Insights into the Functionalization of the Methylsalicyclic Moiety during the Biosynthesis of Chlorothricin by Comparative Kinetic Assays of the Activities of Two KAS III-like Acyltransferases

Article abstract of DOI:10.1002/cjoc.201900134

Chlorothricin (CHL), an archetypal member of the family of spirotetronate antibiotics, possesses a tetronate-containing pentacyclic aglycone that is conjugated with a modified methylsalicyclic acid (MSA) moiety through a disaccharide linkage. MSA is a pol

Full text of DOI:10.1002/cjoc.201900134

Accepted Article
Title: Insights into the Functionalization of the Methylsalicyclic Moiety during the
Biosynthesis of Chlorothricin by Comparative Kinetic Assays of the Activities of Two
KAS III-like Acyltransferases
Authors: Xuan Yi, Qunfei Zhao, Zhenhua Tian, Xinying Jia, Weiguo Cao, Wen Liu*,
and Qing-Li He*
This manuscript has been accepted and appears as an Accepted Article online.
This work may now be cited as: Chin. J. Chem. 2019, 37, 10.1002/cjoc.201900134.
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published online in Early View: http://dx.doi.org/10.1002/cjoc.201900134.
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Insights into the Functionalization of the Methylsalicyclic Moiety during
the Biosynthesis of Chlorothricin by Comparative Kinetic Assays of the
Activities of Two KAS III-like Acyltransferases
Xuan Yi a,c,†, Qunfei Zhao b,†, Zhenhua Tianc, Xinying Jiac, Weiguo Caoa , Wen Liu*c,d, and Qing-Li He*b
aDepartment of Chemistry, Innovative Drug Research Center, Shanghai University, 99 Shangda Road, Shanghai, 200444, China.
bInnovation Research Institute of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, 1200 Cai Lun Road, Shanghai, 201203,
China.
cState Key Laboratory of Bioorganic and Natural Products Chemistry, Center for Excellence on Molecular Synthesis, Shanghai Institute of Organic Chemistry,
University of Chinese Academy of Sciences, 345 Lingling Road, Shanghai, 200032, China.
d Huzhou Center of Bio-Synthetic Innovation, 1366 Hongfeng Road, Huzhou, 313000, China
Cite this paper: Chin. J. Chem. 2019, 37, XXX—XXX. DOI: 10.1002/cjoc.201900XXX
Summary of main observation and conclusion Chlorothricin (CHL), an archetypal member of the family of spirotetronate antibiotics, possesses a
tetronate-containing pentacyclic aglycone that is conjugated with a modified methylsalicyclic acid (MSA) moiety through a disaccharide linkage. MSA is a
polyketide product assembled by the iterative type I polyketide synthase ChlB1. Incorporation of this pharmaceutically important moiety into CHL relies
on the activities of two distinct β-Ketoacyl-ACP synthase III (KAS III)-like acyltransferases, ChlB3 and ChlB6, which function together to coordinate the
transfer of MSA through ChlB2, a discrete acyl carrier protein (ACP). During the maturation of CHL, MSA needs to be further functionalized by
C2-O-methylation and C5-chlorination; however, timing of this functionalization process remains poorly understood. In this study, we report comparative
kinetic assays of the activities of the two KAS III-like acyltransferases ChlB3 and ChlB6 using substrates that vary in substitution extent and ACP carrier.
ChlB3 prefers to transfer the immediately assembled 6-methyl-MSA moiety from ChlB1-ACP to the discrete ACP ChlB2, from which this moiety is
preferred to be transferred directly onto the molecule desmethylsalicyl-CHL prior to C2-O-methylation and C5-chlorination. Consequently, MSA
functionalization appears to occur at the molecule level rather than at the covalently tethered protein level, i.e., ChlB1-ACP or ChlB2. Both ChlB3 and
ChlB6 are flexible in substrate tolerance, holding promise for CHL engineering-based structural diversity by using variable MSA moiety.
