Contributions of conserved serine and tyrosine residues to catalysis, ligand binding, and cofactor processing in the active site of tyrosine ammonia lyase

Article abstract of DOI:10.1016/j.phytochem.2008.02.007

Tyrosine ammonia lyase (TAL) catalyzes the conversion of l-tyrosine to p-coumaric acid using a 3,5-dihydro-5-methylidene-4H-imidazole-4-one (MIO) prosthetic group. In bacteria, TAL is used for production of the photoactive yellow protein chromophore and for caffeic acid biosynthesis in certain actinomycetes. Here we biochemically examine wild-type and mutant forms of TAL from Rhodobacter sphaeroides (RsTAL). Kinetic analysis of RsTAL shows that the enzyme displays a 90-fold preference for l-tyrosine versus l-phenylalanine as a substrate. The pH-dependence of TAL activity with l-tyrosine and l-phenylalanine demonstrates a common protonation state for catalysis, but indicates a difference in charge-state for binding of either amino acid. Site-directed mutagenesis demonstrates that Ser150, Tyr60, and Tyr300 are essential for catalysis. Mutation of Ser150 to an alanine abrogates formation of the MIO prosthetic group, as shown by mass spectrometry, and prevents catalysis. The Y60F and Y300F mutants were inactive with both amino acid substrates, but bound p-coumaric and cinnamic acids with less than 12-fold changes in affinity compared the wild-type enzyme. Analysis of MIO-dithiothreitol adduct formation shows that the reactivity of the prosthetic group is not significantly altered by mutation of either Tyr60 or Tyr300. The mechanistic roles of Ser150, Tyr60, and Tyr300 are discussed in relation to the three-dimensional structure of RsTAL and related MIO-containing enzymes.

Full text of DOI:10.1016/j.phytochem.2008.02.007

Available online at www.sciencedirect.com
PHYTOCHEMISTRY
Phytochemistry 69 (2008) 1496–1506
www.elsevier.com/locate/phytochem
Contributions of conserved serine and tyrosine residues
to catalysis, ligand binding, and cofactor processing
in the active site of tyrosine ammonia lyase
Amy C. Schroeder 1, Sangaralingam Kumaran 1, Leslie M. Hicks, Rebecca E. Cahoon,
Coralie Halls, Oliver Yu, Joseph M. Jez *
The Donald Danforth Plant Science Center, 975 N. Warson Road, St. Louis, MO 63132, USA
Received 20 September 2007; received in revised form 17 December 2007
Available online 17 March 2008
Abstract
Tyrosine ammonia lyase (TAL) catalyzes the conversion of L-tyrosine to p-coumaric acid using a 3,5-dihydro-5-methylidene-4H-imid-
azole-4-one (MIO) prosthetic group. In bacteria, TAL is used for production of the photoactive yellow protein chromophore and for
caffeic acid biosynthesis in certain actinomycetes. Here we biochemically examine wild-type and mutant forms of TAL from Rhodobacter
sphaeroides (RsTAL). Kinetic analysis of RsTAL shows that the enzyme displays a 90-fold preference for L-tyrosine versus L-phenylal-
anine as a substrate. The pH-dependence of TAL activity with L-tyrosine and L-phenylalanine demonstrates a common protonation state
for catalysis, but indicates a difference in charge-state for binding of either amino acid. Site-directed mutagenesis demonstrates that
Ser150, Tyr60, and Tyr300 are essential for catalysis. Mutation of Ser150 to an alanine abrogates formation of the MIO prosthetic
group, as shown by mass spectrometry, and prevents catalysis. The Y60F and Y300F mutants were inactive with both amino acid sub-
strates, but bound p-coumaric and cinnamic acids with less than 12-fold changes in affinity compared the wild-type enzyme. Analysis of
MIO–dithiothreitol adduct formation shows that the reactivity of the prosthetic group is not significantly altered by mutation of either
Tyr60 or Tyr300. The mechanistic roles of Ser150, Tyr60, and Tyr300 are discussed in relation to the three-dimensional structure of
RsTAL and related MIO-containing enzymes.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Enzyme mechanism; Prosthetic group; Mass spectrometry; Fluorescence spectroscopy; Tyrosine ammonia lyase
Tyrosine ammonia lyase (TAL) is a member of the
amino acid ammonia lyase enzyme family, which also
includes histidine ammonia lyase (HAL) and phenylalanine
ammonia lyase (PAL). They catalyze the conversion of a-
amino acids to a,b-unsaturated acids by elimination of
ammonia from tyrosine 3 (Fig. 1), histidine, and phenylal-
´
anine 1 (Fig. 1), respectively (Poppe and Retey, 2005).
These enzymes are found in plants, bacteria, and fungi.
In plants, PAL catalyzes the non-oxidative deamination
of L-phenylalanine 1 to trans-cinnamic acid 2 (Fig. 1A).
This reaction is the first step in the phenylpropanoid path-
way and ultimately leads to multiple classes of phenolic
natural products, such as lignins, flavonoids, and isoflavo-
noids (Winkel-Shirley, 2001). PAL is found in dicot and
monocot plants, but a secondary TAL activity is associated
with the enzyme from monocots (Guerra et al., 1985; Ros-
ler et al., 1997; Khan et al., 2003).
Abbreviations: DTT, D/L-dithiothreitol; HAL, histidine ammonia lyase;
IPTG, isopropyl-1-thio-b-D-galactopyranoside; MES, 2-(N-morpho-
lino)ethanesulfonic acid; MIO, 3,5-dihydro-5-methylidene-4H-imidazole-
4-one; NTA, nitriloacetic acid; PAL, phenylalanine ammonia lyase;
RsTAL, Rhodobacter sphaeroides tyrosine ammonia lyase; TAL, tyrosine
ammonia lyase.
*
Corresponding author. Tel.: +1 314 587 1450; fax: +1 314 587 1550.
TAL catalyzes the conversion of L-tyrosine 3 to p-cou-
maric acid 4 (Fig. 1A). Although bacteria do not synthesize
E-mail address: jjez@danforthcenter.org (J.M. Jez).
Both of these authors contributed equally to the work.
1
0031-9422/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2008.02.007

A.C. Schroeder et al. / Phytochemistry 69 (2008) 1496–1506
1497
of the substrate a-amino group to the methylidene carbon
of the MIO prosthetic group (Hermes et al., 1985). Alterna-
tively, the reaction could occur by a Friedel–Crafts acyla-
tion mechanism in which attack of the electron-rich
aromatic ring of the substrate occurs on the electron-defi-
´
cient methylidene carbon of the MIO (Poppe and Retey,
´
2005; Schuster and Retey, 1995). Mutagenesis studies of
HAL and PAL have probed the contribution of active site
residues to catalysis (Ro¨ther et al., 2001, 2002), but a sim-
ilar analysis of TAL has not been described.
