Paclitaxel Biosynthesis: Adenylation and Thiolation Domains of an NRPS TycA PheAT Module Produce Various Arylisoserine CoA Thioesters

Article abstract of DOI:10.1021/acs.biochem.6b01188

Structure-activity relationship studies show that the phenylisoserinyl moiety of paclitaxel (Taxol) is largely necessary for the effective anticancer activity. Several paclitaxel analogues with a variant isoserinyl side chain have improved pharmaceutical

Full text of DOI:10.1021/acs.biochem.6b01188

Article
pubs.acs.org/biochemistry
Paclitaxel Biosynthesis: Adenylation and Thiolation Domains of an
NRPS TycA PheAT Module Produce Various Arylisoserine CoA
Thioesters
Ruth Muchiri† and Kevin D. Walker*,†,‡
†Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, United States
‡Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824, United States
S
* Supporting Information
ABSTRACT: Structure−activity relationship studies show that
the phenylisoserinyl moiety of paclitaxel (Taxol) is largely
necessary for the effective anticancer activity. Several paclitaxel
analogues with a variant isoserinyl side chain have improved
pharmaceutical properties versus those of the parent drug. To
produce the isoserinyl CoAs as intermediates needed for
enzyme catalysis on a semibiosynthetic pathway to paclitaxel
analogues, we repurposed the adenylation and thiolation
domains (Phe-AT) of a nonribosomal peptide synthetase
(TycA) so that they would function as a CoA ligase. Twenty-
eight isoserine analogue racemates were synthesized by an
established procedure based on the Staudinger [2+2] cyclo-
addition reaction. Phe-AT converted 16 substituted phenyl-
isoserines, one β-(heteroaryl)isoserine, and one β-(cyclohexyl)isoserine to their corresponding isoserinyl CoAs. We imagine that
these CoA thioesters can likely serve as linchpin biosynthetic acyl donors transferred by a 13-O-acyltransferase to a paclitaxel
precursor baccatin III to make drug analogues with better efficacy. It was also interesting to find that an active site mutant [Phe-
AT (W227S)] turned over 2-pyridylisoserine and the sterically demanding p-methoxyphenylisoserine substrates to their CoA
thioesters, while Phe-AT did not. This mutant is promising for further development to make 3-fluoro-2-pyridylisoserinyl CoA, a
biosynthetic precursor of the oral pharmaceutical tesetaxel used for gastric cancers.
Taxus plant cell fermentation is the principal method used
currently to make the antineoplastic drug paclitaxel.1,2 While
this biological method produces paclitaxel at a rate of 500 kg/
year,1 the plant cells have competing specialized metabolic
pathways that divert part of the carbon flux away from
paclitaxel.3 To address these challenges, a sustainable biological
method can be envisioned that directs carbon flux from
baccatin III (4), a pathway intermediate in cells that is 4 times
more abundant than paclitaxel,4 to a desired taxane end product
(3) (Figure 1). This proposed engineered semibiosynthetic
pathway involves transfer of an arylisoserinyl moiety from a
CoA thioester to the C-13 hydroxyl of baccatin III.5 To date, no
naturally occurring arylisoserinyl CoA ligase has been found in
Taxus plants or derived cell cultures where paclitaxel is made.
However, in an earlier study, we repurposed the adenylation
and thiolation domains of tyrocidine synthetase A (Phe-ATE)
from Bacillus brevis ATCC 8185 as a surrogate CoA ligase to
make (2R,3S)-phenylisoserinyl CoA.6 With a suitable CoA
ligase identified, a biosynthetic approach can now be built using
the ligase and acyltransferases from the paclitaxel pathway.7 In
particular, an acyl CoA-dependent 13-O-phenylpropanoyltrans-
ferase (BAPT)8 incubated with an arylisoserinyl CoA and
baccatin III can provide an intermediate N-debenzoylpaclitaxel
analogue (5) (Figure 1).5 The latter incubated with benzoyl
CoA, or another aroyl CoA, and a Taxus N-benzoyltransferase
would complete the biosynthesis of 3 or its derivatives.9
Despite the wide application of paclitaxel in the treatment of
various cancers10 and heart disease,11,12 there are concerns
about adverse physiological effects and drug resistance.13,14
These limitations created an effort to develop paclitaxel
analogues that minimized side effects yet maintained good
pharmacological properties.15 Typically, the analogues have
improved drug potency or increased water solubility versus that
of paclitaxel in cancer cell bioassays.16,17 Several analogues had
a modified isoserinyl side chain at C-13 of the diterpene core to
improve pharmacological effects against various multidrug-
resistant cancer cell lines (Table 1).14,18−21 As important was
the fact that the 2′-hydroxyl and 3′-N-acyl functional groups on
the isoserine moiety of paclitaxel variants were necessary for
drug efficacy. For example, acetylation of the 2′-OH inactivates
the drug,22 or its replacement with a fluoro or thio group
decreases the level of binding of the drug to microtubules.23−25
Combined removal of the 2′-hydroxyl and 3′-N-acyl groups of
Received: November 22, 2016
Revised: January 23, 2017
Published: February 23, 2017
© XXXX American Chemical Society
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companies refocus research efforts on previously approved
drugs, thus likely reducing the number of new taxoids brought
to market.
