The maximum activity of the mutant and native enzymes with l-DOPA was detected at 45 and 40C, respectively, which was 15C lower than when using l-tyrosine as the substrate.. Consistent w
Trang 1reaction and alleviation by T15A mutation on intertwined N-terminal arm
Seung-Goo Lee1, Seung-Pyo Hong2, Do Young Kim1, Jae Jun Song1, Hyeon-Su Ro3and
Moon-Hee Sung2,4
1 Systems Microbiology Research Center, KRIBB, Daejeon, Korea
2 Bioleaders Corporation, Daejeon, Korea
3 Department of Microbiology and Research Institute of Life Science, KyeongSang National University, Chinju, Korea
4 Department of Bio- and Nanochemistry, Kookmin University, Seoul, Korea
Tyrosine phenol-lyase (TPL; EC 4.1.99.2) is a
carbon-carbon lyase that catalyzes the a,b-elimination and
b-replacement of l-tyrosine and its related amino
acids, with pyridoxal-5¢-phosphate (PLP) as the
cofac-tor [1] Meanwhile, at high concentrations of
ammo-nium pyruvate, the enzyme catalyzes the synthesis of
aromatic amino acids from phenolic substrates
through the reverse reaction of a,b-elimination [2,3]
(Scheme 1) Application of the enzyme for the synthe-sis of 3,4-dihydroxyphenyl-l-alanine (l-DOPA) from catechol has also attracted particular attention [4–6], because l-DOPA is used as a general medicine for the treatment of Parkinson’s disease [7]
Investigations on the metabolic fate of l-DOPA in biological fluids have discovered the formation of con-densation adducts with endogenous aldehydes, like PLP,
Keywords
cofactor affinity; L -DOPA; N-terminal arm;
Pictet–Spengler condensation; tyrosine
phenol-lyase
Correspondence
M.-H Sung, Department of Bio- and
Nanochemistry, Kookmin University,
Seoul 136-702, Korea
Fax: +82 2 910 4415
Tel: +82 2 910 4808 ⁄ 5098
E-mail: smoonhee@kookmin.ac.kr
(Received 27 August 2006, revised 16
October 2006, accepted 18 October 2006)
doi:10.1111/j.1742-4658.2006.05546.x
Citrobacter freundii l-tyrosine phenol-lyase (TPL) was inactivated by a Pictet–Spengler reaction between the cofactor and a substrate, 3,4-dihyd-roxyphenyl-l-alanine (l-dopa), in proportion to an increase in the reaction temperature Random mutagenesis of the tpl gene resulted in the genera-tion of a Thr15 to Ala mutant (T15A), which exhibited a two-fold improved activity towards l-DOPA as the substrate The Thr15 residue was located on the intertwined N-terminal arm of the TPL structure, and comprised an H-bond network in proximity to the hydrophobic core between the catalytic dimers The maximum activity of the mutant and native enzymes with l-DOPA was detected at 45 and 40C, respectively, which was 15C lower than when using l-tyrosine as the substrate The half-lives at 45C were about 16.8 and 6.4 min for the mutant and native enzymes, respectively, in 10 mm l-DOPA On treatment with excess pyrid-oxal-5¢-phosphate (PLP), the l-DOPA-inactivated enzymes recovered over 80% of their original activities, thereby attributing the inactivation to a loss of the cofactor through Pictet–Spengler condensation with l-DOPA Consistent with the extended half-life, the apparent Michaelis constant of the T15A enzyme for PLP (Km,PLP) increased slowly when increasing the temperature, while that of the native enzyme showed a sharp increase at temperatures higher than 50C, implying that the loss of the cofactor with the Pictet–Spengler reaction was prevented by the tighter binding and smal-ler release of the cofactor in the mutant enzyme
Abbreviations
AspAT, aspartate aminotransferase; IPTG, isopropyl thio-b- D -galactoside; LDH, lactate dehydrogenase; L -DOPA, 3,4-dihydroxyphenyl- L -alanine; PLP, pyridoxal-5¢-phosphate; TNA, tryptophan indole-lyase; TPL, tyrosine phenol-lyase.
