Transcriptome-wide identification and characterization of Ornithogalum saundersiae phenylalanine ammonia lyase gene family

Article abstract of DOI:10.1039/c4ra03385j

OSW-1 is a promising antitumor glycoside present in the Ornithogalum saundersiae plant. Biosynthesis of the p-methoxybenzoyl group on the disaccharide moiety of OSW-1 is known to take place biochemically by phenylpropanoid biosynthetic pathway, but molecular biological characterization of related genes has been insufficient. Phenylalanine ammonia lyase (PAL, EC 4.3.1.24), which catalyzes the deamination of l-phenylalanine to yield trans-cinnamic acid, plays a key role in phenylpropanoid metabolism. Thus, the study on the characterization of the genes involved in the OSW-1 biosynthetic pathway, particularly the well-documented genes such as PAL, is essential to further the understanding of the biosynthesis of OSW-1. Here, transcriptomic sequencing of O. saundersiae was performed to speed up the identification of a large number of genes related to OSW-1 biosynthesis. De novo assembly of the transcriptome sequence provided 210733 contigs, 104, 180 unigenes, and four unigenes showing high similarities with PALs. Two full-length cDNAs encoding PALs (OsaPAL2 and OsaPAL62) from O. saundersiae were cloned using sequence information from these four unigenes. The PAL and tyrosine ammonia lyase (TAL) activities of recombinant OsaPAL proteins were unambiguously determined by HPLC with UV and MS detection as well as by NMR spectroscopy. Subsequently, a series of site-directed mutants were generated with the aim of improving enzyme activity and investigating the importance of particular residues in determining substrate selectivity. The results reveal that the Phe-to-His mutants, i.e., OsaPAL2F134H and OsaPAL62F128H, exhibited higher TAL activity than the corresponding wild types, providing direct evidence that the Phe residue is responsible for substrate specificity. Mutagenesis studies also demonstrated that the Thr-to-Ser mutants, i.e., OsaPAL2T196S and OsaPAL62T194S, showed significantly higher substrate affinity than the wild types. Furthermore, the Gly-to-Ala mutants, i.e., OsaPAL2G209A and OsaPAL62G207A, showed higher PAL and TAL activities. These findings provide further insight into the genes responsible for OSW-1 biosynthesis and will facilitate the future application of OsaPALs in synthetic biology.

Full text of DOI:10.1039/c4ra03385j

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PAPER
Transcriptome-wide identification and
characterization of Ornithogalum saundersiae
phenylalanine ammonia lyase gene family†
Cite this: RSC Adv., 2014, 4, 27159
*
Zhi-Biao Wang, Xi Chen, Wei Wang, Ke-Di Cheng and Jian-Qiang Kong
OSW-1 is a promising antitumor glycoside present in the Ornithogalum saundersiae plant. Biosynthesis of
the p-methoxybenzoyl group on the disaccharide moiety of OSW-1 is known to take place biochemically by
phenylpropanoid biosynthetic pathway, but molecular biological characterization of related genes has been
insufficient. Phenylalanine ammonia lyase (PAL, EC 4.3.1.24), which catalyzes the deamination of
L-phenylalanine to yield trans-cinnamic acid, plays a key role in phenylpropanoid metabolism. Thus, the
study on the characterization of the genes involved in the OSW-1 biosynthetic pathway, particularly the
well-documented genes such as PAL, is essential to further the understanding of the biosynthesis of
OSW-1. Here, transcriptomic sequencing of O. saundersiae was performed to speed up the identification
of a large number of genes related to OSW-1 biosynthesis. De novo assembly of the transcriptome
sequence provided 210 733 contigs, 104, 180 unigenes, and four unigenes showing high similarities with
PALs. Two full-length cDNAs encoding PALs (OsaPAL2 and OsaPAL62) from O. saundersiae were cloned
using sequence information from these four unigenes. The PAL and tyrosine ammonia lyase (TAL)
activities of recombinant OsaPAL proteins were unambiguously determined by HPLC with UV and MS
detection as well as by NMR spectroscopy. Subsequently, a series of site-directed mutants were
generated with the aim of improving enzyme activity and investigating the importance of particular
residues in determining substrate selectivity. The results reveal that the Phe-to-His mutants, i.e.,
OsaPAL2F134H and OsaPAL62F128H, exhibited higher TAL activity than the corresponding wild types,
providing direct evidence that the Phe residue is responsible for substrate specificity. Mutagenesis
studies also demonstrated that the Thr-to-Ser mutants, i.e., OsaPAL2T196S and OsaPAL62T194S,
showed significantly higher substrate affinity than the wild types. Furthermore, the Gly-to-Ala mutants,
i.e., OsaPAL2G209A and OsaPAL62G207A, showed higher PAL and TAL activities. These findings provide
further insight into the genes responsible for OSW-1 biosynthesis and will facilitate the future application
of OsaPALs in synthetic biology.
Received 14th April 2014
Accepted 21st May 2014
DOI: 10.1039/c4ra03385j
www.rsc.org/advances
OSW-1 (Fig. 1A), which is a natural saponin isolated from
Ornithogalum saundersiae, exhibits exceptionally potent anti-
tumor activity both in vitro and in vivo.3–5 As such, it is a
1. Introduction
Phenylalanine ammonia lyase (PAL, EC 4.3.1.24), a member of
the superfamily of ammonia lyases, catalyzes the conversion of
L-phenylalanine to trans-cinnamic acid by a non-oxidative
deamination. In recent years, considerable attention has been
paid to the presence of PALs in medicinal plants since they are
involved in the biosynthesis of the majority of phenol-contain-
ing secondary metabolites, many of which are pharmacologi-
cally active.1,2
promising lead compound for the development of novel anti-
tumor drugs; however, because of its low content in the plant
and a long and laborious synthesis,6–10 little progress has been
made in developing OSW-1 as a potential drug candidate aer
its discovery in 1992.11 Accordingly, we have initiated a project
to identify an alternative, preparative-scale method to produce
OSW-1 and to elucidate its biosynthetic pathway and the
enzymes involved.
OSW-1 is characterized by a disaccharide moiety attached
to the C-16 position of the steroid aglycone, which contains a
p-methoxybenzoyl (MBz) and an acetyl (Ac) group.11 According
to previous structure–activity relationship (SAR) studies, the
disaccharide moiety is important for the cytotoxicity of OSW-1
and, in particular, the removal of the Ac and MBz groups
decreases the activity of OSW-1 by approximately 1000-fold.3,12,13
Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union
Medical College (State Key Laboratory of Bioactive Substance and Function of
Natural Medicines & Ministry of Health Key Laboratory of Biosynthesis of Natural
Products), Beijing, 100050, China. E-mail: jianqiangk@imm.ac.cn; Tel:
+86-10-63165169
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ra03385j
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Fig. 1 (A) The structure of OSW-1 with the p-methoxybenzoyl group (dashed circle) and the enzymes responsible for OSW-1 biosynthesis. (B)
The proposed pathways for the biosynthesis of the p-methoxybenzoyl group. Dashed circles show the b-oxidation pathway and dashed boxes
show the non b-oxidation pathway. Solid arrows show the established biochemical steps and broken arrows show the hypothetical steps.
Stacked arrows indicate the involvement of multiple enzymatic reactions. (PAL, phenylalanine ammonia lyase; C4H, cinnamate-4-hydroxylase;
TAL, tyrosine ammonia lyase; CA, trans-cinnamic acid; pCA, p-coumaric acid).