Recent structural and functional studies of TAL identi-
fied a histidine in the active site as essential for controlling
substrate preference for L-tyrosine 3 over L-phenylalanine 1
(Watts et al., 2006; Louie et al., 2006). Importantly, the X-
ray structure of TAL gave the first view of an amino acid
ammonia lyase in complex with either a reaction product
or substrate analog (Louie et al., 2006). The structure
shows that the MIO prosthetic group of TAL is formed
from Ser150, and that a number of amino acids, including
Tyr60 and Tyr300, may contribute to binding of substrates
and products (Louie et al., 2006). Tyrosines corresponding
to Tyr60 and Tyr300 of TAL are conserved in PAL and
HAL, and each of these residues has been proposed to
function as a general base in the deamination reactions cat-
alyzed by HAL, PAL, and TAL (Schwede et al., 1999; Rit-
ter and Schulz, 2004; Ro¨ther et al., 2001, 2002; Louie et al.,
2006). The functional roles of Ser150, Tyr60, and Tyr300 in
the TAL active site are the focus of this study.
Fig. 1. Reactions catalyzed by TAL and PAL: (A) TAL generates
p-coumaric acid 4 from L-tyrosine 3. PAL catalyzes the conversion of
L-phenylalanine 1 to cinnamic acid 2. (B) Formation of the 3,5-dihydro-5-
methylidene-4H-imidazole-4-one (MIO) prosthetic group in the TAL
active site.
phenylpropanoids, PAL and TAL provide cinnamic acid 2
and p-coumaric acid 4, respectively, in specialized meta-
bolic pathways. For example, PAL is used in the biosynthe-
sis of the marine natural product enterocin (Xiang and
Moore, 2002, 2005). TAL plays a critical role in synthesiz-
ing the chromophore of photoactive yellow protein in bac-
teria and for caffeic acid biosynthesis in actinomycetes
(Kyndt et al., 2002, 2003; Berner et al., 2006). Multiple
research groups have used TAL for metabolic engineering
of flavonoid and resveratrol biosynthesis pathways that
require p-coumaric acid 4 as a precursor molecule (Watts
et al., 2004; Jiang et al., 2005; Zhang et al., 2006; Qi
et al., 2007; Trotman et al., 2007). Because TAL forms p-
coumaric 4 acid directly from L-tyrosine 3, its use in heter-
ologous expression systems circumvents the need to express
both PAL and 4-coumaric acid hydroxylase, a membrane-
bound cytochrome P450 enzyme, for conversion of L-phen-
ylalanine 1 to p-coumaric acid 4.
Historically, the amino acid ammonia-lysases were
thought to use a prosthetic dehydroalanine as an electro-
phile in the reaction mechanism, but the three-dimensional
structures of HAL and PAL indicated that these enzymes
contain a 3,5-dihydro-5-methylidene-4H-imidazole-4-one
(MIO) group for substrate activation (Fig. 1B) (Schwede
et al., 1999; Calabrese et al., 2004; Ritter and Schulz,
2004). Spontaneous cyclization and dehydration of an ala-
nine-serine-glycine segment of the polypeptide backbone in
the active site of HAL and PAL results in formation of the
MIO group (Fig. 1B) (Poppe, 2001; Baedeker and Schulz,
1. Results
1.1. Expression, purification, and mutagenesis
The TAL gene from Rhodobacter sphaeroides encodes a
523 amino acid protein (Mr = 55042.6 Da; pI = 7.3), which
shares 28.4%, 31.2%, and 35.6% amino acid sequence
identity with Rhodosporidium toruloides PAL, Petroselinum
hortense (parsley) PAL, and Pseudomonas putida HAL
(Zhang et al., 2006). BLAST searching of sequence dat-
abases identify HAL, not PAL, as a closer relative of
TAL. Hexahistidine-tagged RsTAL was purified from
E. coli using Ni2+-affinity and size-exclusion chromatogra-
phies. SDS-PAGE analysis (not shown) showed that the
purified protein migrated as a 55 kDa species, as expected
based on the calculated molecular weight.
To examine the contribution of selected conserved active
site residues to the reaction catalyzed by RsTAL, we gener-
ated site-directed mutants of the enzyme. Based on
sequence comparisons of RsTAL with PAL and HAL,
we mutated Ser150 (S150A) to disrupt formation of the
MIO group in the active site, Tyr300 (Y300F) to examine
the contribution of this residue as a general base in the
reaction mechanism, and Tyr60 (Y60F) to probe the role
of a flexible active site loop (Schwede et al., 1999; Ritter
and Schulz, 2004; Ro¨ther et al., 2001, 2002; Louie et al.,
2006). Each RsTAL mutant was expressed and purified
´
2002a,b; Retey, 2003). Mechanistically, two possible
reaction sequences for HAL and PAL have been proposed.
The first mechanism involves direct nucleophilic addition

1498
A.C. Schroeder et al. / Phytochemistry 69 (2008) 1496–1506
with procedures used for wild-type enzyme. The final yield
of soluble protein for each mutant was comparable to that
obtained with wild-type RsTAL. The Y60F, S150A, and
Y300F mutants behaved the same as RsTAL in SDS–
PAGE and gel-filtration chromatography (not shown).
steady-state kinetic parameters (kcat and Km) of RsTAL
for tyrosine 3 and phenylalanine 1 as substrates were deter-
mined (Table 1). This analysis confirms the identity of
RsTAL because comparison of the catalytic efficiencies
(kcat/Km) indicates a 90-fold preference for tyrosine 3 over
phenylalanine 1 as a substrate.
1.2. Functional analysis of wild-type RsTAL
To obtain information about the protonation state of
the reaction catalyzed by RsTAL, the pH dependence of
the reaction was examined using tyrosine 1 and phenylala-
nine 1 as substrates (Fig. 2 and Table 2). With tyrosine 3 as
a substrate (Fig. 2A), RsTAL showed pH-dependencies of
kcat and kcat/Km with similar pKa values (Table 2). Since
both curves displayed similar inflection points, the
enzyme–substrate complex and either free enzyme or free
substrate may require depronation of a common chemical
group for conversion of tyrosine 3 to p-coumaric acid 4.
In contrast, the pH-dependence of RsTAL with phenylala-
nine 1 as a substrate only had a breakpoint for kcat with a
pKa = 6.8, and with values of kcat/Km being independent of
pH (Fig. 2B and Table 2). This suggests the loss of a bind-
ing interaction with the change in substrate, and likely
reflects the loss of an interaction with the hydroxyl group
of tyrosine 3, which is not present in phenylalanine 1.
To determine if purified recombinant RsTAL was active
using aromatic amino acid substrates, the enzyme-cata-
lyzed conversion of L-tyrosine 3 to p-coumaric acid 4 was
monitored spectrophotometrically. Purified RsTAL was
found to be active under standard assay conditions with
a specific activity of 85 nmol minꢀ1 mg proteinꢀ1. We also
examined the ability of RsTAL to catalyze the transforma-
tion of substrate analogues, including histidine, phenylala-
nine, 3-iodotyrosine, and nitrophenylalanine. Of these,
enzymatic activity was only observed with phenylalanine
1 (specific activity: 15 nmol minꢀ1 mg proteinꢀ1). Multiple
preparations of RsTAL yielded similar specific activities
and steady-state kinetic parameters. The specific activity
of RsTAL with tyrosine 3 as a substrate was 12-fold lower
than that of the R. capsulatus enzyme (Kyndt et al., 2002)
and 5-fold higher than that of TAL from Saccharothrix
espanaensis (Berner et al., 2006).