Trang 2excreting tetrahydroisoquinolines in the urine of patients
after the oral administration of l-DOPA [8,9] The
for-mation of l-DOPA-PLP cyclic adducts has also been
detected in the inactivation of l-DOPA decarboxylase
by a substrate [10,11], eventually leading to the
dissoci-ation of the cofactor However, despite extensive studies
on TPL as a biocatalyst [2–6,12,13], the inhibitory effect
of l-DOPA-PLP adduct formation on the enzymatic
synthesis of l-DOPA has not yet been addressed
Structural studies on the enzymes from Citrobacter
freundii (PDB entries: 1TPL, 2TPL) and Erwinia
herbicola(1C7G) have found them to be composed of
four identical subunits, each with one molecule of PLP
[14,15] Each subunit of C freundii TPL is comprised of
an N-terminal arm (residues 1–19), small domain, and
PLP-binding large domain The active site is located in
a cleft surrounded by one subunit and the large domain
of the adjacent subunit, constituting a catalytic dimer
The two dimers are then tightly combined through a
hydrophobic cluster at the center of the tetramer and
intertwined N-terminal arms (Fig 1)
The above mentioned architecture is conserved in
many a-family PLP-enzymes including tryptophan
indole-lyase (TNA; PDB entry: 1AX4) and aspartate aminotransferases (AspAT; PDB entry: 1ARI) [16–18]
In porcine cytosolic aspartate aminotransferase (AspAT), the N-terminal arm protruding toward the large domain of the other subunit is essential for both the catalytic activity and thermal stability of the enzyme [19–21] Similarly, the AspAT of Bacillus circulans shows a weakened cofactor affinity at the truncation of the N-terminal arm, resulting in a monomeric nonfunc-tional conformation [22] Meanwhile, structural studies
of Proteus vulgaris TNA have revealed an intimate correlation between cofactor binding and the interfacial H-bonds formed on the subunit interface [17]
In this study, a random mutagenesis approach to evolve a robust TPL for l-DOPA synthesis resulted in
an effective mutation, T15A, located on the N-terminal arm of C freundii TPL Biochemical characterization
of the native and mutant enzyme proved the mutation
on the interface increased the stability of the catalytic capability of the enzyme by preventing cyclic conden-sation between l-DOPA and PLP (Fig 1)
Results
Random mutagenesis and structural identification of T15A mutant
An error-prone PCR of C freundii TPL and subse-quent cloning into Escerichia coli XL1-Blue resulted in
a mutant library containing 1–5 mutations that were evenly distributed over the entire TPL sequence About
10 000 colonies from the library were subjected to rapid screening on microtiter plates with l-DOPA
as the substrate To select a highly active mutant from the library, the activity with l-DOPA was divided by the corresponding activity when using l-tyrosine as the substrate, thereby compensating for a variation in the expression levels When comparing the normalized activities, mutant #44 was identified as the most active, with a two-fold increased activity with l-DOPA A sequence analysis of #44 exhibited an amino acid change from Thr15 to Ala, while a structural analysis
of C freundii TPL (1TPL, 2TPL) revealed that Thr15 was located on the intertwined N-terminal arm, com-prising an H-bond network between the catalytic dimers within a proximal distance of the hydrophobic core (Fig 2A) The hydroxyl group of Thr15 was H-bonded to the sidechain of Lys59, and connected to the sidechain of Asp58 via a water molecule, which was also linked to the backbone nitrogen of Thr15 (Fig 2B) In addition, the sidechain of Thr15 was involved in nonbonded interactions with the Lys59 and Glu308 sidechains from the other catalytic dimer
Fig 1 Schematic view of Pictet–Spengler reaction and cofactor
release from holo-TPL enzymes The adductive reaction between
L -DOPA and pyridoxal-5¢-phosphate (PLP) leads to the depletion of
the cofactor in the reaction solution, inactivating the enzyme
depending on the cofactor binding affinity.
Scheme 1 Synthesis reaction by TPL.