Many previous studies have indicated that the p-hydroxybenzoic prokaryotes.24–26 In plants, PALs are fairly ubiquitous; moreover,
acid group is derived either from L-Phe or L-Tyr (Fig. 1B) through they can be found in monocots,27–30 dicots,1,18,19,31 gymno-
the respective PAL-catalyzed formation of trans-cinnamic acid sperms,32–35 ferns,36 lycopods,36 liverworts,36 and algae;37
or p-coumaric acid.14–16 Thus, it is likely that PALs play an however, there are no reports about PALs in the Asparagaceae
important role in this phenylpropanoid biosynthetic pathway species.
for OSW-1; however, to date, none of the PALs in O. saundersiae
Here, a gene family containing two OsaPAL genes was iso-
have been cloned or characterized.
lated for the rst time from an Asparagaceae plant, O. saun-
PAL was rst puried from Hordeum vulgare in 1961,17 and it dersiae. Aer a successful functional characterization, a series
was later found to be widespread in plants,18–20 fungi21–23 and of site-directed mutants were generated with the aim of
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improving enzyme activity and investigating the importance of glycerol, 72 mM K2HPO4, 17 mM KH2PO4) supplemented with
specic residues in determining substrate selectivity. The in appropriate antibiotics for selection.
vitro assays indicated that we successfully improved both the
PAL and TAL deamination activities of OsaPALs using a single
amino acid substitution, a discovery that we believe will provide
additional ways to improve PAL activity of other such enzymes.
We also believe that a greater understanding of how OsaPALs
participate in the biosynthesis of OSW-1 will facilitate future
applications of OsaPALs in synthetic biology.
2.3. Plant materials
O. saundersiae was grown under sterile conditions on 6,7-V
medium38 at a temperature of 22 ꢁC under a 16 h light/8 h dark
cycle. Sterile bulbs were collected and used immediately for
RNA isolation.
2.4. Transcriptome sequencing and analysis
RNA extraction and cDNA library construction were performed
as described in Kong et al.39 The resultant cDNA library was
sequenced using Illumina HiSeq™ 2000. Short nucleotide reads
obtained via Illumina sequencing were assembled using the
Trinity soware (http://www.trinity-soware.com) to produce
error-free, unique contiguous sequences (contigs). These con-
tigs were ligated to obtain non-redundant unigenes, which
could not be extended on either end. Unigene sequences were
aligned by Blast X to protein databases like NCBI nr, Swiss-Prot,
KEGG and COG (e-value < 0.00001), and aligned by Blast N to
nucleotide databases nt (e-value < 0.00001), resulting in
proteins with the highest sequence similarity with the given
unigenes along with their functional annotations.
2. Experimental methods
2.1. Substrates, chemicals and enzymes
Materials (suppliers) used in this study were as follows: L-Phe
and L-Tyr were used in the enzyme assays (Sigma-Aldrich Co.
Ltd., St. Louis, MO, USA); In-Fusion® HD Cloning Kit, restric-
tion enzymes (Takara Shuzo Co. Ltd., Kyoto, Japan) and Super
RT cDNA Kit were used to synthesize full-length cDNAs and
KOD-Plus-Neo DNA polymerases (Toyobo Co. Ltd., Osaka,
Japan); RNeasy Plant Mini Kit was used for RNA extraction
(Qiagen, Dusseldorf, Germany); Ni-Sepharose (Invitrogen,
Carlsbad, CA, USA) and Fast Mutagenesis System kit were used
for the site-directed mutagenesis (TransGen Biotech Co. Ltd.,
Beijing, China). All other chemicals used in this study were of
analytical grade.
2.5. Generation of full-length OsaPALs cDNA and sequence
analysis
Because the assembled sequences were products of de novo
assemblies, they were considered prone to error. To conrm
2.2. Strains and plasmids
Prokaryotic expression vector, i.e. pET-28a (+), which was used whether a sequence represented a true gene product, experi-
for heterologous expression, was purchased from Novagen, mental verication was performed by designing gene-specic
Madison, USA. Expression host strain Transetta (DE3) and the primers (Table 1) for the OsaPAL full-length sequences, and
cloning vector pEASY®-Blunt were purchased from TransGen then verifying the identity of amplied products by sequencing.
Biotech Co. Ltd., Beijing, China. TG1 was used as a bacterial
Total RNA isolated from sterile bulb tissue of O. saundersiae
host for recombinant plasmid amplication. The host was using an RNeasy Plant Mini Kit (Qiagen) was used as a template
grown in Luria–Bertani medium (10 g lꢀ1 Bacto-Tryptone, 5 g lꢀ1 for reverse transcription with primer oligo (dT)20 primers and
Bacto-yeast extract, 10 g lꢀ1 NaCl) or induced in TB medium (12 reverse transcriptase ReverTra Ace (TOYOBO) according to the
g lꢀ1 Bacto-Tryptone, 24 g lꢀ1 Bacto-yeast extract, 4 ml lꢀ1 manufacturer's instructions. The amplication of OsaPAL
Table 1 Primers used in gene cloning and plasmid construction
Primers
Sequences (50–30)
Description
FPAL1
FPAL2
RPAL3
RPAL4
50-CATCATAATCTGACGGTTTTC-30
50-ATGGAGAACGGCAACGGTAA-30
50-CAGAATTATGAAATTCCAGCC-30
50-TCAACATATTGGCAGCGGTGC-30
50-TCGCGGATCCGAATTCATGGAGA
ACGGCAACGGTAAC-30
Forward primer used for OsaPAL2 amplication in the rst round
Forward primer used for OsaPAL2 amplication in the second round
Reverse primer used for OsaPAL2 amplication in the rst round
Reverse primer used for OsaPAL2 amplication in the second round
Forward primer used for pET28a–OsaPAL2 construction
FET28aPAL2
RET28aPAL2
50-GTGCGGCCGCAAGCTTTCAACATA
TTGGCAGCGGTGC-30
Reverse primer used for pET28a–OsaPAL2 construction
FPAL5
FPAL6
RPAL1
RPAL2
50-CAATCAGCCGTTTACGAGACC-30
50-ATGGAATCCCTCCACGCCAAC-30
50-CCGAAGTACTGAATGAAAATC-30
50-CTAGCAAATGGGCAGGGGAG-30
50-TCGCGGATCCGAATTCATGGAAT
CCCTCCACGCCAAC-30
Forward primer used for OsaPAL62 amplication in the rst round
Forward primer used for OsaPAL62 amplication in the second round
Reverse primer used for OsaPAL62 amplication in the rst round
Reverse primer used for OsaPAL62 amplication in the second round
Forward primer used for pET28a–OsaPAL62 construction
FET28aPAL62
RET28aPAL62
50-GTGCGGCCGCAAGCTTCTAGCA
AATGGGCAGGGGAG-30
Reverse primer used for pET28a–OsaPAL62 construction
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cDNAs was performed by a nested PCR method using KOD Plus (Table 1). Aer gel purication, the PCR product was sub-cloned
Taq polymerase and gene-specic primers (Table 1). The into pET-28a (+) according to the In-Fusion® HD Cloning Kit
amplied full-length cDNAs, i.e., OsaPAL2 and OsaPAL62, were protocol. Finally, sequencing was used to verify the integrity of
each inserted into the pEASY®-Blunt vector to generate pEASY- plasmids pET28a–OsaPAL2 and pET28a–OsaPAL62 with both of
OsaPAL2 and pEASY-OsaPAL62, respectively, for sequencing.
them containing a His6 tag.