Mass spectrometry verified that RsTAL produces
p-coumaric acid 4 and cinnamic acid 2 from L-tyrosine 3
and L-phenylalanine 1, respectively (see Section 3.5). The
Table 2
Summary of pH-dependence of steady-state kinetic parameters
L-tyrosine 3
L-phenylalanine 1
kcat (minꢀ1
pKa
Ymax
)
6.82 0.09
8.29 0.31
6.78 0.29
1.43 0.09
Table 1
Steady-state kinetic parameters of RsTAL
kcat/Km (Mꢀ1 sꢀ1
)
Substrate
kcat (minꢀ1
)
Km (lM)
kcat/Km (Mꢀ1 sꢀ1
)
pKa
Ymax
7.02 0.17
225 20
–
L-tyrosine 3
L-phenylalanine 1
4.8 0.1
1.1 0.1
301 48
6170 740
266
2.97
–
Enzyme assays were performed as described under Section 3. All values
are expressed as a mean SE for an n = 3. Units for the pH-independent
values (Ymax) for each parameter are indicated.
Reactions were performed as described under Section 3 All kcat and Km
values are expressed as a mean SE for an n = 3.
Fig. 2. pH-Dependence of steady-state kinetic parameters: (A) V (solid circles) and V/K (open circles) vs. pH profiles with L-tyrosine 3 as substrate. (B) V
(solid circles) and V/K (open circles) vs. pH profiles with L-phenylalanine 1 as substrate. Lines indicate the best fit of data, as described in Section 3.

A.C. Schroeder et al. / Phytochemistry 69 (2008) 1496–1506
1499
Table 3
1.3. Enzymatic activity and ligand binding properties of
active site mutants
Analysis of product binding by fluorescence titration
p-Coumaric acid 4
Kd (lM)
Cinnamic acid 2
Kd (lM)
For comparison with wild-type enzyme, the Y60F,
S150A, and Y300F mutants were assayed for activity using
L-tyrosine 3 and L-phenylalanine 1. No reaction rates above
background were observed with either substrate for any of
the mutant enzymes (100 lg protein, 6 h assay length).
To determine if the active site mutations compromised
ligand binding, a fluorescence titration assay was used
(Fig. 3). Addition of either p-coumaric acid 4 or cinnamic
acid 3 to RsTAL quenches the protein fluorescence
emission signal (Fig. 3A). Titration of RsTAL with p-cou-
maric acid 4 and cinnamic acid 2 (Fig. 3B) yielded Kd val-
ues of 0.5 lM and 5.0 lM, respectively (Table 3). Binding
of p-coumaric acid 4 and cinnamic acid 2 to the Y60F,
S150A, and Y300F mutants using this assay showed that
each mutant bound both ligands with less than 12-fold dif-
ferences compared to RsTAL (Table 3). These results indi-
cate that mutation of Tyr60, Ser150, or Tyr300 did not
introduce major structural changes that drastically affect
ligand binding in the active site.
RsTAL
Y60F
S150A
Y300F
0.48 0.04
2.4 0.2
5.8 0.4
3.9 0.3
5.0 0.6
1.2 0.4
2.1 0.2
1.4 0.3
Fluorescence titrations were performed as described under Section 3. All
Kd values are expressed as a mean SE for an n = 3.
of this species using collisionally activated dissociation pro-
duced a series of fragment ions consistent with this peptide
sequence (Fig. 4B). As shown in Fig. 4C, multiple fragment
ions were assigned to the peptide, while the absence of frag-
mentation in the region containing Ala149-Gly151 gives
further evidence for the presence of the MIO modification.
Additionally, a species of 2195.16 Da was detected that
correlates to the mass of the unmodified form of the tryptic
peptide within 20 ppm (Fig. 4A). Fragmentation of this 3+
species at 733 m/z confirmed the identity of this peptide
species (Figs. 4D & E). Fragment ions in the region
between Ala149 and Gly151 are further evidence that the
peptide is not modified. This suggests that the final sample
contains a mixture of RsTAL containing the MIO cofactor
and protein lacking the active site modification. It should
be noted that the relative intensities of the two forms can-
not be estimated from intensity of the ion fragments.
Analysis of the S150A mutant clearly shows that the
mutation prevents formation of the MIO prosthetic group.
Mass spectrometry of an unfractionated trypsin digestion
of the S150A mutant yielded a peptide species of 2179.15
Da, correlating with the mass of the unmodified tryptic
peptide (Gly145-Arg166) within 20 ppm (Fig. 5A). Direct
fragmentation of this species using collisionally activated
dissociation produced a series of fragment ions consistent
with this peptide sequence (Fig. 5B). As shown in
1.4. Mass spectrometry analysis of the MIO cofactor
The amino acid ammonia lyases typically contain an
MIO cofactor, which is formed post-translationally by
cyclization of an alanine-serine-glyine segment in the active
site (Fig. 1B) (Poppe, 2001; Baedeker and Schulz, 2002a,b).
This consensus sequence in RsTAL includes Ala149,
Ser150, and Gly151. To identify the MIO group in RsTAL,
we used tryptic digestion and ESI-Q-TOF mass spectrom-
etry. Analysis of an unfractionated trypsin digestion of
RsTAL yielded a peptide species of 2159.16 Da (detected
as the 3+ charge-state at m/z 720.7), correlating with the
mass of the MIO-containing tryptic peptide (Gly145-
Arg166) within 20 ppm (Fig. 4A). Direct fragmentation
Fig. 3. Fluorescence titration analysis of product binding: (A) Emission spectra of RsTAL (1.5 lM protein) (solid line) and RsTAL in the presence of
30 lM p-coumaric acid 4 (dashed line) or 30 lM cinnamic acid 2 (dotted line). Excitation wavelength was 290 nm. Emission intensity is reported in
a.u. = arbitrary units. (B) Titration of p-coumaric acid 4 (solid circles) and cinnamic acid 2 (open circles) binding to RsTAL. Data are plotted as the
percentage of fluorescence change versus ligand concentration. Lines indicate the best fit of data, as described in Section 3.

1500
A.C. Schroeder et al. / Phytochemistry 69 (2008) 1496–1506
Fig. 4. Mass spectrometric analysis of wild-type RsTAL: (A) ESI-Q-TOF mass spectrum (m/z 300–1150) of an unfractionated tryptic digestion; inset
(m/z 720–740) highlights the 3+ charge-state corresponding to the MIO-containing peptide of interest (theoretical Mr: 2159.12 Da) and unmodified active
site peptide (theoretical Mr: 2195.12 Da). (B) MS/MS fragmentation of the 3+ charge-state (m/z 720.7). (C) Graphical fragment map correlating the
fragmentation ions to the sequence of the MIO-containing peptide. The active site serine residue is circled. (D) MS/MS fragmentation of the 3+ charge-
state (m/z 732.7). (E) Graphical fragment map correlating the fragmentation ions to the sequence of the unmodified active site peptide.