Trang 3In summary, the proximate interaction of Thr15 with
the other subunits suggested that the effect of T15A
on the catalytic capability was related to changes in
the interdomain architectures of the catalytic dimers
Purification, kinetic parameters, and catalytic
stability withL-DOPA as substrate
The E coli XL-1 Blue cells bearing the plasmid
pHR1001 or pDA44 revealed a thick protein band
with a molecular mass of 52 kDa in an SDS⁄ PAGE
analysis after induction with 1 mm isopropyl
thio-b-d-galactoside (IPTG) Based on ammonium sulfate
precipitation between 50 and 70% saturation, followed
by ion exchange and hydrophobic chromatography,
the native TPL and T15A mutant were purified to
homogeneity with a recovery yield of 45% and 39%,
respectively The purified proteins were preserved in
a refrigerator after being reprecipitated in 70%
(NH4)2SO4, then desalted just before use to recover
their original specific activities of around 1.2 and 0.64
unitsÆmg)1, respectively, with l-tyrosine as the
sub-strate
The kinetic parameters were determined in triplicate
experiments at 30C, with 0.05–1 mm l-tyrosine or
0.5–12 mm l-DOPA as the substrate The catalytic rate
constants (kcat) for the native and mutant enzymes
with l-DOPA were 0.31 s)1 and 0.68 s)1, respectively
(Table 1), while the Michaelis constants with l-tyrosine
were determined as 0.24 and 0.22 mm, respectively,
indicating a conserved geometry at the binding site, and with l-DOPA were determined to be 3.2 and 4.6 mm, respectively, yet with larger error limits Inves-tigations of the substrate range of the TPLs revealed that 3-chloro-l-tyrosine, dl-serine, and dl-cysteine also served as substrates to a lesser extent, whereas
d-tyrosine, d-DOPA, dl-tryptophane, dl-phenylalan-ine, and dl-alanine were all inert towards the enzymes The native and mutant enzymes were then investi-gated for their stability and activity at temperatures between 15 and 80C When heated for 20 min in the standard buffer, both enzymes remained stable up to
55C in a 0.1 m potassium phosphate buffer (pH 8.0) (Fig 3A) The half-inactivation temperatures for the native and mutant enzymes were calculated to be 62.2 and 65.2C, respectively, with a four-parameter sig-moid equation using sigmaplot (Systat Software Inc., Richmond, CA, USA) Plus, the inclusion of two sub-strates for the synthesis of l-DOPA (20 mm catechol and 1.0 m ammonium acetate) increased the half-inac-tivation temperatures to 72.1 and 73.9C, respectively, similar to the stabilization of porcine cytosolic AspAT
by a-ketoglutarate [20] The maximum activity for the a,b-elimination of l-tyrosine was observed at 55 and
60C for the native and mutant enzymes, respectively (Fig 3B) However, when l-DOPA was applied as the substrate, the temperatures producing the maximum activity were down-shifted by 15C for both the native and mutant enzymes to 40 and 45C, respectively (Fig 3C)
Fig 2 Interfacial architectures of catalytic dimers of C freundii TPL (A) Overall struc-ture, extracted from prediction of oligomeric states server at EBI (http://pqs.ebi.ac.uk/) Yellow colored molecules represent 3-(4¢-hydroxyphenyl)propionic acid adopted from 2TPL PDB file (B) Magnified view of red-lined box in overall structure Green lines represent intimate molecular interactions including hydrogen bond networks in vicinity
of Thr15 on intertwined N-terminal arm H-bond information was extracted from entry code 1TPL of Protein Data Bank.
Table 1 Kinetic constants for C freundii TPL and T15A mutant.
Enzymes
Trang 4Inactivation of C freundii TPL by Pictet–Spengler
reaction
Cyclic adducts of l-DOPA with endogenous aldehydes
have been detected in biological solutions for decades
As such, when the C freundii TPL (30 lm) was
incu-bated with 10 mm l-DOPA in a 0.1 m potassium
phosphate buffer (pH 7.5) at 30C, a time-dependant decrease in the absorbance at 400 nm was detected, resulting in a new absorption peak at 330 nm (Fig 4A), corresponding to previous literature on the inactivation of l-DOPA decarboxylases when using
l-DOPA as the substrate [5,6,23] When a pseudo first-order kinetic (low initial concentration of the enzyme) was applied for the decolorization rate of
C freundii TPL with l-DOPA, the rate constant (k1) was calculated as 0.012 min)1 using a kinetic equation, logAt
A 0¼ k1t , where A0 and At are the
Fig 3 Effect of temperature on stability and activity of C freundii
TPL and T15A mutant (A) Stability, (B) activity with L -tyrosine, and
(C) activity with L -DOPA as substrates The stability was evaluated
as the remaining activity after the enzymes were incubated in a
100 m M potassium phosphate buffer (pH 8.0) at the indicated
tem-peratures for 20 min The activity with L -tyrosine and L -DOPA as
the substrates was measured in the standard reaction mixture for
20 min at different temperatures and the amount of pyruvate
pro-duced determined by the salicylaldehyde method Closed symbols
represent native enzyme (d,r) and open symbols represent T15A
mutant (s,e) Diamond symbols indicate improved stability in the
presence of 20 m M catechol and 1.0 M ammonium acetate.