OsaPAL2 and OsaPAL62 were analyzed using online bio-
The expression plasmids pET28a–OsaPAL2 and pET28a–
informatic tools from NCBI and ExPASy. Open reading OsaPAL62 were transformed into the expression host strain
frame (ORF) nding was performed using the on-line program Transetta (DE3) grown in LB agar containing 170 mg mlꢀ1
(http://www.ncbi.nlm.nih.gov/gorf/org.cgi). Various physico- chloromycetin and 50 mg mlꢀ1 kanamycin. Aer overnight
chemical parameters of proteins were evaluated using the incubation at 37 ꢁC, a single clone of each was used to inoculate
ProtParam tool (http://web.expasy.org/protparam/). TMHMM 20 ml of LB/chloromycetin/kanamycin at 37 ꢁC, which was
(http://www.cbs.dtu.dk/services/TMHMM/) was used to predict shaken at 200 rpm until the OD at 600 nm reached a value of
the transmembrane helices of proteins, and SignalP 4.1 (http:// 1.0, diluted 1 : 50 into 50 ml TB supplemented with 170 mg mlꢀ1
www.cbs.dtu.dk/services/SignalP/) was used to predict cleavage chloromycetin and 50 mg mlꢀ1 kanamycin, and then shaken at
ꢁ
sites of signal peptides. Protein subcellular locations were pre- 200 rpm at 37 C until the OD at 600 nm reached 0.8. Subse-
dicted using TargetP 1.1 Server (http://www.cbs.dtu.dk/services/ quentꢁly, 0.1 mM IPTG was added and aer shaking at 150 rpm
TargetP/), and functional sites of proteins were analyzed with at 20 C overnight, the induced cells were harvested by centri-
PROSITE tools (http://prosite.expasy.org/). Concord (http:// fugation (7500g, 2 min) at 4 ꢁC. The pellets were then either
helios.princeton.edu/CONCORD/)40 was used to predict the stored at ꢀ80 C or used directly.
ꢁ
secondary structures of OsaPAL2 and OsaPAL62, and protein
multiple sequence alignment was performed using ClustalX
2.7. Enzyme purication
(version 2.1).41 A phylogenetic tree was constructed using the
For enzyme purication, all steps were performed at 4 ꢁC. First,
E. coli cells were washed and re-suspended in lysis buffer (pH
neighbor-joining method with the MEGA5.1 program.42
8.0, 20 mM sodium phosphate containing 5 mM imidazole and
300 mM NaCl). Cells were then lysed with a high-pressure
2.6. Protein expression
The prokaryotic expression vector pET-28a (+) was digested with homogenizer (800 bar, 3 passes), aer which 1 U per ml DNaseI
restriction endonucleases BamHI and EcoRI to generate the was added, and then the homogenate was incubated at 4 ꢁC for
linearized vector. Plasmids pEASY-OsaPAL2 and pEASY-Osa- approximately 2 h. Aer centrifugation at 10 000g for 15 min,
PAL62 were used as templates for sub-cloning the full-length the supernatant was passed through a 0.2 mm pore-size lter to
sequences of OsaPALs with respective specic primer pairs remove E. coli cell debris and other contaminants, and then
Table 2 Oligonucleotide primer pairs used for site-directed mutagenesis in this study. The bases underlined in each primer are the codons
introduced
Mutant
Template
Primer
Sequence
OsaPAL2F134H
pET28a–OsaPAL2
PAL2F134HF
PAL2F134HR
PAL2T196SF
PAL2T196SR
PAL2V202SF
PAL2V202SR
PAL2G209AF
PAL2G209AR
PAL2G209AF
PAL2DMR
PAL2T196SF
PAL2T196SR
PAL62F128HF
PAL62F128HR
PAL62T194SF
PAL62T194SR
PAL62V200SF
PAL62V200SR
PAL62G207AF
PAL62G207AR
PAL62DMF
50-TGCAGAGAGAGTTGATAAGGCATCTGAATGCCGG-30
50-ATGCCTTATCAACTCTCTCTGCAGGGCGCCCCC-30
50-TACGAGGCACCATCAGCGCCTCCGG-30
OsaPAL2T196S
pET28a–OsaPAL2
50-GCTGATGGTGCCTCGTAGAGGAAGGCA-30
50-GCCTCCGGCGACCTAAGCCCCTTGTCCT-30
50-GCTTAGGTCGCCGGAGGCGGTGATGGTG-30
50-TTGTCCTACATTGCCGCGCTTCTCACCG-30
50-CGCGGCAATGTAGGACAAGGGGACTAGG-30
50-TTGTCCTACATTGCCGCGCTTCTCACCG-30
50-CGCGGCAATGTAGGACAAGGGGCTTAGG-30
50-TACGAGGCACCATCAGCGCCTCCGG-30
OsaPAL2V202S
pET28a–OsaPAL2
OsaPAL2G209A
pET28a–OsaPAL2
OsaPAL2V202S/G209A
OsaPAL2T196S/V202S/G209A
OsaPAL62F128H
OsaPAL62T194S
pET28a–OsaPAL2
pET28a–OsaPAL2V202S/G209A
pET28a–OsaPAL62
pET28a–OsaPAL62
pET28a–OsaPAL62
pET28a–OsaPAL62
pET28a–OsaPAL62
pET28a–OsaPAL62V200S/G207A
50-GCTGATGGTGCCTCGTAGAGGAAGGCA-30
50-TTCAGAAGGAGCTCATCAGACATCTCAACGCGGG-30
50-ATGTCTGATGAGCTCCTTCTGAAGGGCACCACC-30
50-CTCCGCGGCACGATCAGCGCCTCCGGCG-30
50-GCTGATCGTGCCGCGGAGAGGGAGGCAC-30
50-GCCTCCGGCGACCTCAGCCCGTTGTCCT-30
50-GCTGAGGTCGCCGGAGGCGGTGATCGTG-30
50-ATATCGCCGCGATCCTCACCG-30
OsaPAL62V200S
OsaPAL62G207A
50-CGCGGCGATATAGGACAACGG-30
OsaPAL62V200S/G207A
OsaPAL62T194S/V200S/G207A
50-TTGTCCTATATCGCCGCGATCCTCACCG-30
50-CGCGGCGATATAGGACAACGGGCTGAGG-30
50-CTCCGCGGCACGATCAGCGCCTCCGGCG-30
50-GCTGATCGTGCCGCGGAGAGGGAGGCAC-30
PAL62DMR
PAL62T194SF
PAL62T194SR
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loaded onto a pre-equilibrated column containing Ni-NTA
resin. The column was washed with washing buffers (pH 8.0, 20
mM sodium phosphate buffer containing 20–50 mM imidazole
and 300 mM NaCl) to remove non-specically bound proteins,
aer which an elution buffer (pH 8.0, 20 mM sodium phosphate
containing 300 mM imidazole and 300 mM NaCl) was used to
elute the His6-tagged protein.
2.9. Optimum pH of recombinant OsaPALs
To evaluate the effect of pH on enzyme activity, the following
buffers were used: 0.1 M MES, pH 5.5–6.7; 0.1 M HEPES, pH 6.8–
8.2; 0.1 M CHES, pH 8.6–10.0; 0.1 M CAPS, pH 9.7–11.1; and 0.1
M Na2HPO4–NaOH, pH 11.5–12.0. Assays were performed at a
constant temperature of 37 ꢁC for 20 min and monitored
continuously with a multimode reader. Controls without
enzymes were included and each experiment was performed in
triplicate.
To remove small molecules such as imidazole, dialysis was
performed. A semipermeable membrane with a molecular
weight cutoff of 30 kDa was selected and approximately 20 ml
protein sample was dialyzed against 1 l dialysis buffer (pH 8.0,
10 mM sodium phosphate) for 4 h at 4 ꢁC with four changes of
dialysis buffer. Proteins were then dried in a vacuum freeze-
2.10. Optimum temperature of recombinant OsaPALs
For determination of the optimal temperature, reactions were
performed in 0.1 M CHES buffer (pH 9.5) and pre-incubated at
different temperatures in the range 30–60 ꢁC for 10 min.