´
Fig. 5C, multiple fragment ions were assigned to the pep-
tide predicted sequence. There was no evidence for an
MIO cofactor-containing peptide, as would be indicated
by a 2143.12 Da species, nor evidence of any residual
Ser150 in the active site of the mutant preparation
(expected 2195.12 Da species).
properties of the prosthetic group (Schuster and Retey,
1995). In this experiment, we incubated wild-type and
mutant TAL in the presence of various concentrations of
DTT and monitored changes in fluorescence emission over
time (Fig. 6). Kitz–Wilson analysis of the changes in MIO
emission signal resulting from formation of the thiol-
adduct between DTT and the MIO cofactor in wild-type
1.5. MIO–DTT adduct formation in wild-type and mutant
RsTAL
RsTAL showed pseudo-first order kinetics with a kinact =
0.08 hꢀ1 and KI = 14.5 mM (Fig. 6, inset). Modification
of wild-type RsTAL by DTT was also monitored by activ-
ity assays using L-tyrosine as a substrate and showed a loss
in enzymatic activity that paralleled the loss in MIO emis-
sion signal (not shown). Although enzymatic activity could
not be monitored in the mutant enzymes, modification of
MIO by DTT showed changes in the fluorescence emission
signals of the Y60F and Y300F mutants (Fig. 6, inset),
which yielded inactivation constants similar to wild-type
To evaluate if mutation of either Tyr60 or Tyr300
altered the reactivity of the active site MIO group, a cova-
lent modification assay was used. Earlier work describes
the inactivation of PAL by DTT via formation of a thiol-
adduct with the MIO cofactor (Ritter and Schulz, 2004).
Likewise, through a similar mechanism, cysteine forms a
thiol-adduct with MIO in HAL that changes the spectral

A.C. Schroeder et al. / Phytochemistry 69 (2008) 1496–1506
1501
Fig. 5. Mass spectrometric analysis of the S150A mutant RsTAL. (A) ESI-Q-TOF mass spectrum (m/z 300–1150) of an unfractionated tryptic digestion;
inset (m/z 715–740) highlights the 3+ charge-state corresponding to the unmodified S150A active site peptide (theoretical Mr: 2179.12 Da). (B) MS/MS
fragmentation of the 3+ charge-state (m/z 727.4). (C) Graphical fragment map correlating the fragmentation ions to the sequence of the unmodified S150A
active site peptide.
enzyme (Table 4). This suggests that the reactivity of the
MIO cofactor in the wild-type and mutant enzymes are
similar.
2. Discussion
TAL and other enzymes of the amino acid ammonia
lyase family convert a-amino acids to a,b-unsaturated
´
acids by elimination of ammonia (Poppe and Retey,
2005). The X-ray structure of TAL yielded the first view
of an amino acid ammonia lyase in complex with either a
reaction product or substrate analog (Louie et al., 2006).
As in HAL and PAL (Schwede et al., 1999; Calabrese
et al., 2004; Ritter and Schulz, 2004), the active site of
TAL centers around an MIO prosthetic group, which is
formed by the autocatalytic cyclization of a three-amino
acid loop that includes Ser150 (Poppe, 2001;Baedeker
and Schulz, 2002a,b; Louie et al., 2006). Mechanistically,
each of the two alternate reaction sequences proposed for
the amino acid ammonia lyase family requires a general
base for catalysis (Schwede et al., 1999; Calabrese et al.,
2004; Ritter and Schulz, 2004; Baedeker and Schulz,
2002a,b; Louie et al., 2006). Within the active site of
TAL, both Tyr60 and Tyr300 have been proposed to serve
this function. Using RsTAL, we have examined the roles of
Ser150, Tyr60, and Tyr300 in catalysis, ligand binding, and
MIO processing.
Fig. 6. Kinetics of MIO–DTT adduct formation in wild-type and mutant
RsTAL. Time- and concentration-dependent modification of MIO by
DTT. Wild-type RsTAL was incubated in the presence of 0, 2, 5, 10, 20, or
40 mM DTT (top to bottom) with fluorescence emission of the MIO
prosthetic group monitored over time, as described in Section 3. The inset
shows the Kitz-Wilson analysis of DTT modification for wild-type (solid
circle), Y60F (open circle), and Y300F (open triangle) RsTAL. The line
shows the fit of the wild-type data.
Table 4
DTT-modification kinetics of wild-type and mutant RsTAL
kinact (hꢀ1
)
KI (lM)
RsTAL
Y60F
Y300F
0.08 0.01
0.08 0.01
0.09 0.01
14.5 0.1
10.3 0.1
16.7 0.1
Conversion of tyrosine 3 and phenylalanine 1 into p-
coumaric acid 4 and cinnamic acid 2, respectively, by
RsTAL occurs through a common chemical mechanism,
as indicated by the similar pH-dependence of kcat for the
Modification studies were performed as described under Section 3. All
values were obtained by Kitz–Wilson analysis and are expressed as a
mean SE for an n = 3.

1502
A.C. Schroeder et al. / Phytochemistry 69 (2008) 1496–1506
substrates (Fig. 2A and Table 2). Removal of the substrate
hydroxyl group results in a pH-independent kcat/Km profile
for phenylalanine 1 (Fig. 2B and Table 2), which suggests
the loss of a binding interaction in the active site. As ele-
gantly shown by Watts et al. (2006) and Louie et al.
(2006), a histidine in the active site of TAL determines
the substrate preference for tyrosine 3 over phenylalanine
1. In the crystal structure of TAL (Louie et al., 2006), the
histidine side-chain forms a hydrogen bond with the hydro-
xyl group of the tyrosine 3 substrate. The observed pKa in
the kcat/Km rate profile of TAL for tyrosine 3 likely corre-
sponds to the active site histidine.
Substitutions of Tyr60 and Tyr300 with a phenylalanine
1 in the RsTAL active site yield inactive mutant enzymes
that retain the ability to bind aromatic ligands. Previously,
formation of MIO-thiol adducts was used to monitor the
reactivity of the MIO prosthetic group in the active sites
´
of both HAL and PAL (Schuster and Retey, 1995; Ritter
and Schulz, 2004). Here we used a fluorescence-based assay
to monitor DTT adduct formation by changes in the MIO
emission signal of wild-type and mutant RsTAL (Fig. 6
and Table 4). Although generation of the MIO–DTT
adduct is not the optimal reaction catalyzed by the enzyme
´
(Poppe and Retey, 2005; Poppe, 2001), this assay indicates
Site-directed mutagenesis of Ser150, Tyr60, and Tyr300
in the RsTAL active site yields mutant enzymes with no
detectable activity using either tyrosine 3 or phenylalanine
1 as substrate, indicating that each of these conserved
active site residues is essential for catalysis. Mutation of
the residue corresponding to Tyr60 in HAL and PAL
caused 2650- and 75,000-fold reductions in specific activity,
respectively (Ro¨ther et al., 2001, 2002). Likewise, 55- and
235-fold decreases in reaction rate occurred with mutation
of the residue corresponding to Tyr300 in HAL and PAL,
respectively (Ro¨ther et al., 2001, 2002). Previous studies of
HAL and PAL only report changes in reaction rates
(Ro¨ther et al., 2001, 2002). Here we employed additional
methods to evaluate the effect of mutating these residues
on ligand binding and formation of the MIO prosthetic
group. As demonstrated using fluorescence titrations, the
S150A, Y60F, and Y300F mutant enzymes bind p-couma-
ric 4 and cinnamic acids 3 with modest changes in Kd val-
ues (Fig. 3 and Table 3), indicating that the loss of
enzymatic activity upon mutation of these active site resi-
dues does not result from the inability to bind ligands.