Fig 4 Pictet–Spengler adduct formation from C freundii TPL in presence of L -DOPA (A) Spectral analyses of enzyme solution (30 l M ) treated with 10 m M L -DOPA in 0.1 M potassium phosphate buffer (pH 7.5) at 30 C Inset: Time-dependent decrease in absorb-ance at 400 nm (B) HPLC analyses of enzyme mixture After the spectral change was completed, the reaction solution was subjec-ted to centrifugal ultrafiltration (molecular cutoff: 10 000), the fil-trate loaded on a DOWEX 50 W column (pH 3.0), and the eluted solution precipitated with three volumes of isopropanol on ice The precipitates were dissolved in water, then analyzed by HPLC The standard was a 2 : 1 mixture of pyridoxal-5¢-phosphate and
L -DOPA-PLP adduct synthesized in the authors’ laboratory.
Trang 5absorbance at times 0 and t, respectively (inset in
Fig 4A) After the spectral change was completed, the
reaction solution was treated with a cation exchange
resin (DOWEX 50 W), analyzed by HPLC, and found
to include an l-DOPA-PLP adduct with the same
retention time and molecular mass (426 Da) as the
Pictet–Spengler type adduct (Fig 4B) synthesized as
described below Meanwhile, the rate constant (k1) of
free PLP was 0.12 ± 0.02 min)1 under the pseudo
first-order reaction conditions Consequently, the free
cofactor was estimated to be 10-fold more susceptible
to adduct formation than the enzyme-bound PLP
The rate constants also increased with the pH and
temperature, as previously reported for the reaction
between l-DOPA and d-glucose [7] In particular, the
k1 values increased up to 0.5 ± 0.1 min)1when 1.0 m
ammonium chloride (pH 8.2) was included in the
reac-tion solureac-tion
As seen in Fig 3C, the maximum activity of the
mutant and native enzymes with l-DOPA as the
sub-strate was 15C lower than when using l-tyrosine as
the substrate, plus both enzymes were similarly stable
up to 55C Consequently, because the spectral and
kinetic studies on the decolorization of TPL suggested
that the compromised activity was closely related to
the loss of the coenzyme via adduct formation, an
experiment on the stability of the enzyme-bound
cofac-tors was performed in a 1.0 m ammonium chloride
solution (pH 8.2) with 10 mm l-DOPA During
incu-bation at 45C, the enzyme mixtures were withdrawn
intermittently, 100-fold diluted in an assay solution,
and examined for their remaining activity using 1 mm
l-tyrosine as the substrate The remaining activity of
the mutant and native enzymes decreased according to
the incubation time, down to 30% and 6% of the
ini-tial activity (dotted lines in Fig 5) with a half-life of
16.8 and 6.4 min, respectively
However, when the same samples were assayed in
the presence of excess PLP (200 lm), both enzymes
recovered over 80% of their original activity (solid
lines in Fig 5), indicating that the inactivation could
be attributed to the loss of the cofactor through a
con-densation reaction with l-DOPA
Stabilization of cofactor binding by T15A
mutation
The extended lifetime of T15A in 10 mm l-DOPA
sug-gested that the intertwined N-terminal architecture,
where Thr15 is located, was closely related with the
cofactor binding affinity of C freundii TPL To verify
the effect of the T15A mutation on the cofactor
affin-ity, the apparent Michaelis constants for PLP (Km,PLP)
with the native and mutant enzymes were investigated
at temperatures ranging from 30 to 60C
As shown in Fig 6A, the Km,PLPfor C freundii TPL increased slowly below 45C, accompanied by an increase in the catalytic rate constant (kcat) However, above 50C, the binding constants increased very sharply, while the kcat remained at a similar level (Fig 6B) An increase in the Km,PLPwas also detected with the T15A mutant, yet significantly slower than that with the native enzyme (Fig 6A) As such, the cofactor release from the active site was increased rel-ative to the temperature, likely accelerating the adduct formation with l-DOPA, yet this was significantly relieved by the T15A mutation located on the inter-twined N-terminal arm
Finally, the effect of the T15A mutation on l-DOPA synthesis was investigated in a reaction solution (10 mL) including 0.65 m ammonium chloride (pH 8.5),