Substrates were added to initiate the reactions, and, aer
incubation for 15 min, during which time the change in
absorption was measured, glacial acetic acid was added to
terminate the reaction. Controls without enzymes were
included and each experiment was performed in triplicate.
ꢁ
dryer and stored at ꢀ80 C until use.
2.8. Enzyme activity and analysis
Enzyme activity of recombinant puried OsaPALs was unam-
biguously determined by a combination of HPLC-UV, HPLC-MS
1
and H and 13C NMR spectroscopies using L-Phe and L-Tyr as
substrates. The reaction mixture (200 ml), containing 0.1 M CHES
buffer (pH 9.5), 100 mM L-Phe or 10 mM L-Tyr and different
amounts of puried protein, was incubated at 37 ꢁC for 30 min,
and then terminated by adding 200 ml chloroform. Aer centri-
fugation at 15 000g for 10 min, the supernatant was ltered
through an 0.2 mm pore-size lter and analyzed by HPLC-UV on a
HITACHI LaCrom elite L-2000 HPLC system (HITACHI, Toyo-
kawa, Japan) using a C18 column [YMC-Pack ODS-A (5 mm, 12
nm, 250 ꢂ 4.6 mm)] and gradient elution using 0.05% aqueous
triuoroacetic acid (solvent A) and CH3CN (solvent B). Aer pre-
equilibration in 98 : 2 A : B (v/v), the sample was injected and
chromatographed using a linear gradient to 35 : 65 A : B (v/v) over
2.11. Kinetic analysis of recombinant OsaPALs
Kinetic analysis of native enzymes was performed at pH 9.5 and
44 ꢁC using a total volume of 200 ml, which contains various
concentration ranges of substrates (L-Phe 9.76 mM to 20 mM;
L-Tyr 15.6 mM to 8 mM) and puried proteins (for L-Phe: 6.3 mg
OsaPAL2, 15.5 mg OsaPAL62, 47.32 mg OsaPAL2F134H, 36.5 mg
OsaPAL62F128H, 19.6 mg OsaPAL2T196S, 22.8 mg Osa-
PAL62T194S, 23 mg OsaPAL2V202S, 647.2 mg OsaPAL62V200S,
8.8 mg OsaPAL2G209A, 12.1 mg OsaPAL62G207A; For L-Tyr: 135.6
mg OsaPAL2, 136.4 mg OsaPAL62, 236.6 mg OsaPAL2F134H,
182.84 mg OsaPAL62F128H, 175.2 mg OsaPAL2G209A, 120.6 mg
OsaPAL62G207A). Assays for individual substrates were per-
formed in triplicate for 10 min (L-Phe) or 20 min (L-Tyr). The
formation of product was continuously monitored with a
multimode reader. Kinetic constants values were determined
from Lineweaver–Burk plots. All kinetic assays were performed
in triplicate and controls without enzymes or substrates were
included.
26 min at a ow rate of 1 ml minꢀ1
.
The reaction products of L-Phe (or L-Tyr), catalyzed by Osa-
PAL2 and OsaPAL62 [named OsaPAL2-L-Phe-p (or OsaPAL2-L-
Tyr-p) and OsaPAL62-L-Phe-p (or OsaPAL62-L-Tyr-p), respec-
tively], were isolated using a YMC semi-preparative column
[YMC-Pack ODS-A (5 mm, 12 nm, 250 ꢂ 10 mm)] using a linear
gradient elution from 40–65% solvent B in 15 min for L-Tyr
products and a linear gradient elution from 40–80% solvent B in
15 min for L-Phe products, both of them at a ow rate of 2 ml
minꢀ1. UV detection at 275 and 310 nm was used for enzymatic
products of L-Phe and L-Tyr, respectively. HPLC-MS was per-
2.12. Site-directed mutagenesis
formed using an Agilent 1200 RRLC series HPLC system (Agilent Site-directed mutagenesis was used to investigate the impor-
Technologies, Waldbronn, Germany) coupled with a QTRAP tance of various amino acid residues in determining enzyme
tandem mass spectrometer (QTRAP 2000, Applied Biosystems/ activity. Point and double mutations were introduced into
MDS SCIEX) equipped with a Turbo Ion spray source (Concord, OsaPALs by PCR-based amplication of the entire OsaPAL
ON, Canada) operated in the negative ionization mode and expression plasmid (pET28a–OsaPAL2 or pET28a–OsaPAL62)
controlled by Analyst 1.5 soware. Mass spectra were obtained using two mutated oligonucleotide primers (Table 2), each
in the enhanced full mass scan mode in the range of m/z 100– complementary to the opposite strand of the vector. All
1000. NMR spectra of products dissolved in deuterated components necessary for PCR-based mutagenesis were
dimethyl sulfoxide (DMSO-d6) and placed in 5 mm NMR tubes provided in the Fast Mutagenesis System kit and were used
were recorded using Bruker AVIII-600 and Bruker AVIII-500 according to the manufacturer's instructions. All mutants were
NMR spectrometers (Bruker-Biospin, Germany), which were conrmed by sequencing and those plasmids with target
operated at 600 and 500 MHz, respectively. Chemical shis (d) substitutions and without other unwanted mutations were
and coupling constants (J) were provided in ppm and hertz (Hz), retained. Triple mutations were introduced in the same way
respectively.
using already existing mutants as templates.
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Fig. 2 cDNA cloning and wild-type enzyme expression. (A) cDNA cloning of OsaPAL2 and OsaPAL62 and the construction of expression
plasmids. (B) SDS-PAGE analysis of recombinant wild-type enzymes. From right to left: empty vector as the control, OsaPAL2 crude lysate,
purified wild-type OsaPAL2, protein standards, purified wild-type OsaPAL62, OsaPAL62 crude lysate, empty vector and molecular weights
corresponding to protein standards. The arrows indicate the target proteins.
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GenBank with the four unigenes indicated that none of these
unigenes contained a full-length CDS. Of them, unigenes 26880
and 25029 had the equal sequence identity with the same PALs
deposited in GenBank, suggesting that they were situated
within the same candidate PAL. It is the same case with unigene
26221 and 25031. Furthermore, sequence alignment analysis
postulated that unigenes 26880 and 25029 were the 50- and 30-
end of one candidate PAL, while unigenes 26221 and 25031 were
the 50 and 30-end of another candidate PAL, respectively.
Accordingly, a gene family containing two PAL cDNAs was
acquired by transcriptomic analysis.
2.13. Comparison of mutant activities
To compare the activities of wild-type and mutants, mutants
were expressed and puried according to the methods
described above. The purity of proteins was estimated by SDS-
PAGE and pure proteins were dried in a vacuum freeze-dryer
ꢁ
and stored at ꢀ80 C. Prior to use, enzymes were dissolved in
CHES buffer (0.1 M, pH 9.5), and the proteins were determined
using the Bio-Rad protein assay (Bio-Rad, USA). HPLC was used
to compare the catalytic activities of different mutants. Assays
were performed in 1000 ml aqueous buffer containing 100 mM
CHES (pH 9.5) and 25 mg enzyme. Aer pre-incubation at 37 ꢁC
for 10 min, substrates (50 mM L-Phe or 10 mM L-Tyr) were added
and reactions run for 1, 2 or 3 h before being terminated by
boiling for 5 min. Mixtures were then centrifuged (15 000g,
10 min) and 15 ml injected into the HPLC system. Each assay
was performed in triplicate. Kinetic constants of some single
mutants were also determined using the above procedure.