Mutation of Ser150 in RsTAL prevents formation of the
MIO prosthetic group, as shown by ESI-Q-TOF mass
spectrometric analysis of wild-type and mutant enzymes
(Figs. 4 and 5). Formation of the MIO moiety of TAL,
HAL, and PAL occurs through the spontaneous cycliza-
tion and dehydration of an alanine-serine-glycine segment
of the polypeptide backbone in the active site (Fig. 1B)
(Poppe, 2001; Baedeker and Schulz, 2002a,b). This reaction
is analogous to cyclization of the chromophores in green
and red fluorescent proteins (Ormo et al., 1996; Wall
et al., 2000). Mass spectrometry of wild-type RsTAL
(Fig. 4) showed a heterogeneous mixture of the MIO-con-
taining and unprocessed forms of the active site peptides,
even though quantitation could not be surmised in this
experiment. The difference in peak intensities could reflect
difference in ionization efficiencies of the processed (MIO-
containing) and unprocessed (no MIO) active site peptides.
In addition, the cyclized MIO-containing peptide may
interfere with protease digestion, as trypsin binds linear
polypeptide backbones. Nonetheless, mutation of Ser150
to an alanine blocks the autocyclization reaction (Figs.
1B and 5) required for formation of the catalytic MIO
group of RsTAL and results in a catalytically compromised
mutant protein.
that mutations in Tyr60 and Tyr300 do not significantly
alter the reactivity of the MIO prosthetic group.
As shown here, both Tyr60 and Tyr300 are critical for
efficient catalysis in TAL; however, the contribution of
either residue to the TAL reaction mechanism remains
equivocal. Mechanistically, the hydroxyl group of tyrosine
3 requires activation through interactions with other active
site residues to serve as a general acid/base (Blomster et al.,
1995; Jornvall et al., 1995; Jez et al., 1997; Schlegel et al.,
1998; Liu et al., 1999).
In the three-dimensional structure of RsTAL, Tyr300
forms a hydrogen bond with Gln436, which in turn inter-
acts with a water molecule in the active site (Louie et al.,
2006). This set of interactions may reduce the pKa of
Tyr300 allowing it to serve as a general base. Substitution
of a phenylalanine 1 for Tyr300 likely disrupts this network
and affects the local active site structure. In the X-ray crys-
tal structure of HAL containing a phenylalanine mutation
at the corresponding position, differences in the active site
architecture were observed (Baedeker and Schulz, 2002b).
The tyrosine-to-phenylalanine mutation did not shift the
position of the side-chain phenyl group, but eliminated a
hydrogen bond formed with an adjacent glutamate residue
(Glu414 in HAL) that results in a large displacement of the
acidic side-chain into the binding site (Baedeker and
Schulz, 2002b). Moreover, the nitrogen atom of the MIO
prosthetic group contributed from the glycine residue
changed from a pyramidal sp3 hybridization to a planar
sp2 hybridization state (Baedeker and Schulz, 2002b).
Given the similarity of TAL and HAL, analogous struc-
tural changes may alter how the substrate fits in the active
site, such that the loss of activity results from altered posi-
tioning of the substrate relative to the MIO prosthetic
group (without compromising binding affinity), and/or dis-
ruption of the hydrogen bond network interacting with this
residue.
Alternatively, the structure of TAL (Louie et al., 2006)
and mutagenesis of both HAL and PAL (Ro¨ther et al.,
2001, 2002) suggest that Tyr60 could act as the general base
in the reaction catalyzed by the amino acid ammonia
lyases. Within the RsTAL active site, Tyr60 resides in a
highly conserved flexible loop that forms an ‘‘inner-
lid” of the active site (Louie et al., 2006). In earlier
crystallographic studies of HAL and PAL, the analogous
structural regions were either disordered or displayed high

A.C. Schroeder et al. / Phytochemistry 69 (2008) 1496–1506
1503
temperature factors (Schwede et al., 1999; Calabrese et al.,
2004; Ritter and Schulz, 2004). Molecular dynamics simu-
lations of PAL implied that this loop may be functionally
important (Pilbak et al., 2006), but the structures of
RsTAL in complex with products demonstrated for the
first time that Tyr60 moves into close contact with bound
p-coumaric acid 4 (Louie et al., 2006). The experiments
described here show that removal of the hydroxyl group
from this residue in the Y60F RsTAL mutant yields inac-
tive protein that binds p-coumaric and cinnamic acids
and retains a reactive MIO prosthetic group.
Novagen. The QuikChange PCR-based mutagenesis kit
was purchased from Stratagene. Ni2+-nitriloacetic acid
(NTA)-agarose was bought from Qiagen. The Superdex-
200 26/60 FPLC column was from Amersham Biosciences.
All other reagents were purchased from Sigma–Aldrich.
3.2. Bacterial expression vector construction and site-
directed mutagenesis
The coding region of RsTAL was amplified by PCR
from a previously described yeast expression vector (Zhang
et al., 2006) using 50-dTTTGCTAGCATGCTGCCATGA-
30 as the forward primer (NdeI site is underlined; the start
codon is in bold) and 50-dTTTGGATCCTCAGAG-
GGGAGATTGC-30 as the reverse primer (BamHI site is
underlined and the stop codon is in bold). The PCR
product was subcloned into the pET28a vector to yield
pET28a-RsTAL expression vector. Automated nucleotide
sequencing confirmed the fidelity of the expression
construct (Washington University Sequencing Facility).
The Y60F and Y300F site-directed mutants of RsTAL
were generated using the QuikChange (Stratagene) PCR
method with pET28a-RsTAL as template. Presence of
mutations in the resulting vectors was confirmed by
DNA sequencing. Due to the high GC content of the
region around the codon for Ser150, an alternate strategy
was used to generate the S150A RsTAL mutant. The
expression construct of the RsTAL S150A mutant was gen-
erated by amplifying a 227 nucleotide region of the gene
containing the mutation, which was flanked by PstI and
FspI sites. Primers for the amplification of this region were
50-dTTTCTGCAGGCCAATCTTGTCCATCATCTGGC-
CAGCGGC (forward primer, PstI site underlined), and
5’-dTTTTGCGCAAGCGGTGTCAGGTCACCCGCGC-
CCACCG (reverse primer, FspI site underlined and the
mutated basepair for the serine to alanine mutation is indi-
cated in bold italic). PCR products were amplified from the
pET28a-RsTAL template using PfuTurbo polymerase
(Invitrogen), digested with FspI and PstI, and ligated into
FspI/PstI-digested pET28a, where conditions were
adjusted to give partial digestion with FspI due to the pres-
ence of an FspI site in the vector. Automated nucleotide
sequencing of both strands confirmed the fidelity of the
final vector.