50 mm sodium pyruvate, 50 mm catechol, 0.1 mm PLP, 0.1% sodium sulfite, and 15 units of the enzyme
In addition, because alcoholic additives have been shown to be beneficial for the synthesis of l-DOPA by
C freundii TPL [3], 10% ethanol was also included in the reaction solution When the synthesis reaction was carried out for 2.5 h at 45C, the concentration of
l-DOPA increased rapidly up to the maximum level within an hour, then slightly decreased (Fig 7), prob-ably because of the adduct formation between
l-DOPA and remaining pyruvate [2,5] However, the upward curve flattened much earlier with C freundii TPL, consequently the l-DOPA productivity of T15A was at least two-fold higher than that with C freundii
Fig 5 Inactivation of TPL enzymes by L -DOPA and its reactivation
by PLP Timecourse profiles of inactivation and activity recovery in presence of excess pyridoxal 5¢-phosphate An enzyme mixture containing 0.01 unitsÆml)1of TPL, 0.1 m M PLP, and 10 m M L -DOPA
in a 100 m M potassium phosphate buffer (pH 8.0) was incubated at
45 C for different times, and the remaining activity determined in the presence of excess PLP (d,s) or without PLP (r,e) Closed symbols represent native enzyme and open symbols represent T15A mutant.
Trang 6TPL, consistent with the robustness of T15A at
eleva-ted temperatures (Figs 5 and 6C) No oxidation of
l-DOPA was detected while the solutions were flushed
with nitrogen gas
Discussion
The enzymatic synthesis of l-DOPA using E herbicola
TPL is more successful at a low temperature range
from 15 to 24C [5,24] Likewise, with C freundii
TPL, the synthesis was facilitated at 18C [2], although the enzyme activity was about 20% of the maximal activity (Fig 3C) The compromised produc-tivity at high temperatures has been attributed to the formation of byproducts and the oxidative deterior-ation of catechol or pyruvate during the reaction, all
of which are accelerated by the temperature [2,5]
In this study, it was postulated that the lowered pro-ductivity of C freundii TPL at elevated temperatures partly resulted from a decolorization reaction in the enzyme mixture, which eventually led to the depletion
of the cofactor PLP, accompanied by the inactivation
of the enzyme HPLC and 1H NMR analyses of the purified adduct revealed that the inactivation resulted from Pictet–Spengler type condensation between
l-DOPA and PLP Notwithstanding previous reports
on the inactivation of PLP enzymes, aromatic decarb-oxylases, by l-DOPA [10,23], this is the first time the rapid inactivation of TPL has been explained based on
a Pictet–Spengler reaction
Consistent with the observation that a Pictet–Spen-gler reaction is accelerated relative to the reaction temperature [8], the optimal temperature for enzyme activity in the presence of l-DOPA was 15C lower than that with l-tyrosine as the substrate (Fig 3B,C) The inactivation profile of the enzyme with 10 mm
l-DOPA (Fig 5) also agreed with the optimal tem-perature results Meanwhile, the incubation of TPL with d-DOPA, a stereoisomer of l-DOPA that does
Fig 6 Effect of temperature on kinetic constants for C freundii
TPL and T15A mutant (A) Apparent binding constant (K m,PLP ) for
PLP, (B) catalytic rate constant (kcat), and (C) ratio of kcat⁄ K m ,PLP.
The kinetic constants were determined from double reciprocal plots
of the reaction rate versus the PLP concentration at different
tem-peratures Closed symbols represent Km,PLP values for C freundii
TPL, while open symbols represent Km,PLPvalues for T15A mutant.
Fig 7 Synthesis of L -DOPA by C freundii TPL and T15A mutant The synthetic reaction was carried out using partially purified enzymes in a solution (10 mL) containing 0.65 M ammonium chlor-ide (pH 8.5), 50 m M sodium pyruvate, 50 m M catechol, 0.1 m M
PLP, 0.1% sodium sulfite, 10% ethanol and 15 units of enzyme The reaction bottle was flushed with nitrogen gas, tightly sealed with a rubber stopper, and incubated at 45 C Samples were with-drawn using a syringe in a stream of nitrogen gas to prevent oxida-tion of the ingredients Closed symbols represent native enzyme and open symbols represent T15A mutant.