Other alkyl/alkenylisoserine taxoids include the cyclo-
hexylisoserinyl analogue 13, which has activity superior to
that of paclitaxel in a microtubule disassembly inhibition assay
and is cytotoxic against a doxorubicin-resistant murine leukemia
cell line (P388/Dox).32 Ortataxel (6) (Spectrum Pharmaceut-
icals), with an isobutyl substituent on the isoserinyl moiety,33
was last known to be in phase II clinical trials34 showing activity
against paclitaxel- and docetaxel-resistant breast and non-small
cell lung cancers.33
During the mid-1990s to early 2000s when these paclitaxel
analogues were semisynthesized, there was little advancement
of the biosynthesis of the natural product paclitaxel or its
nonnatural analogues from baccatin III. Limitations against
developing a biosynthetic approach were largely caused by a
lack of identified paclitaxel genes from which a biocatalytic
platform could be built.35 The first few enzymes identified on
the paclitaxel pathway in Taxus plants were a cyclase, a few
hydroxylases, and five acyl CoA-dependent acyltransferases.8
None of the CoA ligases needed to make key acyl CoA
intermediates had been identified, and this omission provided
motivation for our earlier study.6 We repurposed a tridomain
TycA (Phe-ATE), on a nonribosomal peptide synthetase
pathway, as a CoA ligase36 to convert phenylisoserine to
phenylisoserinyl CoA, a linchpin pathway intermediate.
Encouraged by this finding, we used a truncated Phe-AT
didomain in this study to biosynthesize other isoserinyl CoA
analogues. The selection of the isoserine substrates synthesized
herein for our biosynthetic study was based in part on the
various isoserine variants found in bioactive paclitaxel analogues
semisynthesized in previous studies (Table 1). The biocatalyzed
CoA thioesters can potentially be incorporated into a
semibiosynthetic scheme to make not only paclitaxel from
baccatin III but also various precursors toward paclitaxel
analogues, some of which are in clinical trials.11,35,37−39 In
addition, the scope of this document covers the stereospecificity
of Phe-AT for racemic isoserines and evaluates how a single
variable that changes steric and/or electronic properties of
monosubstituted arylisoserines affects the turnover rate of the
catalyst.
Figure 1. Representative multistep semisynthesis of paclitaxel (R =
variable phenyl analogues, heteroaromatics, and alkyl derivatives): (i)
(a) Et3SiCl, pyridine; (b) acetyl chloride, pyridine; (ii) (a) n-
butyllithium, compound A; (b) HF, pyridine. Proposed biosynthesis of
paclitaxel (and its analogues): (iii) 10-deacetylbaccatin III acetyl-
transferase, acetyl CoA; (iv) 13-O-phenylpropanoyltransferase, an
isoserinyl CoA (27); (v) N-debenzoyltaxane-N-benzoyltransferase,
benzoyl CoA. Structure designations: 10-deacetylbaccatin III (1), 7-O-
triethylsilylbaccatin III (2), paclitaxel (R = phenyl) (or an analogue)
(3), baccatin III (4), and N-debenzoylpaclitaxel (R = phenyl) (or an
analogue) (5).
paclitaxel results in an inactive N-debenzoyl-2′-deoxypaclitax-
el.25
Various semisynthesized second-generation paclitaxel with a
modified isoserinyl side chain includes, for example, addition of
an m-hydroxyl on the phenylisoserinyl ring (8) (Table 1) that
allows construction of a conformationally constrained analogue
with improved binding to its β-tubulin target.26 Another
bioassay showed that a p-fluoro analogue (10) had a potency
(IC50) ∼20-fold greater than that of docetaxel against multiple
drug-resistant human leukemia cells.27 In addition, pyridyl
analogues (16 and 17)16,17 were found to be more potent than
paclitaxel or docetaxel in vincristine-resistant cancer cell lines
expressing P-glycoprotein drug-efflux pumps. Fluoro-pyridyl
compound tesetaxel (18), in phase I clinical trials, is an oral
taxane used as a single agent or in combination with the
antineoplastic pharmaceutical Xeloda (Genentech; generic
name capecitabine) in patients with solid tumors.28 Simotaxel
(12) was developed as a more potent analogue by replacement
of the phenylisoserinyl group with a polar thienylisoserinyl
group for oral bioavailability.29 Phase I clinical trials with 12,
however, were terminated for undisclosed reasons.30 It should
be noted that several taxoids that stalled in clinical trials for
“undisclosed reasons” are not cited for drug failure
(clinicaltrials.gov). It is perceived that the cost of screening
efficacious new taxoid compounds in a slow economy has
forced pharmaceutical companies to curtail development of
new drug analogues.31 Driven by economics only, these
MATERIALS AND EXPERIMENTAL DETAILS
■
Expression, Purification, and Characterization of Phe-
AT Proteins. Plasmids pET28phe-at and pET28phe-atS563A
(see the Supporting Information) were separately used to
transform Escherichia coli BL21(DE3) cells. Five 10-mL cultures
of the two E. coli transformants were separately grown in LB
medium supplemented with 50 μg mL−1 kanamycin at 37 °C
for 12 h. A 10-mL aliquot of each seed culture expressing Phe-
AT/His and Phe-AT(S563A)/His was used to inoculate LB
medium (5 × 1 L for each transformant). The bacteria were
grown at 37 °C to an OD600 of ∼0.6; then isopropyl β-D-1-
thiogalactopyranoside was added to a final concentration of 0.5
mM, and the culture was grown for 18 h at 16 °C. The cells
were pelleted by centrifugation (30 min at 4000g) at 4 °C,
resuspended in binding buffer [20 mM Tris-HCl buffer
containing 0.5 M NaCl and 5 mM imidazole (pH 7.8)],
lysed by sonication, and then centrifuged at 15000g for 0.5 h.
The supernatant was decanted and centrifuged at 135000g for
1.5 h to remove cell wall debris and light membranes.