How Tyr60 is activated to function as a general base in
the TAL reaction mechanism is unclear. All of the reported
structures for the amino acid ammonia lyases are either for
apoenzyme or product/inhibitor complexes; no view of a
substrate or substrate-analog bound to the enzyme is avail-
able to show the positions of active site residues in the
Michaelis complex. Interestingly, a mechanism for activa-
tion of Tyr60 to serve as a general base is suggested by
recent structural studies of a related enzyme, i.e., tyrosine
aminomutase. Christianson et al. (2007) used a mecha-
nism-based inactivation strategy to obtain a crystal struc-
ture of the MIO-containing tyrosine aminomutase in
complex with a substrate analog covalently attached to
the MIO group. The tyrosine aminomutase active site is
structurally similar to those of TAL, PAL, and HAL,
and contains an MIO-prosthetic group and residues analo-
gous to Tyr60 and Tyr300 in positions similar to RsTAL.
In the tyrosine aminomutase structure, the residue corre-
sponding to Tyr60 is positioned near the a- and b-carbons
of the substrate analog in a position suggesting its function
as a general base. The hydroxyl group of the tyrosine 3
interacts with the nitrogen of an adjacent glycine residue,
which is conserved in TAL and other amino acid ammonia
lyases. This interaction and the contribution of helical
dipoles in the active site are suggested to lower the pKa
of this tyrosine. Moreover, mutation of this residue in tyro-
sine aminomutase yields inactive enzyme (Christianson
et al., 2007).
2.1. Conclusion
In conclusion, the details of the reaction mechanism and
the roles of specific active site residues in the amino acid
ammonia lyase family remain contested and unresolved.
Ultimately, definitive evidence for the mechanistic roles
of the two active site tyrosine residues requires further
structural and functional studies.
3.3. Protein expression and purification
Expression constructs were transformed into E. coli
Rosetta (DE3) cells. Transformed cells were grown at
37 °C in Terrific Broth containing 50 lg mlꢀ1 kanamycin
and 34 lg mlꢀ1 chloramphenicol until the A600 nm was
0.6–0.8. Protein expression was induced by addition of iso-
propyl-1-thio-b-D-galactopyranoside (IPTG) (1 mM final).
Cells were grown 4–6 h at 20 °C before harvesting by cen-
trifugation. Cell pellets were resuspended in 50 mM Tris
(pH 8), 500 mM NaCl, 20 mM imidazole, 10% (v/v)
glycerol, and 1% (v/v) Tween-20 and then sonicated. After
3. Experimental
3.1. Materials
Oligonucleotides were synthesized by Integrated DNA
Technologies. The pET28a bacterial expression vector
and the Escherichia coli Rosetta (DE3) cells were from

1504
A.C. Schroeder et al. / Phytochemistry 69 (2008) 1496–1506
centrifugation, the supernatant was passed over a Ni2+
-
tandem mass spectrometer (Applied Biosystems) outfitted
with an electrospray ion source. Information-dependent
analysis of samples and authentic standards was operated
in positive mode with the source voltage of 5.5 kV and
source temperature of 500 °C. In the enhanced MS scan
mode, scans were carried out between m/z 100 and 1000.
In the enhanced product ion scan mode, parent ions were
fragmented with collision energy +25 kV. Fragments were
monitored between m/z 50 and 600. The observed m/z
values of the parent ion and fragmentation pattern for each
product were determined, as follows: p-coumaric acid,
[C9O3H8+H]+ 165.09, [C9O2H7+H]+ 149.02, [C8OH7+
H]+ 121.06; cinnamic acid, [C9O2H8+H]+ 149.05,
[C9OH7+H]+ 132.04, [C8H7+H]+ 104.06, which were con-
firmed with authentic standards.
NTA column equilibrated in the same buffer as above.
After washing the column in 50 mM Tris (pH 8),
500 mM NaCl, 20 mM imidazole, and 10% (v/v) glycerol,
bound protein was eluted using the same buffer containing
250 mM imidazole. To remove the N-terminal His-tag,
thrombin was added to eluate, which was then dialyzed
overnight (4 °C) against 50 mM Tris (pH 8.0), 500 mM
NaCl, 20 mM imidazole, and 10% (v/v) glycerol. The sam-
ple was depleted of thrombin and uncut protein by running
over a mixed benzamidine-sepharose/Ni2+-NTA column.
Flow-through from this step was loaded onto a Super-
dex-200 26/60 size-exclusion FPLC column equilibrated
in 50 mM Tris (pH 8) and 100 mM NaCl. Fractions con-
taining RsTAL were pooled and stored at ꢀ80 °C. Protein
concentrations were determined using Bradford reagent
(Bio-Rad) with bovine serum albumin as standard.
3.6. Analysis of MIO formation by mass spectrometry
3.4. Enzyme assays
Proteolysis was performed by addition of TPCK-treated
trypsin (Promega) to purified wild-type or S150A RsTAL
(100 lg) at a protease to substrate ratio of 1:10 (w/w) in
50 mM NH4HCO3 (pH 7.8) and incubated at 30 °C for
15 min. Reactions were quenched by the addition of 20%
(v/v) HCO2H (10 lL) and subjected to C18 Zip-Tip (Milli-
pore) purification for desalting before analysis by electro-
spray ionization quadrupole time-of-flight (ESI-Q-TOF)
mass spectrometry. Sample analysis proceeded with an
ABI QSTAR XL (Applied Biosystems/MDS Sciex) hybrid
QTOF MS/MS mass spectrometer equipped with a nano-
electrospray source (Protana XYZ manipulator). Positive
mode nanoelectrospray was generated from borosilicate
nanoelectrospray needles at 1.5 kV. TOF mass spectra were
obtained using the Analyst QS software with an m/z range
of 300–2000. The m/z response of the instrument was cali-
brated with standards from the manufacturer. Species of
interest were selected using the quadrupole and subjected
to collisionally activated dissociation (MS/MS) for confir-
mation of peptide identification. The fragmentation data
were interpreted using Analyst BioTools software and Pro-
sight PTM for correlation of fragment ions to the peptide
primary amino acid sequence.