Trang 7not serve as a substrate for the enzyme reaction,
pro-duced a similar effect to l-DOPA, indicating that
the adduct-forming reaction was independent of the
enzyme reaction and a chemical reaction between
l-DOPA and the free PLP released from the active
site The release of PLP from the enzyme was
acceler-ated at an elevacceler-ated temperature, as shown by the
Km,PLPversus temperature profile of the native enzyme
(Fig 6A) The enzyme-bound PLP reacted with the
l-DOPA in the reaction mixture to form an
l-DOPA-PLP adduct at a rate of 0.012 min)1, as shown by the
inset in Fig 4A, which was 10 times slower than
the rate with the free PLP and l-DOPA (Fig 1) The
removal of PLP by release and the subsequent Pictet–
Spengler reaction may have been responsible for the
rapid decrease in the kcat⁄ Km,PLP value of the native
enzyme at temperatures above 45C (Fig 6C) Note
that the kcat⁄ Km,PLPvalue was the catalytic rate in the
presence of a limited concentration of PLP
In contrast, the Km,PLP-value for T15A was less
sensitive to the temperature (Fig 6A), suggesting a
tight binding of the cofactor at the enzyme active
site Therefore, the mutation on the intertwined
N-terminal arm stabilized the cofactor binding
affin-ity, thereby improving the catalytic properties at
elevated temperatures (Fig 7), as indicated by the
higher stability of the kcat⁄ Km,PLPvalue for the T15A
enzyme (Fig 6C)
Citrobacter freundii TPL has a 50% sequence
iden-tity with the tryptophanase from P vulgaris, which
degrades tryptophan to indole, ammonia, and pyruvate
[14,25] The secondary, tertiary, and quaternary
struc-tures are also highly conserved, plus a hydrophobic
cluster and intertwined N-terminal arms are formed on
the intersubunit interface, contributing to its stability
The network of hydrogen bonds and salt bridges
formed upon the binding of PLP is known to influence
the quaternary structure of tryptophanases [17]
There-fore, when considering the common structural features
of a-family PLP enzymes [26,27], the T15A mutation
on the N-terminal arm may have increased the rigidity
of the cofactor binding architecture of C freundii TPL
through adjusting the quaternary interfaces One
poss-ible communication between the N-terminal arm and
the active site is through Tyr71, which belongs to the
adjacent subunit of the catalytic dimer Tyr71 is
known to be essential for activity, as a general acid
catalyst for the elimination of the leaving group from
a quinonoid intermediate, and also for PLP binding
[28] The PLP binding constant for the Y71F mutant
of C freundii TPL was estimated to be 1 mm, while
the wildtype TPL showed a binding constant of 0.6 lm
based on spectrophotometric titration Consistently,
the equivalent Tyr70 in aspartate aminotransferase also has a PLP binding function [29]
Thus, this study demonstrated that the deterioration
of the cofactor through a Pictet–Spengler reaction with
l-DOPA appeared to be a significant interference with the biotechnological production of l-DOPA when using C freundii TPL The T15A mutation improved the cofactor binding affinity at high temperatures, along with the apparent turnover rate when using
l-DOPA as the substrate, through an interfacial inter-action between the N-terminal arm and the cleft active site However, l-DOPA synthesis at a high temperature also increases the adduct formation between l-DOPA and a substrate pyruvate [2,5], eventually decreasing the l-DOPA concentration during a prolonged reaction
at a high temperature, as observed in Fig 7 Thus, despite the increased catalytic efficiency and stability of the T15A mutant, l-DOPA synthesis at a high temperature should be further scrutinized to minimize the adduct formation between l-DOPA and pyruvate For example, a continuous limited supply of pyruvate into the reaction solution could be used to maintain the pyruvate concentration at a minimal level, thereby decreasing the adduct formation rate In addition, based on the effect of alcohols [3], the reaction ingre-dients could also be optimized to increase the l-DOPA synthesis and relieve the adduct formation