B
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Table 1. Examples of Paclitaxel, Docetaxel, and Variously Modified Analogues
Crude soluble protein isolated from bacteria expressing Phe-
AT/His and Phe-AT(S563A)/His was estimated by the
Bradford protein assay at ∼50 and ∼75 mg of total protein,
respectively. These fractions were independently loaded onto a
Ni-NTA affinity column (Qiagen, Valencia, CA) and eluted
according to the protocol described by the manufacturer. The
column was eluted with an increasing concentration of
imidazole (from 20 to 250 mM) in binding buffer. Fractions
containing the His-tagged enzymes were identified by sodium
dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−
PAGE) analysis and Coomassie Blue staining. Fractions eluting
in 50−100 mM imidazole were combined and showed >95%
pure protein corresponding to a molecular weight consistent
with that of the calculated molecular weight for Phe-AT/His
and Phe-AT(S563A)/His at 69.6 kDa. Each enzyme solution
(100 mL) was separately concentrated to 1 mL by size-selective
centrifugation (Centriprep 30000 MWCO unit, Millipore,
Billerica, MA). The buffer was exchanged with assay buffer
[50 mM HEPES containing 100 mM NaCl and 1 mM EDTA
(pH 8.0)] over five dilution/concentration cycles. Enzyme
purity was estimated by SDS−PAGE with Coomassie Blue
staining. The purified proteins were stored at −80 °C. The
concentration of each protein [35 and 28 mg/mL for Phe-AT
and Phe-AT(S563A), respectively] was determined on a
NanoDrop ND1000 spectrophotometer (Thermo Scientific,
Wilmington, DE). The estimated extinction coefficient (ε280) of
Phe-AT and Phe-AT(S563A) was set at 71740 M−1 cm−1,
based on the approximation that spectral contributions of the
tyrosine, tryptophan, and cysteine at A280 do not differ
significantly in the native protein and the denatured form.61,62
Kinetic Evaluation of Phe-AT and Phe-AT(S563A) for
CoA and Amino Phenylpropanoids. (R)-β-Phenylalanine
and (2R,3S)-phenylisoserine (each at 1 mM) were separately
incubated in 1-mL reaction mixtures containing 100 mM
HEPES (pH 8.0), ATP (1 mM), MgCl2 (3 mM), CoA (1 mM),
and Phe-AT or Phe-AT(S563A) (100 μg) to establish steady-
state conditions with respect to protein concentration and time
at 31 °C. Under steady-state conditions, (R)-β-phenylalanine
and (2R,3S)-phenylisoserine at 5−2000 μM were separately
incubated with Phe-AT and Phe-AT(S563A) (20 μg) for 15
min. The assays were quenched by acidifying to pH ∼2 (10%
formic acid). Acetyl CoA (1 μM) was added as an internal
standard to each sample to correct for losses during workup.
The biosynthetic products were quantified by liquid chroma-
tography electrospray multiple-reaction monitoring (LC−ESI-
MRM) mass spectrometry on a Quattro-Premier XE mass
spectrometer coupled to an Acquity UPLC system fitted with a
C18 Ascentis Express column (2.5 mm × 50 mm, 2.7 μm) at 30
°C. An aliquot (5 μL) of each sample was loaded onto the
column, and the analytes were eluted with a solvent gradient of
acetonitrile (solvent A) and 0.05% triethylamine in distilled
water (solvent B) (held at 2.5% solvent A for 3.17 min,
increased to 100% solvent A over 5 s with a 2 min hold, and
then returned to 2.5% solvent A over 5 s with a 50-s hold) at a
flow rate of 0.4 mL/min. The effluent from the column was
directed to the mass spectrometer where the first quadrupole
mass analyzer (in negative ion mode) was set to select for the
C
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Table 2. Synthesis of Isoserine Analogues
a
(i) (a) Dichloromethane, 4 Å molecular sieves, 12 h, 23 °C; (b) triethylamine, acetoxyacetyl chloride, dichloromethane, 0−23 °C, 2−5 h; (ii) (a)
ceric ammonium nitrate in H2O, acetonitrile, 0 °C, 1−3 h (up to this step, X = acetyl in 22t and 22u); (b) 7 N HCl, reflux, 24 h (after this step, X =
b
H in 22t and 22u). The entries in parentheses in groups of four are the percent isolated yield after steps i-a and i-b, the percent isolated yield after
step ii-a, the percent isolated yield after step ii-b, and the overall percent yield of the isoserine product with respect to the aldehyde (bold) from left
c
to right, respectively. The entries in parentheses in groups of three for compounds 22v, 22w, and 22x are the percent isolated yield after step i-b in
Figure 2, the percent isolated yield after step ii-b in Figure 2, and the overall percent yield of the pyridylisoserine product with respect to the
aldehyde (bold) from left to right, respectively.
molecular ion of a biosynthesized acyl CoA product. The
selected ion was then directed to a collision gas chamber
wherein the collision energy was optimized to maximize the
abundance of a signature fragment ion (m/z 408.31, derived by
a fragmentation reaction in the CoA moiety of the acyl CoA)
monitored in the second quadrupole mass analyzer in negative
ion mode. The peak area under the curve of the monitored
fragment ion at m/z 408.31 corresponding to each biosynthetic
phenylpropionyl CoA thioester was converted to concentration
by comparison to a standard curve of authentic CoA (0.048−
100 μM). The initial velocity (v0) of production of (R)-β-
phenylalanyl and (2R,3S)-phenylisoserinyl CoA made in
separate enzyme catalysis assays was plotted against substrate
concentration and fit by nonlinear regression to the Michaelis−
Menten equation (R2 was typically between 0.90 and 0.99) to
calculate the apparent KM and kcat (see Figure S40).
The apparent KM values of Phe-AT and Phe-AT(S563A) for
CoA were assessed by incubating each enzyme separately with
(R)-β-phenylalanine (1 mM), MgCl2 (3 mM), ATP (1 mM),
and CoA (5−2000 μM) at 31 °C for 15 min. Acetyl CoA (1
μM) was added as an internal standard to each sample. The
products of the enzyme-catalyzed reactions were quantified by
LC−MRM mass spectrometry, and the monitored fragment ion
(m/z 408.31) derived from the CoA thioester analytes in the
effluent was quantified in a manner identical to the procedure
described previously.6 The initial velocity (v0) of production of
(R)-β-phenylalanyl CoA made in separate assays was plotted
against substrate concentration and fit by nonlinear regression
to the Michaelis−Menten equation (R2 was 0.9) to calculate
KM.