Standard assays of TAL activity were performed at
25 °C in 0.5 mL volume containing 50 mM Tris (pH 8)
and 2 mM L-tyrosine for 10 min. Production of p-coumaric
acid 4 (A310 nm; e = 17,423 Mꢀ1 cmꢀ1) from L-tyrosine 3
was monitored using a Beckman DU800 spectrophotome-
ter. Conversion of L-phenylalanine 1 to trans-cinnamic acid
2 (A290 nm; e = 10,000 Mꢀ1 cmꢀ1) was measured spectro-
photometrically under similar conditions. Steady-state
kinetic parameters were determined by initial velocity
experiments with L-tyrosine 3 and L-phenylalanine 1. The
untransformed data were fit to the Michaelis–Menten
equation using Kaleidagraph (Syngery Software). pH-rate
profiles were obtained using a triple buffer system
(50 mM sodium acetate, 50 mM 2-(N-morpholino)ethane-
sulfonic acid (MES), and 50 mM Tris, final concentrations)
in place of Tris buffer (Ellis and Morrison, 1982). Data
were fit to the equation for increasing activity vs. pH,
logY = log (c/1 + H/Ka), where Y is the measured param-
eter (kcat or kcat/Km), Ka is the dissociation constant for the
titrated group, and c is the pH-independent value of Y
(Cleland, 1979).
3.5. Product identification
3.7. Fluorescence titration analysis of product binding
Purified RsTAL (1 lg) was incubated with 2 mM L-tyro-
sine 3 or L-phenylalanine 1 for 20 min in 10 mM Tris (pH
8.5) at 30 °C. The reaction mixture was then extracted with
EtOH2 and separated using an LC-20AD HPLC system
(Shimadzu, Tokyo, Japan) equipped with a Spherisorb
ODS-2 reversed phase C-18 column (5 lm; 250 ꢁ 4.6
mm). Compounds were loaded with solvent A (1% (v/v)
AcOH), followed by elution with a linear gradient of sol-
vent B (5–70% (v/v) CH3CN) for 40 min at a flow rate of
0.5 mL minꢀ1. Product identity was first confirmed by
comparison of retention time with standards. Mass
spectrometric analysis used a Q-TRAP 4000 quadrupole
TAL was dialyzed in 50 mM Tris (pH 8) and 150 mM
NaCl. Fluorescence measurements were made on a Varian
Cary fluorimeter (kexcitation = 295 nm and kemission
=
345 nm; slit widths = 5 nm). Titrations were performed at
25 °C by addition of either p-coumaric acid or cinnamic
acid to 0.5 mL of 50 mM Tris (pH 8) and 150 mM NaCl
containing 1.5 lM of wild-type or mutant protein. Control
titrations with buffer alone did not produce any change in
emission signal. The Kd value was calculated using Kale-
idagraph with the data fit to DF = (DFmax[L])/([L] + Kd),
where DF is the change in emission signal in the presence
of ligand (L) and DFmax is the maximal change in signal.

A.C. Schroeder et al. / Phytochemistry 69 (2008) 1496–1506
1505
Ellis, K.J., Morrison, J.F., 1982. Buffers of constant ionic strength for
studying pH-dependent processes. Methods Enzymol. 87, 405–426.
Guerra, D., Anderson, A.J., Salisbury, F.B., 1985. Reduced phenylalanine
ammonia-lyase and tyrosine ammonia-lyase activities and lignin
synthesis in wheat grown under low pressure sodium lamps. Plant
Physiol. 78, 126–130.
Hermes, J.D., Weiss, P.M., Cleland, W.W., 1985. Use of nitrogen-15 and
deuterium isotope effects to determine the chemical mechanism of
phenylalanine ammonia-lyase. Biochemistry 24, 2959–2967.
Jez, J.M., Bennett, M.J., Schlegel, B.P., Lewis, M., Penning, T.M., 1997.
Comparative anatomy of the aldo-keto reductase superfamily. Bio-
chem. J. 326, 625–636.
Jiang, H., Wood, K.V., Morgan, J.A., 2005. Metabolic engineering of the
phenylpropanoid pathway in Saccharomyces cerevisiae. Appl. Environ.
Microbiol. 71, 2962–2969.
Jornvall, H., Persson, M., Krook, M., Atrian, S., Gonzalez-Duarte, R.,
Jeffrey, J., Ghosh, D., 1995. Short-chain dehydrogenases/reductases.
Biochemistry 34, 6003–6013.
3.8. Assay of MIO-modification by DTT
Wild-type or mutant RsTAL was incubated in 50 mM
Tris (pH 8) and 150 mM NaCl in the presence of 0 to
40 mM D/L-dithiothreitol (DTT) at 25 °C. The change in
fluorescence emission signal of the MIO prosthetic group
(kexcitation = 310 nm and kemission = 350 nm; slit widths =
5 nm) was monitored over time. Control incubations
showed no change in the emission signal of MIO over the
time range used. The resulting data was plotted as log (per-
cent quenching of signal) versus time, which were fit to the
first-order equation, ꢀdQ/dt = k[I], where the change in
emission signal (Q) over time is related to the concentration
of DTT, [I], multiplied by k, a rate constant. Using this
equation, the half-life for modification of the MIO cofactor
(t1/2) was determined for each DTT concentration. Kitz–
Wilson analysis of the data yielded the limiting rate con-
stant for inactivation (kinact) and KI (Kitz and Wilson,
1962).
Khan, W., Prithiviraj, B., Smith, D.L., 2003. Chitosan and chitin
oligomers increase phenylalanine ammonia-lyase and tyrosine ammo-
nia-lyase activities in soybean leaves. J. Plant Physiol. 160, 859–863.
Kitz, R., Wilson, I.B., 1962. Acceleration of the rate of reaction of
methanesulfonyl fluoride and acetylcholinesterase by substituted
ammonium ions. J. Biol. Chem. 237, 3235–3239.
Kyndt, J.A., Meyer, T.E., Cusanovich, M.A., Van Beeumen, J.J., 2002.
Characterization of a bacterial tyrosine ammonia lyase, a biosyn-
thetic enzyme for the photoactive yellow protein. FEBS Lett. 512,
240–244.
Kyndt, J.A., Vanrobaeys, F., Fitch, J.C., Devreese, B.V., Meyer, T.E.,
Cusanovich, M.A., Van Beeumen, J.J., 2003. Heterologous production
of Halorhodospira halophila holo-photoactive yellow protein through
tandem expression of the postulated biosynthetic genes. Biochemistry
42, 965–970.
Liu, Y., Barrett, J.E., Schultz, P.G., Santi, D.V., 1999. Tyrosine 146 of
thymidylate synthase assists proton abstraction from the 5-position of
2’-deoxyuridine 5’-monophosphate. Biochemistry 38, 848–852.
Louie, G.V., Bowman, M.E., Moffitt, M.C., Baiga, T.J., Moore, B.S.,
Noel, J.P., 2006. Structural determinants and modulation of substrate
specificity in phenylalanine-tyrosine ammonia-lyases. Chem. Biol. 13,
1327–1338.
Ormo, M., Cubitt, A.B., Kallio, K., Gross, L.A., Tsien, T.Y., Remington,
S.J., 1996. Crystal structure of the Aequorea victoria green fluorescent
protein. Science 273, 1392–1395.