release from pantetheinyl group for the absence of thioesterase
Background and Originality Content
During the biosynthesis of bioactive molecules, different
starter units and modified units immensely contribute to the
(TE) ,[12] Claisen cyclase thioesterase (CLC-TE),[13] or thioester
reduction(TR) domain.[14] Conversely two KAS III-like
acyltransferases (ChlB3 and ChlB6) centered on the acyl-S-carrier
structural diversity of those bioactive molecules.[1] Chlorothricin
protein (ChlB2) intermediates have been verified to be involved in
the biosynthesis of 1.[15]
（CHL, Scheme 1）, produced by Streptomyces antibioticus DSM
40725, belongs to a growing family of spirotetronate antibiotics
Our previous biochemical characterization in vitro has
elucidated that ChlB3 could transfer 6-methylsalicyl group
anchored on the ChlB1-ACP to holo-ChlB2, a discrete ACP,
with broad biological activities by inhibiting pyruvate carboxylase
and
malate
dehydrogenase.[2]
CHL
comprises
the
tetronate-containing aglycone and two D-Olivose saccharide
chains with the 3’-hydroxyl position of the secondary sugar
decorated by a 5-chloro-6-methyl-O-methylsalicyclic acid moiety
(1, Scheme 1). 5-chloro-6-methyl-O-methylsalicyclic acid (1,
Scheme 1) as a modified unit is crucial to the antibacterial activity
and stability of CHL.[3] After the biosynthetic gene cluster of CHL
was cloned and characterized, the gene clusters of a series of
resulting 6-methylsalicyl-S-ChlB2, and ChlB6 could transfer
5-chloro-6-methylsalicyl
loaded
on
ChlB2
to
desmethylsalicyl-CHL(DM-CHL), producing the final product of
CHL.[15] The only remaining question was the elusive reaction
timing of ChlB4 and ChlB5, which were assumed to be responsible
for the C5-chlorination and C2-O-methylation of
1
by
bioinformatics analysis. The gene knockout of chlB4 and chlB5 led
to producing deschloro-chlorothricin (DCM-CHL) and DM-CHL,
respectively, which couldn’t provide any clues to the reaction
timing of ChlB4 and ChlB5. Theoretically, these two different
modifications may occur on 6-methylsalicyl moiety still loaded on
the acyl carrier protein of ChlB1-ACP and ChlB2, or ChlB4 and
ChlB5 might directly modify 6-methylsalicyl-chlorothricin (M-CHL)
(Scheme 1).
Which pathway really exists in nature? Due to the failure to
get soluble ChlB4 (for chlorination) and ChlB5 (for O-methylation),
we will attempt to figure out this puzzle by the kinetic constants
of ChlB3 and ChlB6 for different substrates in this report.
compounds
with
spirotetramate
or
spirotetronate
structure(pyrroindomycins
A
and B,[4] Abyssomicin C,[5]
Quartromicin D,[6] Tetrocarcin A,[7] Kijanimicin,[8] Lobophorins[9])
have been discovered subsequently, leading to discover some
novel natural enzymes catalyzing unique reactions.[10]
According to the functional analysis of the gene cluster of CHL,
ChlB1-ChlB6 were assigned to be responsible for the synthesis of
1. The iterative type I polyketide synthases (iPKSs) have been
showed to synthesize 6-methylsalicyl from one acetyl CoA and
three malonyl CoA and install it onto the iterative PKS(iPKS)-ACP
domain,[3a, 3c, 11] but 6-methylsalicyl produced by ChlB1 didn’t
*E-mail: qinglihe@shutcm.edu.cn, wliu@mail.sioc.ac.cn
†These authors contributed equally to this work.
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which
may lead to differences between this version and the Version of Record. Please cite this
article as doi: 10.1002/cjoc.201900134
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Yi et al.
Concise Report
5-chloro-6-methyl-O-methylsalicyl-ChlB1-ACP were subsequently
determined (Figure 2 and Table S2). The Km values for
Results and Discussion
Depending on the previous hypothesis, methylation and
chlorination could happen on the aromatic acyl group loaded on
ChlB1-ACP, ChlB2 or DM-CHL. To detect the kinetic parameters of
ChlB3 for different small molecules loaded on ChlB1-ACP,
6-methylsalicyl-S-ChlB1-ACP, 6-methyl-O-methylsalicyl-S-ChlB1-
ACP and 5-chloro-6-methyl-O-methylsalicyl-S-ChlB1-ACP were
generated enzymatically by the sfp phosphopantetheinyl
transferase (PPTase).[16] Similar to 6-methylsalicyl group, both
5-chloro-6-methyl-O-methylsalicyl and 6-methyl-O-methylsalicyl
groups could be transferred to holo-ChlB2 by ChlB3, and neither
of them could be transferred by ChlB6 (Figure 1a, 1b), further
confirming the function of ChlB3 and ChlB6. Another two
substrates, O-methylsalicyl-S-ChlB1-ACP and salicyl-S-ChlB1-ACP
which should not exit in Streptomyces antibioticus DSM 40725,
could also be catalyzed by ChlB3 as we expected (Figure 1c, 1d),
indicating the excellent substrate tolerance of ChlB3. All the new
modified proteins had an identical retention time to default
substrates confirmed by MALDI-TOF-MS (Table S1).