Kinetic Evaluation of Phe-AT for CoA and Phenyl-
isoserine Stereoisomers. The apparent kinetic parameters of
Phe-AT for (2R,3R)-phenylisoserine were determined to
provide a basis of comparison against the catalytically active
(2R,3S)-phenylisoserine isomer. (2R,3R)-Phenylisoserine (at 1
mM) was incubated in a 1 mL reaction mixture containing 100
mM HEPES (pH 8.0), ATP (1 mM), MgCl2 (3 mM), CoA (1
mM), and Phe-AT (100 μg) to establish steady-state conditions
with respect to protein concentration and time at 31 °C.
Aliquots (100 μL) were taken at various time points between 0
and 2 h and their reactions quenched with 10% formic acid (5
μL). Acetyl CoA (1 μM) was added as the internal standard,
and the samples were analyzed as described above. The kinetic
assays with (2R,3R)-phenylisoserine were performed in a
manner similar to the procedure described for (2R,3S)-
phenylisoserine. The resulting (2R,3R)-phenylisoserinyl CoA
biosynthetic product was analyzed by the LC−ESI-MRM
method used before. The peak area under the curve of the
monitored fragment ion at m/z 408.31 derived from a
molecular ion consistent with that of biosynthetic (2R,3R)-
phenylisoserinyl CoA was converted to concentration by
comparison to a standard curve of authentic CoA (0.048−
100 μM). The initial velocity (v0) of production of (2R,3R)-
phenylisoserinyl CoA made in separate assays was plotted
versus substrate concentration and fit by nonlinear regression
to the Michaelis−Menten equation (R2 was typically between
0.90 and 0.99) to calculate the apparent KM and kcat.
Activity Assay for the Determination of the Substrate
Specificity of Phe-AT. To identify productive isoserine
substrates, each isoserine (2 mM) was incubated separately
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with CoA (1 mM), ATP (1 mM), MgCl2 (3 mM), and purified
Phe-AT enzyme (1 mg) in 100 mM HEPES (pH 8.0) in a 1 mL
reaction mixture. The reaction mixtures were incubated for 8 h
at 31 °C. Aliquots (100 μL) were withdrawn initially at 10, 20,
and 40 min and then at 1, 2, 4, 6, and 8 h and transferred to 96-
well plates, the reactions quenched immediately with 10%
formic acid (20 μL), and the samples analyzed on the LC-
Quattro-Premier XE mass spectrometer as described above. An
aliquot (5 μL) of each sample was loaded onto the column, and
the analytes were eluted with a solvent gradient of acetonitrile
(solvent A) and 0.05% triethylamine in distilled water (solvent
B) (held at 2.5% solvent A for 3.17 min, increased to 100%
solvent A over 5 s with a 2-min hold, and then returned to 2.5%
solvent A over 5 s with a 50-s hold) at a flow rate of 0.3 mL/
min.
Apparent Rates of Phe-AT with the Isoserine
Substrates. The conversion rate of Phe-AT for thioester-
ification of the isoserine analogues to their isoserinyl CoA
thioesters was determined by incubating each isoserine (2 mM)
separately with ATP (1 mM), CoA (1 mM), MgCl2 (3 mM),
and purified Phe-AT enzyme (1 mg) in 100 mM HEPES (pH
8.0) at 31 °C for 2 h in triplicate. Time course assays performed
using Phe-AT (1 mg) with the isoserine substrates provided a
justification for the estimation of the relative rates to be at
steady state at 2 h. At the end of each reaction, acetyl CoA (1
μM) was added as the internal standard to each sample to
correct for losses during workup. The biosynthetic thioester
products were quantified by liquid chromatography−multiple-
reaction monitoring (MRM) mass spectrometry on a Quattro-
Premier XE mass spectrometer with an elution gradient as
described above. The peak area of the monitored fragment ion
at m/z 408.31 corresponding to each biosynthetic isoserinyl
CoA thioester was converted to concentration by comparing
the peak area of the same ion generated by authentic CoA using
linear regression analysis.
procedure, differences in isolated yield likely resulted from the
electronic effects of the substituent on the aryl ring. The
substituent effects from the aryl aldehyde had a significant effect
on the overall yield of the lactam. Aryl aldehydes with electron-
withdrawing substituents (m-halogen, o- and p-fluoro, or NO2)
or no substituent, and branched alkyl aldehydes, generally
yielded 9−47% of the lactam in the one-pot reaction (Table 2),
far lower than the average (∼58%) isolated yield for this step.
To contrast, aryl aldehydes with moderate to strong electron-
donating substituents (methyl, methoxy, and acetoxy) typically
yielded 51−95% of the lactam. These observations suggested
that electron-donating substituents stabilized either the electro-
philic carbon in the imine reaction, the cycloaddition reaction,
or both, priming them for effective nucleophilic attack.
Oxidative removal of the anisole protecting group in the next
step averaged an ∼71% yield for 25 N-dearylation reactions
described herein, and the electronics of the substituent did not
seemingly influence the yields. The hydrolysis step with 7 N
HCl to liberate the isoserine-HCl salt was generally
quantitative, giving on average an ∼92% isolated yield relative
to the lactam (Table 2).