Acknowledgments
This work was supported by grants from the National
Science Foundation (MCB0519634), the U.S. Department
of Agriculture (2005-05190), and the Missouri Soybean
Merchandising Council (02-242) to O.Y. Funding for the
Q-TRAP 4000 mass spectrometer was provided through
a National Science Foundation grant (DBI-0521250).
A.C.S. was supported by a National Science Foundation
Research Experiences for Undergraduates grant (DBI-
0244155) and an American Society of Plant Biologists
Summer Undergraduate Research Fellowship. Prosight
PTM, used by L.M.H., was developed in the laboratory
of Prof. Neil Kelleher (Department of Chemistry, U. Illi-
nois-Urbana-Champaign).
´
Pilbak, S., Tomin, A., Retey, J., Poppe, L., 2006. The essential tyrosine-
containing loop conformation and the role of the C-terminal multi-
helix region in eukaryotic phenylalanine ammonia-lyases. FEBS J. 273,
1004–1019.
References
Poppe, L., 2001. Methylidene-imidazolone:
a novel electrophile for
Baedeker, M., Schulz, G.E., 2002a. Autocatalytic peptide cyclization
during chain folding of histidine ammonia-lyase. Structure 10, 61–67.
Baedeker, M., Schulz, G.E., 2002b. Structures of two histidine ammonia-
lyase modifications and implications for the catalytic mechanism. Eur.
J. Biochem. 269, 1790–1797.
Berner, M., Krug, D., Bihlmaier, C., Vente, A., Muller, R., Bechthold,
A., 2006. Genes and enzymes involved in caffeic acid biosynthesis
in the actinomycete Saccharothrix espanaensis. J. Bacteriol. 188,
2666–2673.
substrate activation. Curr. Opin. Chem. Biol. 5, 512–524.
´
Poppe, L., Retey, J., 2005. Friedel–Crafts-type mechanism for the
enzymatic elimination of ammonia from histidine and phenylalanine.
Angew. Chem. Int. Ed. 44, 3668–3688.
Qi, W.W., Vannelli, T., Breinig, S., Ben-Bassat, A., Gatenby, A.A.,
Haynie, S.L., Sariaslani, F.S., 2007. Functional expression of pro-
karyotic and eukaryotic genes in Escherichia coli for conversion of
glucose to p-hydroxystyrene. Metab. Eng. 9, 268–276.
´
Retey, J., 2003. Discovery and role of methylidene imidazolone, a
Blomster, M., Wetterholm, A., Mueller, M.J., Haeggstrom, J.Z., 1995.
Evidence for a catalytic role of tyrosine 383 in the peptidase reaction of
leukotriene A4 hydrolase. Eur. J. Biochem. 231, 528–534.
Calabrese, J.C., Jordan, D.B., Boodhoo, A., Sariaslani, S., Vannelli, T.,
2004. Crystal structure of phenylalanine ammonia lyase: multiple helix
dipoles implicated in catalysis. Biochemistry 43, 11403–11416.
Christianson, C.V., Montavon, T.J., Festin, G.M., Cooke, H.A., Shen, B.,
Bruner, S.D., 2007. The mechanism of MIO-based aminomutases in b-
amino acid biosynthesis. J. Am. Chem. Soc. 129, 15744–15745.
Cleland, W.W., 1979. Statistical analysis of enzyme kinetic data. Methods
Enzymol. 63, 103–138.
highly electrophilic prosthetic group. Biochim. Biophys. Acta 1647,
179–184.
Ritter, H., Schulz, G.E., 2004. Structural basis for the entrance into the
phenylpropanoid metabolism catalyzed by phenylalanine ammonia-
lyase. Plant Cell 16, 3426–3436.
Rosler, J., Krekel, F., Amrhein, N., Schmid, J., 1997. Maize phenylalanine
ammonia-lyase has tyrosine ammonia-lyase activity. Plant Physiol.
113, 175–179.
´
Ro¨ther, D., Poppe, L., Viergutz, S., Langer, B., Retey, J., 2001.
Characterization of the active site of histidine ammonia-lyase from
Pseudomonas putida. Eur. J. Biochem. 268, 6011–6019.

1506
A.C. Schroeder et al. / Phytochemistry 69 (2008) 1496–1506
´
Ro¨ther, D., Poppe, L., Morlock, G., Viergutz, S., Retey, J., 2002. An
active site homology model of phenylalanine ammonia-lyase from
Petroselinum crispum. Eur. J. Biochem. 269, 3065–3075.
Watts, K.T., Lee, P.C., Schmidt-Dannert, C., 2004. Exploring recombi-
nant flavonoid biosynthesis in metabolically engineered Escherichia
coli. Chembiochem 5, 500–507.
Schlegel, B.P., Jez, J.M., Penning, T.M., 1998. Mutagenesis of 3a-
hydroxysteroid dehydrogenase reveals a ‘‘push–pull” mechanism for
proton transfer in aldo-keto reductases. Biochemistry 37, 3538–3548.
Watts, K.T., Mijts, B.N., Lee, P.C., Manning, A.J., Schmidt-Dannert, C.,
2006. Discovery of a substrate selectivity switch in tyrosine ammonia-
lyase, a member of the aromatic amino acid lyase family. Chem. Biol.
13, 1317–1326.
´
Schuster, B., Retey, J., 1995. The mechanism of action of phenylalanine
ammonia-lyase: the role of prosthetic dehydroalanine. Proc. Natl.
Acad. Sci. USA 92, 8433–8437.
Winkel-Shirley, B., 2001. Flavonoid biosynthesis. A colorful model for
genetics, biochemistry, cell biology, and biotechnology. Plant Physiol.
126, 485–493.
´
Schwede, T.F., Retey, J., Schulz, G.E., 1999. Crystal structure of histidine
ammonia-lyase revealing a novel polypeptide modification as the
catalytic electrophile. Biochemistry 38, 5355–5361.
Xiang, L., Moore, B.S., 2002. Inactivation, complementation, and
heterologous expression of encP, a novel bacterial phenylalanine
ammonia-lyase gene. J. Biol. Chem. 277, 32505–32509.
Trotman, R.J., Camp, C.E., Ben-Bassat, A., Dicosimo, R., Huang, L.,
Crum, G.A., Sariaslani, F.S., Haynie, S.L., 2007. Calcium alginate
bead immobilization of cells containing tyrosine ammonia lyase
activity for use in the production of p-hydroxycinnamic acid.
Biotechnol. Prog. 23, 638–644.
Wall, M.A., Socolich, M., Ranganathan, R., 2000. The structural basis for
red fluorescence in the tetrameric GFP homolog DsRed. Nature
Struct. Biol. 7, 1133–1138.
Xiang, L., Moore, B.S., 2005. Biochemical characterization of
a
prokaryotic phenylalanine ammonia lyase. J. Bacteriol. 187, 4286–
4289.
Zhang, Y., Li, S.Z., Pan, X., Cahoon, R.E., Jaworski, J.G., Wang, X., Jez,
J.M., Chen, F., Yu, O., 2006. Using unnatural protein fusions to
engineer resveratrol biosynthesis in yeast and mammalian cells. J. Am.
Chem. Soc. 128, 13030–13031.