3.2. Cloning and analysis of the full-length cDNAs encoding
OsaPALs
Two full-length members of the PAL gene family were isolated
from O. saundersiae by nested PCR using gene-specic primers
(Fig. 2A and Table 1). Identity between the cDNA sequences and
the results of transcriptome sequencing, which were veried by
sequencing, indicated the presence of a bona de PAL gene
family in plants. Sequence information for the two cDNAs,
designated OsaPAL2 and OsaPAL62, was deposited in the Gen-
3. Results and discussion
3.1. Transcriptome analysis of OsaPAL homology
OSW-1 is a cholestane saponin featuring a novel 3b,16b,17a- Bank database (OsaPAL2 accession number KF741222; Osa-
trihydroxycholest-5-en-22-one aglycone with an acylated disac- PAL62 accession number KF741223). OsaPAL2 was derived from
charide attached to the 16-hydroxyl group (Fig. 1A). Biogenetic the unigenes 26221 and 25031 and contained an ORF of 2136
analysis showed that there were at least six kinds of enzymes bp. OsaPAL62 overlapped with unigenes 26880 and 25029 at the
responsible for OSW-1 biosynthesis, including terpenoid 50 and 30 ends, respectively, and it was 2130 bp in length.
biosynthetic enzymes and steroid pathway enzymes, resulting Sequencing identication proved the two cDNAs to be bona de
in OSW-1 aglycone formation. Moreover, cytochrome P450 PALs of O. saundersiae, which is consistent with the fact that
hydroxylase is able to add hydroxyl groups to the C-3, 16 and 17 PALs are encoded by gene families in plant species such as
positions of OSW-1 aglycone, and glycosyltransferase is involved Rubus idaeus,43Arabidopsis44 and Populus trichocarpa.45 More-
in disaccharide moiety attachment to 16-OH of OSW-1 aglycone. over, due to a lack of the genome sequence, we could not
Note that acyltransferases catalyze the introduction of the acetyl determine if the two cDNAs were derived from alternative
and the 4-methoxybenzoyl groups on the disaccharide moiety, splicing.
while enzymes involved in nucleotide sugar donors biosynthesis
The proteins encoded by OsaPAL2 and OsaPAL62 were pre-
provide glycosyl donors in glycosylation reactions and phenyl- dicted by ProtParam tool to be polypeptides containing 711 and
propanoid biosynthetic pathway enzymes convert aromatic 709 amino acids with molecular weights of 77 103.9 and
amino acids to 4-methoxybenzoyl group (Fig. 1A). A total of 76 570.6 Da, respectively. The instability indices (II) indicated
more than 40 enzymes were deduced to be involved in the that the two proteins were stable (II < 40), and their low grand
biosynthesis of OSW-1. It will take much more time to isolate average of hydropathicity (GRAVY) values of ꢀ0.139 (OsaPAL2)
and further characterize the function of all these genes using and ꢀ0.112 (OsaPAL62) indicate their hydrophobicity.46 The
conventional molecular biology technologies. Thus, it is theoretical pI values of 5.64 (OsaPAL2) and 6.15 (OsaPAL62)
particularly important to apply a high-throughput method, were <7, revealing their acidic nature. The TMHMM tool indi-
which allows for drastically quicker and cheaper gene discovery cated that OsaPAL2 or OsaPAL62 are not transmembrane
and leads towards a far more comprehensive view of the OSW-1 proteins, and SignalP 4.1 showed no signal peptide or signal
biosynthetic pathway. The advent of next-generation peptide cleavage sites, indicating that they are not secreted
sequencing approaches such as transcriptomic analysis proteins. These results are consistent with the predictions of
provides a platform that has been proven to be critical in TargetP 1.1 that the two proteins are not located in chloroplasts,
speeding up of the identication of a large number of related mitochondria or secretory pathways.
genes of secondary products. In the previous investigation,
According to the results provided by PROSITE tools, both
about 40 contigs and unigenes were retrieved and identied to OsaPALs contain a 17-amino acid motif (GTITASGDLVPLSYIAG),
be responsible for phenylpropanoid biosynthetic pathway from which is a conserved signature in the superfamily of ammonia
transcriptome sequence data of O. saundersiae.39 Furthermore, lyases, including PAL, tyrosine ammonia lyase (TAL, EC 4.3.1.23)
batch alignment results revealed that there are four unigenes and histidine ammonia lyase (HAL, EC 4.3.1.3). The motif
showing high similarities with PALs. These four unigenes, contains an active Ala–Ser–Gly (A–S–G) triad, which can auto-
namely, 25029, 25031, 26221 and 26880, were 2150, 2130, 1409, catalytically be converted into a MIO (4-methylidene-imidazole-5-
and 317 bp in length, respectively. A batch BlastX search of the one) ring that serves as a prosthetic group and activates
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Fig. 3 Amino acid sequence alignment of OsaPAL2 and OsaPAL62. Asterisks, dots and colons indicate identical, semi-conserved and conserved
substitutions, respectively. The open and filled triangles indicate the residues individually substituted in this study; moreover, the filled triangles
indicate the positions of residues believed to be responsible for substrate selectivity. Rectangles and arrows represent the predicted a-helices
and b-strands, respectively. The highly conserved phenylalanine ammonia lyase signature is circled and the conserved active site motif (A–S–G)
is indicated by the bracket.
substrates by means of electrophilic interactions.47–49 With that the deduced polypeptide of OsaPAL2 is highly homologous
respect to the predicted secondary structure, both proteins had 8 to that of MbPAL (BAG70992.1) in Musa balbisiana. OsaPAL62 is
b-sheets and 25 a-helices with the residues differing between the highly homologous to PALs of some species, e.g., it shares 85%
two proteins found primarily in random coils (Fig. 3).
homology with the PAL sequence of Cinnamomum osmophloeum
CoPAL (AFG26322.1), while the OsaPAL2 and OsaPAL62
sequences are 83.6% homologous.
Phylogenetic analysis revealed that the PALs of angiosperm
plants are divided into two clusters: those from dicotyledonous
3.3. Multiple sequence alignment and phylogenetic analysis
BlastP in NCBI (http://www.ncbi.nlmnih.gov/BLAST) and multi-
alignment analysis according to CLUSTAL X algorithm indicate
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3.4. Expression of recombinant proteins and catalytic
product analysis
The two ORFs were sub-cloned into E. coli vector pET-28a (+) by
the In-Fusion method, which resulted in the heterologous
plasmids pET28a–OsaPAL2 and pET28a–OsaPAL62 (Fig. 2A).
Recombinant His6-tagged OsaPAL2 and OsaPAL62 were
expressed in E. coli and puried on Ni-NTA resin. SDS-PAGE
analysis showed that both OsaPAL2 and OsaPAL62 were
expressed and had molecular weights of approximately 75 kDa
(Fig. 2B).
Enzymes were incubated with L-Phe or L-Tyr, and the prod-
ucts were analyzed by HPLC-UV, HPLC-MS, and NMR. The data
showed that the products of L-Phe catalyzed by OsaPAL2 and
OsaPAL62 (OsaPAL2-L-Phe-p and OsaPAL62-L-Phe-p, respec-
tively) had the same UV spectra as that of trans-cinnamic acid.
In HPLC-MS, the most intense peaks of the two products were
located at m/z 147 due to the loss of a hydrogen radical. The
structure of these two products was further conrmed by NMR
aer semi-preparative isolation to be trans-cinnamic acid
(Fig. 5).
For the corresponding products of L-Tyr, HPLC-UV analysis
showed that both enzymes converted L-Tyr to the new products,
OsaPAL2-L-Tyr-p and OsaPAL62-L-Tyr-p, which exhibit the same
UV-spectra as that of p-coumaric acid. Both OsaPAL2-L-Tyr-p
and OsaPAL62-L-Tyr-p exhibited an abundant ion at m/z 163 due
to the loss of a hydrogen atom with subsequent loss of a
carboxyl group to form an ion at m/z 119. NMR analysis further
conrmed OsaPAL2-L-Tyr-p and OsaPAL62-L-Tyr-p to be p-cou-
maric acid (Fig. 6).