6-methylsalicyl-S-ChlB1-ACP
and
5-chloro-6-methyl-O-
methylsalicyl-ChlB1-ACP with fixed concentration of holo-ChlB2
(at 150μM holo-ChlB2) were 0.043 mM and 1.0 mM, and the kcat
values are about 72.7 min-1 and 0.045 min-1 respectively. Notably,
when the catalytic efficiencies (kcat/Km) are compared,
6-methylsalicyl-S-ChlB1-ACP as substrate was superior to
5-chloro-6-methyl-O-methylsalicyl-S-Ch1B1-ACP by more than
3,000 times. The kinetical data showed that 6-methylsalicyl was
immediately transferred to ChlB2 after synthesis by ChlB1,
excluding the possibility that ChlB4 and ChlB5 modified
6-methylsalicyl immediately when it was still loaded on the
Ch1B1-ACP (Table S2).
Figure 1 HPLC analysis of acyltransferase activities of ChlB3 and ChlB6
from ChlB1-ACP to holo-ChlB2. a) 5-chloro-6-methyl-O-methylsalicyl
transfer by ChlB3 and ChlB6 for 1hr and 8hr. b) 6-methyl-O-methylsalicyl
transfer by ChlB3 and ChlB6 for 1hr and 8hr. c) O-methylsalicyl transfer by
ChlB3 for 10min, 30min and 1hr. D) Salicyl transfer by ChlB3 for 1hr and
8hr.
5000
4000
3000
2000
1000
0
400
300
200
100
0
0
10
20
30
40
50
60
70
80
90
0
10
20
30
40
50
60
70
80
90
[6-m ethylsalicyl-S-ChlB 1-A CP](M )
[5-chloro-6-m ethyl-O -m ethylsalicyl-S -C hlB 1-A C P ](M )
Scheme 1 Possible reaction order of 5-chloro-6-methyl-O-methylsalicyclic
Acid in Chlorothricin biosynthesis
Figure 2 Michaelis-Menten curves of ChlB3 for （a）6-methylsalicyl-S-
ChlB1-ACP and （b）5-chloro-6-methyl-O-methylsalicyl-S-ChlB1-ACP
As mentioned above, many different substrates could be
catalyzed by ChlB3 in vitro. In fact, only one substrate was
preferred or really utilized by the acyltransferase in vivo. Our
previous data have showed ChlB3 catalyzed the conversion of
Subsequently, we sought to explore the substrate spectrum of
ChlB6, which could synthesize CHL by transferring the cargo of
5-chloro-6-methyl-O-methylsalicyl on ChlB2 to DM-CHL. Another
four substrates, 6-methyl-O-methylsalicyl-S-ChlB2, 6-methylsalicyl
-S-ChlB2, O-methylsalicyl-S-ChlB2 and salicyl-S-ChlB2 were
generated enzymatically by the sfp phosphopantetheinyl
transferase (PPTase). Interestingly, ChlB6 could transfer each of
these acyl groups to DM-CHL to produce Deschloro-chlorothricin
(DCM-CHL), 6-methylsalicyl-chlorothricin (M-CHL), O-methylsalicyl
-chlorothricin (MS-CHL) and salicyl-chlorothricin (S-CHL) (Figure
3a-d), indicating that ChlB6 also has excellent substrate tolerance
in vitro. All the new compounds were confirmed by LC-MS (Table
S3).
6-methylsalicyl-S-ChlB1-ACP to 6-methylsalicyl-S-ChlB2 in
time-dependent reaction with a specific activity
a
[15]
of 7.33min-1.
With the same experimental conditions, ChlB3 catalyzed the
transfer of 6-methyl-O-methylsalicyl and 5-chloro-6-methyl-O-
methylsalicyl groups with the specific activities of 0.006 min-1 and
0.008 min-1 respectively (Figure 1a, 1b). The obvious difference in
specific activities for different substrates proved that
6-methylsalicyl-S-ChlB1-ACP was the optimum substrate of ChlB3.