The β-(pyridyl)isoserines were synthesized with modifica-
tions of the method used to make the substituted phenyl-
isoserines (Figure 2). To form the pyridyl-substituted lactam
RESULTS AND DISCUSSION
■
Synthesis of Isoserine Analogues. Several methods for
the synthesis of phenylisoserine analogues proceed through
reactions involving asymmetric epoxidation,37 dihydroxyla-
tion,38 chemoenzymatic resolution,39 aminohydroxylation, or
the conrotatory Staudinger [2+2] cycloaddition of an imine and
ketene that makes a key β-lactam intermediate.30,31 We used
the latter electrocyclic cycloaddition reaction with readily
available reagents for the synthesis of racemic phenylisoserine
analogues substituted with ortho-(F, Cl, Br, CH3, OCH3, NO2),
para-(F, Cl, Br, CH3, OH, OCH3, NO2), or meta-(F, Cl, Br,
CH3, OH, OCH3, NO2) (Table 2). This procedure was also
used to make β-(thiophenyl)isoserine and various β-(alkyl)-
isoserines. Following a previously described procedure,40 we
reacted benzaldehyde analogues with p-anisidine to form a
Schiff base. In a one-pot sequence, the Schiff base reacted with
acetoxyacetyl chloride and triethylamine to form the cis-β-
lactam racemate. Oxidation with ceric ammonium nitrate
removed the p-methoxyphenol group to afford the N-
dearylated β-lactam. Reflux in 7 N HCl hydrolyzed the O-
acetyl group and converted the lactam ring to the isoserines in a
single step (Table 2). Incorporating an early one-pot reaction
sequence and accomplishing the ester hydrolysis and ring
opening in a single step shortened the previously described
synthesis of isoserine racemates from five to three steps.40 The
overall yields of these arylisoserines ranged from 4% (22aa) to
90% (22j). Because each Schiff base was isolated by an identical
Figure 2. Synthesis of pyridylisoserine isomers: (i) (a) dichloro-
methane, 4 Å molecular sieves, 12 h, 23 °C; (b) triethylamine,
acetoxyacetyl chloride, dichloromethane, 0 °C, 3 h; (ii) acetonitrile, 7
N HCl, reflux, 24 h.
rings (22v, 22w, and 22x) through the Staudinger reaction,
benzhydrylamine was used instead of p-anisidine because
oxidative removal of the anisole with ceric ammonium nitrate
in the presence of the pyridyl ring prevented the N-deprotected
lactam products from forming. Likely, the pyridyl rings
dimerized through a radical cation mechanism on treatment
with ceric ammonium nitrate, similar to the mechanism of
dimerization for 2-(furan-3-yl)pyrroles mediated by ammonium
cerium(IV) nitrate described in an earlier study.41 After making
the benzhydryl lactams, we proposed a two-step sequence of
acid hydrolysis to open the lactam ring and remove the O-acetyl
group, followed by hydrogenolysis to eliminate the benzhydryl
moiety. Surprisingly, reflux of the protected lactam under acidic
conditions made the β-(pyridyl)isoserines directly, and the
hydrogenolysis step was unnecessary (Figure 2). The
E
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Table 3. Steady-State Kinetic Parameters of TycA Variants
a
a
Phe-AT
Phe-ATE
(R)-β-Phe
1.6 0.3
51
Phe-AT(S563A)
22.0 4.1
Phe-ATE(S563A)
k
(min−1
)
14.6 0.5
46
3.0 0.04
62
app
cat
KM (μM)
3
8
33
5
1
kacaptp/KM (s−1 M−1
)
)
)
5308 119
527 129
(2R,3S)-PheIso
0.25 0.02
89 15
11200 500
803 17
app
cat
k
(min−1
)
1.5 0.2
440 62
57.4 1.7
1.1 0.7
0.43 0.01
190 10
KM (μM)
kacaptp/KM (s−1 M−1
327 106
53.3 16.7
46.7 8.7
37.5 2.1
b
CoA
app
cat
k
(min−1
)
10
2
0.75 0.05
2000 200
6.3 0.7
29
8
0.9 0.08
804 26
18.7 1.8
KM (μM)
kacaptp/KM (s−1 M−1
208 57
838 74
562 149
853 198
(2R,3R)-PheIso
(min−1
)
0.78 0.13
380 70
app
k
cat
KM (μM)
(2S,3R)-PheIso
c
(min−1
)
nd
app
k
cat
c
KM (μM)
nd
a
b
Data from ref 6. Kinetic measurements for CoA were taken in the presence of (R)-β-phenylalanine, ATP, and MgCl2 at apparent steady state. All
c
values are expressed as means standard deviations of triplicate results. CoA thioester product was below the LC−ESI-MS detection limit.
benzhydrylacetamide byproduct made in the reaction suggested
an SN1 mechanism that released a benzhydryl carbocationic
intermediate, which was captured by the acetonitrile solvent.
Subsequent acid hydrolysis of the acetonitrile moiety produced
the amide byproduct as confirmed by H nuclear magnetic
resonance (NMR) and LC−ESI-MS analysis (Figures S38 and
S39, respectively).
Phe-AT and Phe-AT(S563A) were faster than the wild-type
Phe-ATE in turning over the two test substrates (Table 3). The
app
cat
apparent kcat (k ) of Phe-AT for (R)-β-phenylalanine was
improved 7-fold compared to that of Phe-ATE, whereas the KM
values of truncated and wild-type catalysts were virtually of the
same order of magnitude. Similarly, Phe-AT (6-fold increase)
and Phe-AT(S563A) (2.5-fold increase) were faster than Phe-
ATE and Phe-ATE(S563A), respectively, at turning over
1
Construction and Expression of Phe-AT and Phe-
AT(S563A). An earlier study showed that the wild-type tycA
cDNA encoding the adenylation, thiolation, and epimerization
domains (Phe-ATE) functioned as a CoA ligase with β-amino
β-phenylpropanoates.6 Here, Phe-ATE was truncated to encode
only the adenylation and thiolation domains (Phe-AT) and
thus eliminate recombinant expression of the 400-amino acid
epimerization domain. As important was the fact that the
possibility of epimerization of aminoacyl CoA products was
eliminated. In addition, a Phe-AT(S563A) mutant derived from
Phe-AT was expressed without S563, which was done with Phe-
ATE in an earlier study.6 This mutation prevented natural O-
phosphopantetheinylation of Ser563 of the enzyme and thus
also abrogated subsequent enzyme-linked thioesterification of
the phenylpropanoate substrates in the E. coli expression host.