Fig. 4 Phylogenetic analysis of OsaPAL2, OsaPAL62 and other PALs.
The phylogenetic tree was constructed using the neighbor-joining
method available in the MEGA5.1 program. It shows a comparison of
the sequences from the dicotyledons: Paeonia lactiflora PlPAL
(AFI71896.1), Rhus chinensis RcPAL (AGH13333.1), Populus trichocarpa
PtPAL (ACC63887.1), Hibiscus cannabinus HcPAL (AFN85669.1),
Trifolium pratense TpPAL (AAZ29733.1), Scutellaria baicalensis SbPAL
(ADN32768.1), Melissa officinalis MoPAL (CBJ23826.1), Salvia miltior-
rhiza SmPAL (ABD73282.1), Quercus suber QsPAL (AAR31107.1), Rubus
idaeus RiPAL (AAF40224.1), Platycodon grandiflorus PgPAL
(AEM63671.1) and Coffea canephora CcPAL (AEO92028.1); mono-
cotyledons: Musa balbisiana MbPAL (BAG70992.1), Lycoris radiate
These results strongly suggest that OsaPAL2 and OsaPAL62
have both PAL and TAL activity, consistent with the activity of
PALs present in other monocotyledonous plants such as Zea
mays51 and Phyllostachys edulis.52
LrPAL
(ACM61988.1)
and
Dendrobium
candidum
DcPAL
(AGC23439.1); gymnosperms: Ginkgo biloba GbPAL (ABU49842.1) and
Ephedra sinica EsPAL (BAG74772.1); the ferns: Blechnum spicant
BsPAL (AAW80643.1), Equisetum arvense EaPAL (AAW80639.1) and
Ophioglossum reticulatum OrPAL (AAW80642.1); and yeasts: Rhodo-
torula mucilaginosa RmPAL (CAA31486.1), Rhodosporidium toruloides
RtPAL2 (AAA33883.1), Rhodotorula glutinis ATCC 204091 RgPAL2
(EGU13302.1), Rhodotorula glutinis RgPAL1 (ABB04148.1) and Rho-
dosporidium toruloides RtPAL1 (CAA35886.1).
3.5. Biochemical analysis of OsaPALs
In many previous studies, the formation of trans-cinnamic acid
was usually monitored at 275 or 280 nm.26,51,53 However, at this
wavelength, the substrate L-Phe shows UV absorption (Fig. 5);
thus, the determination of trans-cinnamic acid will be disturbed
when using a multimode reader. Therefore, to eliminate the
effect of the substrate, we monitored absorbance at 300 nm.
The optimal pH and temperature for efficient enzyme activity
of puried enzymes, OsaPAL2 and OsaPAL62, were found to be
similar to those for L-Phe and L-Tyr deamination (Fig. 7). Both
enzymes functioned better in the pH range of 8.5–10.0 and
showed only low activity at pH <7.0 or >11.0. This optimum pH
range is comparable to that of PALs isolated from other
monocotyledonous species such as PePAL from Phyllostachys
edulis (pH 8.5–9.0),52 SmPAL1 from Salvia miltiorrhiza (pH 8.7)2
and BoPAL4 from Bambusa oldhamii (pH 9.0).50 The effect of
temperature on the activity of OsaPAL2 and OsaPAL62 was
similar with both showing highest activity at 44 ꢁC. Similar
temperatures have been observed for maximum activity of PALs
in Rhus chinensis (45 ꢁC)1 and Arabidopsis (46–48 ꢁC)44 with a
slightly lower temperature being optimum for AvPAL in
plants [e.g. PlPAL (AFI71896.1) from Paeonia lactiora and
RcPAL (AGH13333.1) from Rhus chinensis] and those from
monocotyledon plants [e.g. MbPAL (BAG70992.1) from Musa
balbisiana] (Fig. 4). Amino acid sequences of OsaPAL2 and
OsaPAL62 belong to the latter cluster. It has been reported that
PALs from monocotyledons always possess TAL activity,50,51
suggesting that OsaPAL2 and OsaPAL62 may have PAL/TAL
activities. In the resulting phylogeny, OsaPAL62 and PALs from
Lycoris radiate (LrPAL) and Dendrobium candidum (DcPAL) were
clustered in the same clade, whereas OsaPAL2 was alone in a
neighboring branch, suggesting that it belongs to a different
subfamily. In addition, PALs of ferns and gymnosperms formed
independent clusters, a result consistent with the evolution of
plants. PALs of plants and yeasts have a common ancestor but
are separated by a large evolutionary distance with only 36%
homology between OsaPAL2 and RmPAL (CAA31486.1) from
Rhodotorula mucilaginosa.
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Fig. 5 Analysis of the products of OsaPALs in vitro assays with L-Phe. (A) HPLC analysis: traces 1 and 4 show the substrate (L-Phe) and product
(trans-cinnamic acid) standard, respectively; traces 2 and 3 are the controls showing the results for boiled enzymes incubated with the substrate
(L-Phe); traces 5 and 6 are the results for purified wild-type enzymes incubated with L-Phe and the resulting products have similar UV-spectra
with trans-cinnamic acid. (B) Mass spectra (MS) analysis: both OsaPAL2-L-Phe-p and OsaPAL62-L-Phe-p exhibit strongest ion peaks at 147 m/z.
(C) NMR analysis results indicate that OsaPAL2-L-Phe-p and OsaPAL62-L-Phe-p have the same structure.
A. variabilis (40 ꢁC)26 and a higher tꢁemperature being optimum these two wild-type enzymes for L-Phe were lower than those of
for Pal in Helianthus annuus L. (55 C).54
the PALs in Zea mays (3.9 ꢂ 104 Mꢀ1 sꢀ1
)
51 and Jatropha curcas L.
Assays were performed with L-Phe or L-Tyr as the substrate at (1.384 ꢂ 104 Mꢀ1 sꢀ1).56
an optimum pH and temperature to evaluate the kinetic
The higher L-Phe and lower L-Tyr deamination activity of
constants of OsaPAL2 and OsaPAL62 (Fig. 7,8 and Table 3). Both OsaPAL2 and OsaPAL62 is consistent with previous studies on
wild-type enzymes displayed marked kinetic preference for PALs found in monocotyledons.51 Moreover, the OsaPALs were
L-Phe as Km of OsaPAL2 for L-Phe (371.5 ꢃ 26.7 mM) was about able to catalyze the reversible reaction whereby trans-cinnamic
30-fold lower than that for L-Tyr (11690 ꢃ 1410 mM) and kcat/Km acid is converted to L-Phe, an ability that was also observed with
for L-Phe (2160 Mꢀ1 sꢀ1) was substantially higher than that for L- some other PALs (data not shown).
Tyr (39.65 Mꢀ1 sꢀ1). A similar situation was found as a Km of
OsaPAL62 for L-Phe (593.1 ꢃ 48.6 mM) was about 60-fold lower
3.6. Identication of specicity determining residues
than that for L-Tyr (39 280 ꢃ 472 mM) and kcat/Km for L-Phe
(934.9 Mꢀ1 sꢀ1) was higher than that for L-Tyr (16.69 Mꢀ1 sꢀ1).