Consequently, with its enhanced l-DOPA synthesis activity and stability, the T15A enzyme of this work could be used for the development of a new bioconver-sion strategy for the efficient production of l-DOPA
at high temperatures, where it can catalyze the reaction more actively
Experimental procedures
Materials The PLP was purchased from Sigma (St Louis, MO, USA) and the l-DOPA purchased from Boehringer Mannheim (Mannheim, Germany) The restriction endonucleases and T4 DNA ligase were purchased from New England Biolabs (Beverly, MA, USA) and the Taq DNA polymerase from PerkinElmer (Branchburg, NJ, USA) The oligonucleotides were synthesized at Bioneer Co (Daejeon, Korea) and the DNA sequencing performed at Solgent Co (Daejon, Korea) The l-DOPA-PLP adduct was synthesized by mixing
l-DOPA (0.32 g) and PLP (0.2 g) in a 50 mm sodium phosphate buffer (80 mL, pH 8.0) at 45C for 30 min The reaction product was purified on a DOWEX 50 W column (pH 3.0, Sigma) and the eluted solution precipitated with isopropanol (240 mL) on ice for 2 h The precipitates were then washed on a sintered glass filter with acetone and
Trang 8stored in a deep freezer after vacuum-drying The molecular
mass of the adduct was 426 Da on a ESI-MS spectrometer,
and the chemical shift values in D2O determined by
300 MHz 1H NMR experiments were as follows: d 2.36
(3 H, s, H-2¢), 3.2 (2 H, m, H-b), 4.0 (1 H, m, H-a), 4.91
(2 H, d, H-5¢), 5.77 (1 H, s, H-4¢), 6.20 (1H, s, H-5¢¢), 6.69
(1 H, s, H-2¢¢), and 7.74 (1 H, s, H-6) The chemical
struc-ture of the adduct was identified as shown in Fig 1 All
other chemicals used were chemical reagent grade
Random mutagenesis and screening
on microtiter plates
The plasmid pHR1001 harboring the C freundii tpl gene
(gene bank accession no DQ907529) [3] was used as the
template for an error-prone PCR with the following
prim-ers: 5¢-AATTATCCGGCAGAACCCTT-3¢ (forward) and
5¢-GATCAAGCTTTTAGATATAGTCAAAGCGTGC-3¢
(reverse, underlined HindIII) The thermal cycling was
per-formed using a DNA Thermal Cycler (PerkinElmer): 5 min
at 95C, a subsequent 25 cycles of 1 min at 95 C, 2 min at
50C, 3 min at 72 C, and a final extension of 7 min at
72C The amplified PCR products were digested with
Hin-dIII to yield a 1.37 kb DNA fragment The plasmid
pTrc99A was then digested with NcoI, blunt-ended by
Klenow treatment, and digested with HindIII The resulting
plasmid was ligated with the HindIII-treated PCR product
by blunt-cohesive ligation at 16C with a T4 DNA ligase
E coliXL1 Blue cells were then transformed with the ligate
by electroporation and spread on LB-ampicillin plates After
being incubated overnight at 37C, the evolved colonies
were transferred by toothpick to fresh LB-ampicillin plates
The mutant library was inoculated into an
LB-ampicillin-IPTG medium (500 lL) contained in a deep 96-well plate,
and cultivated in a wellplate culture system, MegagrowTM
(Bioneer Co.) The cultivated cells were centrifuged at
5000 g for 20 min with a wellplate centrifuge Union
5KRTM, rotor type TM96-65 (Hanil Sci Ind., Inchon,
Korea), washed in a 50 mm Tris⁄ HCl buffer (pH 8.0), and
treated with 200 lL CellyticTMB (Sigma) for 1 h at 37C
The cell lysate (100 lL) was then transferred into 96-well
PCR plates and mixed with the same amount of substrate
solutions, including 10 mm l-DOPA or 1 mm l-tyrosine,
and 20 lm PLP in a 50 mm potassium phosphate buffer
(pH 8.0) After being incubated at 37C for 20 min, the
reaction solutions were heated for 3 min at 95C,
centri-fuged at 5000 g for 20 min with Union 5KRTMcentrifuge to
remove any insoluble aggregates, and analyzed for pyruvate
formation using the salicylaldehyde method [25] to compare
the enzyme activities towards l-DOPA and l-tyrosine
Expression and purification
Escerichia coli XL-1 Blue cells harboring pHR1001 or