The solubly-expressed enzymes Phe-AT and Phe-AT(S563A)
were isolated from bacterial lysates and purified by Ni-affinity
chromatography for use in the CoA ligase activity assays.
Activity and Kinetic Analyses of Phe-AT. The activities
of Phe-AT and Phe-AT(S563A) were compared to those of the
corresponding tridomain cognates of Phe-ATE measured in an
earlier study.6 Phe-AT and its mutant were incubated with
CoA, Mg2+, ATP, and separately with substrates (R)-β-
phenylalanine and (2R,3S)-phenylisoserine. (R)-β-Phenylala-
nine was the superior phenylpropanoid substrate of Phe-ATE in
an earlier study6 and thus was used here to help benchmark the
activity of the Phe-AT mutants. The identity of the biosynthetic
thioester products was verified by LC−ESI-MS/MS and
comparison against mass fragmentation profiles of authentic
amino acyl CoA thioesters. An LC−ESI/MRM technique was
used to quantify the biosynthetic products for kinetic analysis.
(2R,3S)-phenylisoserine to its CoA thioester.
app
The k of Phe-AT(S563A) for CoA was 32-fold higher
cat
compared to that of its wild-type counterpart, Phe-ATE-
app
cat
(S563A), while the k of Phe-AT for CoA improved 13-fold
versus that of Phe-ATE. The catalytic efficiency (kacaptp/KM) of
Phe-AT for CoA improved 2 orders of magnitude over that of
Phe-ATE due mainly to a lower KM value. Phe-AT and Phe-
AT(S563A) also had catalytic efficiencies for (R)-β-phenyl-
alanine increased 10- and 14-fold, respectively, versus those of
their ATE counterparts due mainly to a 7-fold increase in the
app
cat
k
values of both catalysts. It was interesting to find that the
catalytic efficiencies of Phe-AT and Phe-AT(S563A) for
(2R,3S)-phenylisoserine increased only slightly (23 and 42%,
respectively) over those of their full-length versions, largely
because of improved turnover rates (Table 3). Guided by the
slightly better catalytic efficiency of Phe-AT over Phe-
AT(S563A) for phenylisoserine, we selected the former for
the substrate specificity assays with various isoserine analogues.
Stereospecificity of Phe-AT for Phenylisoserine Ster-
eoisomers. Earlier structure−activity relationship studies show
that the natural (2R,3S)-phenylisoserine side chain isomer at C-
13 of paclitaxel is important for its activity.22,42 A computational
study that identified electronic and quantum features that
defined bioactive molecules predicted that the (2S,3R)-
stereoisomer in a paclitaxel variant was ∼4-fold more effective
than natural paclitaxel.43 It should be noted that no empirical
biological assays have been reported to date that verify this
theoretical finding. Thus, here we explored the stereospecificity
of Phe-AT, by separately incubating substrates (2R,3S)-,
(2R,3R)-, or (2S,3R)-phenylisoserine with CoA, Mg2+, and
ATP at apparent saturation. The (2S,3S)-isomer is in principle
F
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Table 4. Biosynthesis of Isoserine Analogues
a
b
(i) Mg2+ (3 mM), ATP (1 mM), CoA (1 mM), Phe-AT (1 mg), 22 (1 mM), pH 8.0, 31 °C, 2 h. The entries in parentheses in groups of two are
apparent velocity (vapp, in nanomoles per hour) and vrel as a percentage with respect to 27a from left to right, respectively. The vapp values of Phe-
AT(W227S) are shown in bold and italics for 27p and 27v. ND indicates the biosynthetic product was below the detection limit of the mass
spectrometer.
commercially available from specialized sources; however, it
was cost prohibitive to obtain this isomer through custom
synthesis at the time of this study. Our formidable efforts to
synthesize the rare isomer fell beyond the scope of this study.
The phenylisoserinyl CoA products were detected by electro-
spray ionization mass spectrometry (ESI-MS). Phe-AT turned
over the (2R,3S)-isomer, having the same stereochemistry of
the natural side chain of paclitaxel, 2-fold faster than it did the
(2R,3R)-diastereomer. Under the same assay conditions, the
CoA thioester of (2S,3R)-phenylisoserine was below the limits
of detection (Table 3).
Relative Rates of Phe-AT for Isoserine Substrate
Analogues. In this study, the substrate scope of Phe-AT was
assessed with racemic isoserine substrates synthesized via the
Staudinger reaction. Therefore, we estimated the magnitude
and mode of inhibition on Phe-AT by the nonproductive
CH3, OH, CH3O, NO2) substituent were converted to their
acyl CoA by Phe-AT. The enzyme activity was also observed
with the nonaromatic β-(cyclohexyl)isoserine and heteroar-
omatic β-(thiophenyl)isoserine analogues, but not with the
isopropyl- and tert-butylisoserines or (pyridyl)isoserine ana-
logues (Table 4). The rate (7 nmol h−1) at which Phe-AT
converted phenylisoserine (prepared as a racemate) to its CoA
thioester was set at 100% and used for comparison against the
relative rates of Phe-AT for the other racemic isoserine
substrates at apparent saturation (1 mM) (Table 4).
