationally from the ASG sequence motif at the active site of aromatic ammonia lyases (31, 38–40) and S-tyrosine aminomutase (21) is sensitive to borohydride reduction and chemical modification by cyanide. When PAM was pretreated for 15 min with either 1 mM potassium cyanide or 1 mM sodium borohydride before adding 1 mM 2S-{alpha}-phenylalanine to initiate the reaction, mutase activity was completely abolished. However, partial protection against inactivation by potassium cyanide (60% protection) and sodium borohydride (30% protection) was obtained when saturating levels of substrate were added prior to the addition of the chemical modifiers compared with a control assay without inhibitors.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
Amino acid {alpha},{beta}-aminomutases, which catalyze the exchange of the amino group and an adjacent hydrogen, include lysine 2,3-aminomutase (32), leucine 2,3-aminomutase (15), arginine 2,3-aminomutase (41), and TAM (21, 42). Functionally expressed in E. coli, the PAL1981 cDNA acquired from a T. cuspidata library has demonstrated that this newly acquired clone encodes PAM, which is required for the biosynthesis of R-{beta}-phenylalanine, an obligatory intermediate for the assembly of the C-13 N-benzoyl phenylisoserine side chain of Taxol. A comparison of the deduced amino acid sequence of PAM from Taxus with that of TAM (56% similarity) and ammonia lyases (50–70% similarity) revealed that there is significant homology and that PAM shares the signature 175ASG177 sequence motif found in TAM and in the histidine ammonia lyase/PAL family. These similarities suggest a common mechanism in the vicinal interchange catalyzed by the mutases PAM and TAM and of the elimination of ammonia catalyzed by the related lyases. The active site ASG motif undergoes posttranslational modification to an electronegative MIO moiety. The accepted mechanistic model for MIO catalysis proposes nucleophilic attack as a 1,4-Michael addition of the aromatic ring ortho-carbon on the exocyclic methylidene of the MIO (21, 42) (see Fig. 1B). Electron flow promotes carbocation formation at the ipso-carbon of the aromatic ring, thus dramatically lowering the pKa of the benzylic hydrogens at C-3. Stereospecific removal of a hydrogen then initiates propanoate side chain conversion to the corresponding trans-olefinic acid via the elimination of ammonia (43–45) or to the corresponding {beta}-amino acids (42) through hydrogen-amino group interchange. Potassium cyanide and sodium borohydride, both of which are known to abolish selectively the function of the MIO in ammonia lyases (43), completely eliminated PAM activity, thus suggesting that this aminomutase is MIO-dependent. Also suggestive of the function of the MIO prosthetic group of PAM is the inability to convert the ring saturated analog 2S-{alpha}-aminocyclohexanepropanoic acid to the corresponding {beta}-isomer; this substrate lacks reactive nucleophilic ring electrons required to initiate catalysis by attack on the MIO moiety.

Other aminomutases that have been subjected to stereochemical analysis include lysine 2,3- (46), {beta}-lysine 5,6- (46), arginine 2,3- (41), and tyrosine 2,3-aminomutase (21, 42), each of which has been shown to invert stereochemistry at the carbon to which the amino group migrates. The stereochemical course of the reaction catalyzed by the recombinant PAM (and by the native enzyme from T. brevifolia (6, 7)) is different in that enantiospecific isomerization of 2S-{alpha}-phenylalanine to 3R-{beta}-phenylalanine (the only detectable product under standard assay conditions) is accomplished with retention of configuration and of the pro-3R-hydrogen at C-3. Jointly, the pro-3S hydrogen is removed from C-3 and migrates partially (50%) to C-2 of the phenylpropanoid substrate (6); as yet, the stereochemistry of the pro-3S hydrogen attachment at C-2 has not been evaluated. The {beta}-phenylalanine enantiomer produced by PAM has the identical amino group conformation as that of the phenylisoserine side chain of Taxol. 2R-{alpha}-Phenylalanine (the unnatural enantiomer), 2S-{alpha}-tyrosine, and S-{alpha}-aminocyclohexanepropanoic acid are not detectably isomerized by PAM, indicating that this enzyme has very strict structural and/or chemical requirements for the phenylpropanoid substrate.

Interestingly, PAM also catalyzed the enantio-specific reverse reaction, as demonstrated by conversion of both 3R- and 3S-{beta}-phenylalanine to the same 2S-{alpha}-phenylalanine enantiomer. In this reverse reaction, the deuterium at C-3 of both 3R- and 3S-[ring-3-2H6]{beta}-phenylalanines was retained completely when the amino group formerly at C-3 was exchanged with a hydrogen; thus, it appears that PAM can remove the amino group from either the si or re face of the {beta}-amino acid with reattachment to the same re face to form 2S-{alpha}-phenylalanine. Intriguingly, the enzyme selectively converted the 3S-{beta}-substrate (the "unnatural" isomer) 20 times faster than the 3R-{beta}-enantiomer to 2S-{alpha}-phenylalanine. This observation may be physiologically relevant in light of the observation that {beta}-phenylalanine extracted from stem tissue of T. brevifolia saplings is a mixture of R- and S-stereoisomers, with the unnatural S-isomer of unknown origin predominating (6). Thus, by a combination of reverse and forward reactions, the mutase could effectively epimerize the 3S-{beta}-isomer to the 3R-configuration needed for Taxol biosynthesis. Notably, TAM from Streptomyces produces both R- and S-{beta}-tyrosine in a 1:1 ratio from 2S-{alpha}-tyrosine during a 4–5-h incubation (42). By contrast, recombinant PAM from Taxus appeared to produce only the R-{beta}-enantiomer even after 24-h incubation; however, the rapid reverse conversion of S-{beta}-phenylalanine back to substrate may preclude observation of this unnatural product in the assay.