OsaPAL2 and OsaPAL62 also have TAL activity, allowing direct
Assays performed to determine the kinetic constants of
OsaPAL2 and OsaPAL62 for L-Tyr were unable to saturate the
enzymes due to the lower appetency and solubility limitations
of L-Tyr.44 The Km values of these enzymes for L-Phe were similar
to those of other PALs such as the PAL of Oryza sativa (500 mM)55
and the BoPAL2 of B. oldhamii (333 mM).50 The kcat/Km values of
conversion of L-Tyr to p-coumarate, and thus eliminating the
requirement for the hydroxylation step. This activity is valuable
for designing heterologous pathways for producing target
metabolites. One underlying mechanism of bi-functional PAL is
based on the presence of a Phe residue controlling the substrate
selection.57 Although several experiments have supported this
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Fig. 6 Analysis of the products of OsaPALs in vitro assays with L-Tyr. (A) HPLC analysis: traces 1 and 4 show the substrate (L-Tyr) and product (p-
coumaric acid) standard, respectively; traces 2 and 3 are the controls showing the results for boiled enzymes incubated with the substrate (L-Tyr);
Traces 5 and 6 are the results of purified wild-type enzymes incubated with L-Tyr and the resulting products have similar UV-spectra with p-
coumaric acid (note that the wavelength was set to 275 nm to visualize the substrate L-Tyr). (B) Mass spectra (MS) analysis: both OsaPAL2-L-Tyr-p
and OsaPAL62-L-Tyr-p have the same peaks at m/z 163 and 119. (C) NMR analysis results and the structure of the product.
hypothesis, various questions remain unaddressed. In a report L-Tyr deamination activity, which is improved by substituting
by Watts et al.,57 when Phe144 residue was replaced by His in A. the two residues with H to give OsaPAL2F134H and Osa-
thaliana PAL1, the mutant displayed a marked reduction (80- PAL62F128H (Table 3, Fig. 8–10). The optimum pH and
fold) in kcat/Km value together with an 18-fold increase in cata- temperature for the two mutants are nearly the same as those of
lytic efficiency towards tyrosine. This result clearly showed that the wild-type proteins (ESI Fig. 1–3†). The kinetic parameters,
Phe144 controlled substrate selection;57 however, Hsieh et al.50 however, varied markedly when F was mutated to H. For Osa-
did not obtain the same results. When Phe133 was substituted PAL2F134H, the values of Km and kcat/Km for L-Tyr were 16.9-fold
with His, only a slightly increased kcat/Km value toward tyrosine lower and 7.0-fold higher, respectively, than those for the wild-
was observed in the F133H mutant. The authors inferred that type enzyme. Aer F / H mutation, the activity toward L-Phe
other residues also contribute to the substrate selectivity of decreased with Km being 3-fold higher than that for OsaPAL2
PALs. In view of this result, they proposed that further site- and kcat/Km being reduced by 86% (Table 3). OsaPAL62F128H
directed mutagenesis experiments would be necessary for exhibited remarkable TAL activity compared with the wild-type
elucidation of the underlying mechanism of substrate selec- enzyme (Table 3, Fig. 8 and 10) with values of Km and kcat/Km for
tivity.50 This situation poses the question of how exactly the Phe L-Tyr were approximately 30-fold lower and 6-fold higher,
residue functions in the substrate selectivity switch. Site- respectively, than those for the wild-type enzyme. The substi-
directed mutagenesis of Phe residue was accordingly performed tution of F with H resulted in a signicantly reduced activity for
in OsaPALs with the aim of identifying this function.
L-Phe with lower affinity (Km 2.8-fold higher than that of wild-
TAL and PAL have different substrate specicities associated type) and lower catalytic efficiency (kcat/Km 4.7-fold lower than
with a difference in a residue corresponding to F134 in OsaPAL2 that of the wild-type) (Table 3).
and F128 in OsaPAL62 (Fig. 3). This residue gives rise to low
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Fig. 7 Biochemical characterization and comparison of recombinant enzymes. The optimum temperatures and pH for catalyzing L-Phe (A and
C) and L-Tyr (B and D) of both enzymes were determined. (E and F) are overlays of substrate saturation plots of the two enzymes using L-Phe and
L-Tyr as substrates performed in CHES buffer (0.1 M, pH 9.5) at 44 ꢁC by continuously monitoring changes in absorbance at 300 and 310 nm,
respectively. (Note that at a wavelength of 275 nm, the substrate L-Phe shows UV absorption, which was eliminated by monitoring absorbance at
300 nm).
The fact that TAL activity of the OsaPALs is signicantly biosynthetic pathway and allows the scale-up of the production
increased in the F / H mutations proves that this F residue is of OSW-1 using more active enzymes.
associated with substrate specicity as has been reported in
Following the successful functional characterization, the two
some previous studies.1,58 The compound, MIO, shown to be sequences were aligned with three known PALs from yeasts
present in the crystal structure of PAL,59 is believed to be together with other three PALs from plants (Fig. 11). The reason
involved in the PAL reaction as a cofactor attacking the carbon for selection of these PALs was that PALs from yeasts61–63 appear
atom at the C2 position of the aromatic ring of L-Phe in a to have higher catalytic activity than those from plants.1,51,64
Friedel–Cras-type reaction.47 H134 and H128 of Osa- Examination of the sequence alignment revealed three residues
PAL2F134H and OsaPAL62F128H, respectively, interact with the around the ASG triad that were not absolutely conserved. The
hydroxyl group of L-Tyr, resulting in an increase in affinity.58 The three amino acids are T, V, and G in plant PALs, whereas, in
hydroxyl group of L-Tyr also acts as an electron-donating group yeast PALs, the equivalent residues were S, S, and A. This is an
to increase the electron density at the C1, C3 and C5 positions of interesting phenomenon that has not been reported to date in
the aromatic ring,60 thereby interfering with the attack by MIO; the literature. To identify the function of the three amino acids,
thus, the mutants retained higher catalytic activities towards site-directed mutagenesis was performed based on the
L-Phe.61
consensus approach.65–67
The mutants were OsaPAL2T196S and OsaPAL62T194S, in
which T196 of OsaPAL2 and T194 of OsaPAL62 were replaced
with S; OsaPAL2V202S and OsaPAL62V200S, in which V202 of
OsaPAL2 and V200 of OsaPAL62 were replaced with S;
and OsaPAL2G209A and OsaPAL62G207A, in which G209 of
OsaPAL2 and G207 of OsaPAL62 were replaced with A.
In addition, two double mutants, OsaPAL2V202S/G209A
and OsaPAL62V200S/G207A, and two triple mutants,
3.7. Residue substitution successfully improves enzymes
activity
The enzyme-kinetic properties of the puried proteins veried
their biochemical function as bona de OsaPALs, a nding
which is expected to clarify the biosynthetic pathway of OSW-1.
In addition, the successful characterization of the OsaPALs
provides useful information for reconstructing the OSW-1
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Fig. 8 Overlays of substrate saturation plots of different enzymes. (A andB) Plots of initial velocity against L-Phe concentration for reactions of
wild-type and mutant enzymes. (C and D) Plots of initial velocity against L-Tyr concentration for reactions of wild-type and mutant enzymes.
Wild-type enzymes did not reach enzyme saturation due to the limited solubility of L-Tyr.