In general, the vapp values of Phe-AT for substrates with meta
substituents on the aryl ring were higher than those for the para
and ortho isomers (Figure 3). For example, the vapp was ∼1.5-
and 6-fold lower for p- and o-fluoro isomers, respectively, than
for the m-fluoro isomer. Similarly, the vapp for the methyl
substituent on the phenyl ring of the isoserinyl substrate was
lower for the o-methyl (vapp below the detection limit) and p-
methyl (vapp = 1.13 nmol h−1) isomers than for the m-methyl
substrate (vapp = 2.61 nmol h−1) (Table 4). A model of Phe-AT
was built using gramicidin synthetase A [Protein Data Bank
(PDB) entry 1AMU] in complex with phenylalanine as the
reference. We added chloro group(s) to the docked substrate to
serve as representative substituent(s) for the other regioiso-
meric substrates used herein and to help identify the steric
interactions that may affect turnover (Figure 3).
(2S,3R)-enantiomer. In the presence of (2S,3R)-phenyl-
app
cat
isoserine at 250 μM, the k
of Phe-AT for (2R,3S)-
phenylisoserine was 0.12 min−1, and the KM was 500 μM,
which was nearly identical to the KM (440 μM) of Phe-AT in
the absence of the inhibitor (Figure S41). The kacaptp, however,
decreased 15-fold from 1.51 min−1, and the KI was calculated to
be 92.0 μM. The results indicated noncompetitive inhibition of
Phe-AT by the nonproductive (2S,3R)-phenylisoserine enan-
tiomer, suggesting that the enantiomer bound allosterically and
affected turnover. These results showed that obtaining accurate
apparent Michaelis parameters for each racemic isoserine
substrate tested herein was in part confounded by cosubstrate
inhibition. Thus, the approximate relative steady-state rate of
Phe-AT for each racemic isoserine substrate was estimated
through analysis of the biosynthesized isoserinyl CoA by
quantitative ESI-MS−MRM.
The model showed that residue Trp227 (i.e., W227) of Phe-
AT is near (∼3.6 Å) the para carbon of the aryl ring of the
substrate (Figure 3A). Thus, a rationale for the slower turnover
of the para-substituted than of the meta-substituted substrates is
steric compression between the para substituents and W227,
preventing the latter from adopting a productive catalytic
conformation (Figure 3B). By contrast, meta substituents likely
avoid the more encumbered space near Ala224 and Phe566 and
occupy an active site space closest to residues Ile318 and
All substrates in which the phenyl ring had an ortho-(F, Cl,
NO2), para-(F, Cl, Br, CH3, OH, NO2), or meta-(F, Cl, Br,
G
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Figure 3. (A) Model of the Phe-AT active site built from gramicidin synthetase A (PDB entry 1AMU) in complex with phenylalanine. Shown are the
AMP structure (green) and residues (lighter blue) within ∼5 Å of the ring of the α-phenylalanine (yellow) complex. We used a chloro group as a
representative substituent because its nominal van der Waals radius lies between those of second-row (C, O, and F) and fourth-row (Br) atoms of
the various substituents in the substrates used here. Chloro substituents (green spheres) are added to the aryl ring of the docked substrate at the (B)
para, (C) meta, and (D) ortho positions. The latter two positional isomers are shown as disubstituted to capture the steric interactions of both
rotameric forms of the monosubstituted meta and ortho isomers used in this study. The chloro substituent also provides a good approximation of
steric compression between the various substituents and nearby active site residues based on relative distance. Numbers next to dotted lines indicate
distances of ≤4.70 Å; amino acid residues are listed as their three-letter codes followed by their position. Heteroatoms are nitrogen (darker blue),
oxygen (red), sulfur (yellow), and phosphorus (orange).
Cys319 (Figure 3C). In the latter orientation, the meta
substituent is estimated to be closest to the carbonyl oxygen
of Gly312 (Figure 3C), but it should be noted that there is
enough leeway for the phenyl ring to twist from its depicted
plane to relieve steric interactions. In addition, we estimate
from the model that ortho substituents are sterically compressed
by Phe566 and likely are positioned closer to the carbonyl of
Gly312 and the side chain of Ala310, and away from the
carbonyl of Ile318 (Figure 3D). While this open cavity may
provide sufficient space for the ortho substituents to reside, the
substituents can still interact intramolecularly with the
propanoate side chain of the substrate. This intramolecular
interaction likely affects catalytic turnover by disrupting the
motion of the propanoate during the carboxylate adenylation
and thiolation reactions, as suggested for a homologous benzoyl
CoA ligase in a recent study.44
The vapp values of Phe-AT also correlated with the size of the
halogen substituent, regardless of the position on the phenyl
ring. This trend is demonstrated by substrates with the smaller
fluoro substituent being converted fastest to their CoA
thioesters in the halogen series: o-F > o-Cl > o-Br (vapp
=
0.98, 0.11, and 0.0 nmol h−1, respectively), p-F > p-Cl > p-Br
(vapp = 3.98, 0.45, and 0.22 nmol h−1, respectively), and m-F >
m-Cl > m-Br (vapp = 6.1, 1.7, and 1.4 nmol h−1, respectively). It
is worth noting that the activity of Phe-AT with m- and p-Cl
isoserinyl isomers is potentially prophetic toward making a
(3,4-dichloro)phenylisoserinyl analogue of paclitaxel. The latter
was shown in an earlier study to have microtubule assembly
activity 7-fold greater than and melanoma cytotoxicity 40-fold
greater than that of paclitaxel.45
In general, the steric bulk of the substituent negatively
affected turnover likely because of their inability to bind Phe-
H
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Article
AT in a catalytically competent conformation. The vapp for the
production of the isoserinyl CoA analogues by Phe-AT
decreased with an increasing size of the para substituent (H
> F > Cl > Br > OH ∼ NO2 > OCH3), ranging from 3.98 nmol
h−1 for p-fluoro to a vapp that was below the detection limit of
the mass spectrometer for the p-methoxy analogue. Among the
heteroaromatic isoserine analogues, only thiophen-2-ylisoserine
was converted to its CoA thioester by Phe-AT at a vapp of 0.89
nmol h−1. Phe-AT did not turn over the pyridylisoserine
isomers or the branched alkyl isopropyl- and tert-butylisoserine
analogues to their CoA thioesters.