pDA44 were cultivated at 37C for 16 h in 1 litre of an LB
medium containing 100 lgÆmL)1ampicillin Protein expres-sion was induced by the addition of 1 mm IPTG when the absorbance at 600 nm reached 0.5 The harvested cells were then disrupted by sonification in a standard buffer, inclu-ding 0.01% 2-mercaptoethanol, 0.05 mm PLP, and 50 mm Tris⁄ HCl (pH 8.0) The centrifugation supernatant was col-lected, and subjected to ammonium sulfate fractionation between 50% and 70% saturation The enzyme dissolved in the standard buffer was then loaded on to a Resource Q ion exchange (Pharmacia, Uppsala, Sweden), washed with the standard buffer, and eluted using a KCl gradient from
0 to 0.5 m Most of the active fractions were then pooled, adjusted to include 1.7 m (NH4)2SO4, and loaded on to a Phenyl Superose (Pharmacia) The elution from the hydro-phobic column was performed using a reverse gradient of (NH4)2SO4 from 1.7 m to 0 m, then the active fractions were dialyzed against a 100 mm Tris⁄ HCl buffer (pH 8.0) containing 0.2 m KCl, reprecipitated in 70% saturated (NH4)2SO4, and stored in a refrigerator All the column procedures were carried out using an AKTA system (Amer-sham Bioscience, Uppsala, Sweden) at room temperature
Determination of kinetic parameters and cofactor binding affinity
The kinetic constants for l-DOPA and l-tyrosine as sub-strates were determined using a lactate dehydrogenase (LDH)-coupled assay of the pyruvate formation rate The reaction was started by the addition of 0.05–1.0 mm l-tyro-sine or 0.5–12 mm l-DOPA as the substrate, and the decrease in A340monitored at 30C using a spectrophoto-meter, Ultrospec3000 (Pharmacia Biotech, Uppsala, Sweden), equipped with a Peltier cuvette-heating system The pyruvate formation rate was calculated using the extinction coefficient of NADH (6200 m)1Æcm)1) from the slope between 0.5 and 5.0 min, after the early perturbation
of the absorbance was settled
The apparent binding constants of PLP to the enzymes were presumed as the concentration of PLP for half the maximal activity of the enzyme The assay mixture with dif-ferent PLP concentrations (0.5–200 lm) and 2.5 mm l-tyro-sine was equilibrated to different temperatures for 5 min in
a thermo-controlled spectrophotometer, and the enzyme activity measured using an LDH coupling assay, as des-cribed above The apparent binding constants for PLP (Km,PLP) were calculated from a double reciprocal plot
of the reaction rate (v) versus the PLP concentration: m
Vmax¼K ½PLP
m;PLP þ½PLP, where Vmax is the maximum reaction rate at saturating PLP concentrations All the kinetic experiments were performed in triplicate
Enzyme assay and analysis The a,b-elimination activity of TPL was calculated from the pyruvate formation rate determined by a coupling assay
Trang 9with LDH (Roche Diagnostics, Bazel, Switzerland) or using
the salicylaldehyde method [25] The standard reaction
mix-ture contained 10 mm l-DOPA or 1 mm l-tyrosine as the
substrate, 50 lm PLP, 0.2 mm NADH, 10 lgÆmL)1 LDH,
and TPL in a 0.1 m potassium phosphate buffer (pH 8.0)
One unit of enzyme was defined as the activity to catalyze
the formation of 1 lmol of pyruvate per min at 30C The
protein concentration was determined using a Bradford
rea-gent (Bio-Rad, Hercules, CA, USA) with bovine serum
albumin as the standard
The analysis of the l-DOPA-PLP adduct was performed
on a HPLC system (Young-in Co., Seoul, Korea) equipped
with an ODS18 column (Shimazu, Kyoto, Japan) and
UV-detector (295 nm) The elution was carried out using a
co-solvent consisting of a 50 mm potassium phosphate buffer
with 2 mm sodium dodecylsulfate (pH 3.0), methanol, and
acetonitrile (volumetric ratio¼55 : 40 : 5) at a flow rate of
0.6 mLÆmin)1
Acknowledgements
This project was supported by a grant from the
Clea-ner Production Program 10007946 of NCPC, the
KRIBB Research Initiative Program, and the 2006
research fund of Kookmin University, Korea
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