Comparison of PAM from T. cuspidata with that acquired previously from T. chinensis (24) revealed that these enzymes are 98.9% identical at the amino acid level and that they share, as expected, the signature active site motif 175ASG177. They also share conserved substrate binding residues (see Fig. 2) (functional assignment based on positional inference from the crystal structure of histidine ammonia lyase from Pseudomonas (22)); that is, PAM Arg325 and Tyr80 are presumed to be involved in binding the carboxylate of the substrate, Asn231 likely binds the amino group, and Asp460 or Tyr322 are suitably oriented for deprotonation of the benzylic carbon to initiate isomerization.

The native aminomutase reported previously from T. chinensis (PAM_chin) possesses a catalytic efficiency of kcat/Km = 2.1 mM–1 s–1 (calculated from reported Km and kcat values at 1.1 mM and 2.3 s–1 (24), respectively) that is substantially higher than that calculated here for the recombinant PAM (PAM_ cusp) at kcat/Km = 0.333 mM–1 s–1; however, the latter is remarkably similar to the efficiency reported for the tyrosine aminomutase at kcat/Km = 0.360 mM–1 s–1. Examination of the encoded amino acid sequences reveals that the PAM_chin cDNA clone acquired previously (24) is slightly shorter (687 amino acids) than the aminomutase reported here (698 amino acids) by 11 residues at the C terminus (Fig. 2, compare results). If the native PAM is derived from the reported 3' truncated allele isolated from T. chinensis cell cultures, then the difference in kinetic efficiency between the native enzyme (24) and the recombinant aminomutase reported here is perhaps a consequence of the C-terminal truncation, thus suggesting a critical role for the 11 C-terminal amino acids in moderating activity.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
Taxus PAM is a multifunctional enzyme that catalyzes the isomerization of 2S-{alpha}-phenylalanine to 3R-{beta}-phenylalanine with retention of configuration at C-3, the reverse reaction for the conversion of both R- and S-{beta}-phenylalanine to the same 2S-{alpha}-phenylalanine product, and the {alpha},{beta}-elimination of ammonia from the {alpha}-isomer to form trans-cinnamic acid (no cis-cinnamate was detected as a reaction product). The aminomutase has a Km = 45 µM and a kcat = 0.015 s–1, which are similar to those for mechanistically related tyrosine aminomutase (42) and phenylalanine ammonia lyase (37). PAM represents a second MIO-dependent aminomutase that is similar in primary structure and mechanism to related ammonia lyases (21, 42). The functional interplay of the various PAM catalytic routes makes this gene an important target for mutational analysis to assess which residues define the reaction coordinate for each catalytic outcome, with the goal of minimizing the elimination reaction to optimize the expression of PAM for increased yield of {beta}-phenylalanine for transgenic application in increasing the production of Taxol and its analogs in cell culture (10). Investigations of the tertiary structure by x-ray crystallographic analysis and assessment of the subunit architecture of PAM are presently being pursued.

In addition to the two acyl-CoA transferases noted previously (8, 9), the PAM cDNA represents the third defined gene required for Taxol side chain assembly. At least two additional steps represented by the coenzyme A ligase for {beta}-phenylalanine and the N-debenzoyl-2'-deoxytaxol 2'-hydroxylase remain to be elucidated and the corresponding genes acquired.


   FOOTNOTES
 
* This work was supported by startup funds from the Michigan State University College of Natural Sciences and by National Institutes of Health Grant CA-55254. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| Present address: Div. of Food Additives, National Institute of Health Sciences, Tokyo 158-8501, Japan. Back

§ To whom correspondence should be addressed: 320 Chemistry, Dept. of Chemistry, Michigan State University, East Lansing, MI 48824. Tel.: 517-355-9715; Fax: 517-353-1793; E-mail: walke284{at}msu.edu.

1 The abbreviations used are: PAM, phenylalanine aminomutase; PAL, phenylalanine ammonia lyase; PLP, pyridoxal 5'-phosphate; MIO, 4-methylideneimidazol-5-one; TAM, tyrosine 2,3-aminomutase; GC, gas chromotograpy; MS, mass spectrometry; CAPSO, 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid; RACE, rapid amplification of cDNA ends. Back

2 The selectivity of PAM for the 2S-antipode of phenylalanine suggests that the 2S-isomer is the only productive substrate in the (2SR)-[ring-2-2H6]-, (2SR)-[ring-3,3-2H7]-, and (2SR,3RS)-[ring-3-2H6]{alpha}-phenylalanine mixtures. Back



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 

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