In general, Phe-AT has similar relaxed substrate specificity as
observed for other biocatalysts on the pathways of specialized
natural products.47−55 This is a desirable feature in synthetic
biology upon selection of biocatalysts to engineer a biosynthetic
pathway that can recognize surrogate substrates. Phe-AT has
significant potential to biosynthesize an array of aminoacyl
CoAs that can be used by different acyl CoA-dependent
acyltransferases to construct rare bioactive natural product
analogues. In particular, Phe-AT has application for the design
of a biosynthetic route to produce paclitaxel analogues that
contain modified isoserine side chains. There are few bona fide
CoA ligases identified that can activate an amino acid to its
CoA thioester.56 A query of “nonribosomal peptide synthetase”
on the NCBI database captures ∼200000 entries,57 with each
synthetase containing multiple AT domains that transiently
thioesterify amino acids or polypeptides.58 Repurposing the
Phe-AT didomain of TycA as a CoA ligase suggests that other
tandem AT domains of different NRPS enzymes may catalyze
similar ligase activity. In addition, Phe-AT is a prime candidate
for protein engineering through directed evolution59,60 with a
goal of increasing its specificity and turnover profile, for
example, for pyridylisoserine substrates to make oral forms of
taxane drugs, such as tesetaxel. This would broaden the catalytic
space of NRPS-loading modules to include an area where they
can biosynthesize nonnatural acyl CoAs. It is imagined that the
resulting thioesters can be used potentially by broad specificity
CoA-dependent transferases to acylate and thus diversify
bioactive specialized metabolites isolated from various other
species.
Expanding the Substrate Scope of Phe-AT for
(Pyridyl)isoserines (27v, 27w, and 27x) and p-Methox-
yphenylisoserine (27p). We were intrigued that the N
heteroatom, bioisostere of carbon, prevented turnover of the
pyridylisoserines by Phe-AT to their CoA thioesters. We
imagine the pyridyl nitrogen likely H-bonds through an
unknown bond network in the active site, placing the substrate
in a nonproductive trajectory for catalysis. While a structural
model of Phe-AT in complex with phenylalanine places W227
closest to the para position of the aryl ring of the substrate
(Figure 3), the model also showed that second-shell Phe
residues (F201, F202, and F206) border W227. The trifecta of
Phe residues likely aligns W227 for favorable π-stacking with
the substrate. Thus, W227 of Phe-AT was mutated alone or in
tandem with the second-tier Phe triad. The triad was replaced
by positionally equivalent residues found to occur naturally in
homologous enzymes in an attempt to broaden the substrate
specificity. W227 of Phe-AT was exchanged separately with
polar residue Ser [labeled as Phe-AT(W227S)] that could
potentially H-bond with the heteroatom of the pyridyl
substrates. A different mutant, Phe-AT(W227A), was imagined
to weaken steric interactions between the substituent on the
substrate and the active site. F201 and F202 of Phe-
AT(W227S) were also mutated to A201 and Q202, respectively
[designated Phe-AT(W227S) (AQF)], to match the sequence
of a nonribosomal peptide synthetase from Anabaena species
(accession number D5J708) with a serine residue positioned
like W227 in Phe-AT. In addition, F201, F202, and F206 of
Phe-AT(W227A) were changed to A201, R202, and Y206,
respectively [designated Phe-AT(W227A) (ARY)], to match
the sequence of an amino acid adenylation enzyme from
Methylomicrobium album BG8 (accession number H8GJ90)
with an alanine residue positioned like W227 in Phe-AT.
We found that the solubly expressed Phe-AT(W227S)
mutant slowly catalyzed the conversion of 2-pyridylisoserine
to its CoA thioester (27v) (vapp ∼ 0.004 nmol h−1) but was not
active with 3- or 4-pyridylisoserine. The latter CoAs were below
the limits of detection. This turnover is a promising first-
principles result for further development of the Phe-AT mutant
to potentially make 3-fluoro-2-pyridylisoserinyl CoA for
semibiosynthesis of the pharmaceutical tesetaxel46 (see Table
1). Moreover, Phe-AT(W227S) turned over the sterically
demanding p-methoxyphenylisoserine substrate to its thioester
(27p), which was not made by Phe-AT (Table 3). Likely, the
W227S replacement relieved the steric crowding near the para
carbon of the substrate, allowing turnover of the larger
substrate. We also determined that mutants Phe-AT(W227A),
Phe-AT(W227S) (AQF), and Phe-AT(W227A) (ARY) did not
turn over 2-, 3-, or 4-pyridylisoserine or p-methoxyphenyliso-
serine to its CoA thioester. The reason for the latter
observations is unknown at this time.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.bio-
chem.6b01188.
■
S
Experimental methods, LC−MS/MS, kinetics, and NMR
data (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: walker@chemistry.msu.edu.
■
ORCID
Kevin D. Walker: 0000-0001-5208-6692
Funding
The authors are grateful to the Michigan State University Office
for Inclusion and Intercultural Initiatives for supporting the
MSU Chemistry 4-Plus Bridge to the Doctorate Program
(GA017081-CIEG) and for Michigan State University
AgBioResearch Grant RA078692-894.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors thank undergraduate Aaron Barto for his technical
assistance.
■
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