OsaPAL2T196S/V202S/G209A
G207A, were constructed.
and
OsaPAL62T194S/V200S/ 3†). Then, the catalytic activities of these mutants were evalu-
ated (Table 3 and Fig. 8–10), which reveal that although all
Aer protein expression and purication (Fig. 12), the the mutations lay near the PAL motif, their effects were
optimum pH and temperature for these mutants were deter- diverse (Table 3). Using L-Phe as a substrate, both T / S
mined to be the same as those for the wild proteins (ESI Fig. 1– mutations (OsaPAL2T196S and OsaPAL62T194S) and V / S
Table 3 Kinetic constants of OsaPAL2, OsaPAL62 and their mutants with L-Phe and L-Tyr as substrates. All assays were performed in aqueous
100 mM CHES (pH 9.5) at 44 ꢁC. The formation of trans-cinnamic acid and p-coumaric acid were monitored continuously by multimode reader
at 300 and 310 nm, respectively
Substrate
L-Phe
Enzyme
Vmax (nkat per mg protein)
Km (mM)
kcat (sꢀ1
)
kcat/Km (Mꢀ1 sꢀ1
)
OsaPAL2
51.48 ꢃ 0.82
23.40 ꢃ 0.23
33.78 ꢃ 0.36
12.21 ꢃ 0.17
119.3 ꢃ 2.2
371.5 ꢃ 26.7
1241 ꢃ 44
0.8025
0.3646
0.5264
0.1903
1.861
0.5545
0.3328
0.8105
0.0003105
0.9332
0.4635
0.1925
0.4690
0.6557
0.1332
0.3143
2160
OsaPAL2F134H
OsaPAL2T196S
OsaPAL2V202S
OsaPAL2G209A
OsaPAL62
OsaPAL62F128H
OsaPAL62T194S
OsaPAL62V200S
OsaPAL62G207A
OsaPAL2
OsaPAL2F134H
OsaPAL2G209A
OsaPAL62
OsaPAL62F128H
OsaPAL62G207A
293.8
794.1
610.5
2354
934.9
198.2
655.7
2.612
1763
39.65
278.6
132.6
16.69
100.9
33.35
662.9 ꢃ 28.8
311.7 ꢃ 21.1
790.4 ꢃ 57.54
593.1 ꢃ 48.6
1679 ꢃ 43
35.82 ꢃ 0.70
21.5 ꢃ 0.2
52.27 ꢃ 0.46
0.08034 ꢃ 0.00130
60.26 ꢃ 0.61
29.73 ꢃ 2.41
12.35 ꢃ 0.23
30.08 ꢃ 1.33
42.35 ꢃ 4.40
8.603 ꢃ 0.161
20.29 ꢃ 1.56
1236 ꢃ 39
118.9 ꢃ 10.4
529.2 ꢃ 23.0
11690 ꢃ 1410
691.0 ꢃ 46.0
3538 ꢃ 365
39280 ꢃ 472
1320 ꢃ 74
L-Tyr
9423 ꢃ 1148
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Fig. 9 Comparison of OsaPAL2 mutant and wild-type activities. (A and D) HPLC was used to compare OsaPAL2 wild-type and mutant activities
using the same amount of purified enzyme and substrate L-Phe or L-Tyr in an aqueous buffer. The data is for a reaction lasting 3 h with the various
heights indicating the different amounts of products (Note that the UV absorption of the product of L-Phe was high at 275 nm producing flat-
topped peaks. To improve the precision of the results, we also monitored the absorbance at 300 nm.) (B and E) Partially enlarged views of
2V202S/G209A and 2T196S/V202S/G209A. (C and F) Relative activity was determined based on an average rate of 9 independent experiments;
scale was set to 100% for wild-type OsaPAL2. Results are presented as means ꢃ SD.
mutations (OsaPAL2V202S and OsaPAL62V200S) exhibited 0.129%, respectively, of the wild-type activities for L-Phe
lower catalytic efficiency with values of kcat/Km lower than those (Fig. 9 and 10). The introduction of a third mutation to
of the wild-type enzymes, in particular of OsaPAL62V200S afford the triple mutants OsaPAL2T196S/V202S/G209A
whose kcat/Km value was only 0.33% of that of OsaPAL62.
and OsaPAL62T194S/V200S/G207A restored some degree
Interestingly, the T / S mutation, where one hydrophilic of activity for L-Phe (0.016–0.339%) (Fig. 9 and 10). The
amino acid replaces another, showed lower affinity with Km relative activities of other mutants were also evaluated
values 2-fold higher and lower catalytic efficiency compared to by HPLC and shown to be consistent with their
the wild-type. In contrast, the V / S mutation, where a kinetic constants. In brief, the activity of OsaPAL2G209A
hydrophilic amino acid replaces a hydrophobic one, showed and OsaPAL62G207A was highest, followed by the wild-type,
higher affinity than the wild-type. Furthermore, the G / A followed by OsaPAL2T196S and OsaPAL62T194S with
mutants were did not exhibit signicant difference in affinity; slightly lower activity than the wild-type, and nally Osa-
however, they exhibited higher catalytic efficiency than the wild PAL2V202S and OsaPAL62V200S. Activities of the double
type.
mutants OsaPAL2V202S/G209A and OsaPAL62V200S/G207A
The double mutants OsaPAL2V202S/G209A and Osa- and triple mutants OsaPAL2T196S/V202S/G209A and Osa-
PAL62V200S/G207A exhibited activities too low to be detected PAL62T194S/V200S/G207A were negligible. Similar results
by the multimode reader; however, by HPLC analysis, they were obtained by HPLC for L-Tyr deamination activity (Fig. 9
were shown to have activities that were only 0.006% and and 10).
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Fig. 10 Comparison of OsaPAL62 mutant and wild-type activities. (A and D) HPLC was used to compare OsaPAL62 wild-type and mutant
activities using the same amount of purified enzymes and substrate L-Phe or L-Tyr in an aqueous buffer. The data is for a reaction lasting 3 h with
the various heights indicating the different amount of products. (Note UV absorption of the product of L-Phe was high at 275 nm producing flat-
topped peaks. To improve the precision of the results, we also monitored the absorbance at 300 nm). (B and E) Partially enlarged views of
62V200S, 62V200S/G207A and 62T194S/V200S/G207A. (C and F) Relative activity is presented as a mean ꢃ SD of 9 independent experiments,
scale was set to 100% for the wild-type OsaPAL62.
In both of the two proposed reaction mechanisms for
PAL,48,60,68,69 the A–S–G triad in the motif can rearrange to a MIO
ring to attack substrates. The importance of the triad has been
demonstrated in many studies but little research has focused on
other conserved amino acids around it.
The results described above indicate that residues adjacent
to the A–S–G triad may play different roles. In considering the
Km values of single mutants in relation to the positions of
mutations, the higher affinity but lower catalytic efficiency of
OsaPAL2V202S and OsaPAL62V200S suggests that the S202 in
the former and S200 in the latter may form hydrogen bonds
Fig. 11 Multiple alignments of PAL signatures of plants and yeasts. The
red box highlights the active site motif (A–S–G), which undergoes
autocatalysis to form MIO. The residues that differ in yeasts and plants
are shown in black boxes. The sequences used are OsaPAL2, Osa-
with the b-proton of the substrate and hinder its elimination.
However, the elucidation of the exact mechanism needs further
investigation.
PAL62, Rhus chinensis RcPAL (AGH13333), Zea mays ZmPAL
(NP_001105334), Bambusa oldhamii BoPAL2 (ACN62413.1), Rhodo-
torula glutinis JN-1 RgPAL (see ref. 61), Rhodosporidium toruloides
RtPAL3 (P11544), Rhodosporidium toruloides RtPAL2 (AAA33883.1).
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case in the report published by Hsieh et al.51 When Phe133 was
substituted with His, a slightly increased kcat/Km value toward
tyrosine was found in the F133H mutant. They inferred that
other residues also contribute to the substrate selectivity of
PALs. To further clarify the function of phenylalanine, two
mutants OsaPAL2F134H and OsaPAL62F128H were produced
by site-directed mutagenesis. The results corroborated that Phe
does indeed control substrate selection.
Acknowledgements
This work was supported by National Mega-project for Innova-
tive Drugs (no. 2012ZX09301002), Open Foundation of State Key
Laboratory of Bioactive Substance and Function of Natural
Medicines (B-2011-4) and PUMC Youth Fund (2012J21,
3332013112).
Fig. 12 SDS-PAGE analysis of purified mutants of OsaPAL2 and Osa-
PAL62. M stands for protein standards with corresponding molecular
weights shown on the right; small black arrows indicate target proteins
with similar molecular weights.
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