To obtain the accurate enzymatic information of ChlB3, the kinetic
parameters of ChlB3 for 6-methylsalicyl-S-ChlB1-ACP and
ChlB6 transferred 6-methylsalicyl groups on ChlB2 with the
2
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Insights into the Functionalization of the Methylsalicyclic Moiety
Chin. J. Chem.
highest specificities, i.e. 1.06 min-1, than 5-chloro-6-methyl-O-
synthase I, II, III (KAS I, KAS II, KAS III, also known as FabB, FabF,
FabH ) and ketosynthases (KS) in the same phylogenetic tree
(Figure 5c). It was obvious that these acyl transferases used in the
natural products biosynthesis were divided into two different
clades. One clade, including ChlB3 and ChlB6, was
phylogenetically related to KAS III which catalyzed claisen
condensation, and known as KAS III-like acyl transferase. The
methylsalicyl (0.008 min-1) [15]and
6-methyl-O-methylsalicyl
(0.0006 min-1) (Figure 3a and 3b), showing that
6-methylsalicyl-S-ChlB2 should be the best substrate of ChlB6.
To obtain the accurate enzymatic information, the kinetic
parameters of ChlB6 for 6-methylsalicyl-S-ChlB2 and
5-chloro-6-methyl-O-methylsalicyl-S-ChlB2 were subsequently
determined (Figure 4 and table S4). The Km values for
6-methylsalicyl-S-ChlB2 and 5-chloro-6-methyl-O-methylsalicyl-S-
ChlB2 with fixed concentration of DM-CHL (150μM) are 0.24 mM
and 3.8 mM; and the kcat Values are 2.79 min-1 and 0.12 min-1.
The relative specificity constant (kcat/Km) of 6-methylsalicyl-S-
ChlB2 was superior to that of 5-chloro-6-methyl-O-methylsalicyl-S
-ChlB2 by more than 300 times. The kinetic data demonstrated
that 6-methylsalicyl was immediately used to synthesize M-CHL
after being transferred to ChlB2 by ChlB3, indicating that ChlB4
and ChlB5 only could modify M-CHL to form DCM-CHL and CHL,
both of which could be produced in wild-type Streptomyces
antibioticus DSM 40725.
other clade, including CouN7[17], CmaE[18], SyrC[19] and MdpB3[20]
,
was in close proximity to malonyl-CoA: [acyl-carrier-protein]
S-malonyltransferases (cis-AT, FabD and trans-AT), and named
CouN7-like acyltransferase.
Figure 3 HPLC analysis of acyltransferase activity of ChlB6 from holo-ChlB2
to DM-CHL. (a) 6-methyl-O-salicyl transfer by ChlB6 for 1hr, 4hr, 8hr, 14hr
and 24hr. (b) 6-methylsalicyl transfer by ChlB6 for 5min, 20min and 1hr. (c)
O-methylsalicyl and salicyl transfer by ChlB6 for 1hr. (d) The structure of
MS-CHL and S-CHL.
70
700
C-S Bond
C-C Bond
60
600
50
500
40
400
30
300
20
200
10
100
0
0
0
20 40 60 80 100 120 140 160 180 200 220
0
20 40 60 80 100 120 140 160 180 200 220
[6-m ethylsalicyl-S-C hlB 2](M )
[5-chloro-6-m ethyl-O -m ethylsalicyl-S -C hlB 2]( M )
Figure 4 Michaelis-Menten curves of ChlB6 for (a) 6-methylsalicyl-S-ChlB2
and (b) 5-chloro-6-methyl-O-methylsalicyl-S-ChlB2.
Many acyltransferases, similar to ChlB3 and ChlB6, have been
found in the gene clusters of various natural products (Figure S1),
and most of them were KAS III-like acyl transferases responsible
for the formation of C-O, C-N and C-S bonds, with the similar
mechanism of C-S bond formation catalyzed by FabD, however,
KAS III was supposed to catalyze the C-C formation (Figure 5a, 5b).
To further obtain the phylogenetic origin of ChlB3 and ChlB6, we
compared some representative acyl transferases from the
biosynthesis of natural products with cis-acyl transferases (cis-AT),
Figure 5 C-C/O/S/N bond formation catalyzed by KSs and ATs. (a)Possible
mocecular mechanism of C-C bond formation catalyzed by FabH.(b)
Possible mocecular mechanism of C-S bond formation catalyzed by FabD.(c)
Phylogenetic tree analysis of ChlB3, ChlB6 with selected KSs and ATs.
ChlB3 and ChlB6 as members of KAS III-like acyltransferases,
similar to CerJ[21] with catalytic triad of Cys-His-Asp (Figure S2),
used His-Asp to increase the nucleophilic ability of the catalytic
Cys residue, and catalyzed transacylation twice to produce the
final products. But CouN7-like proteins still need X-ray structure of
trans-acyl transferases (trans-AT)
malonyl-CoA–acyl carrier
protein transacylases（FabD）, β-Ketoacyl-acyl carrier protein
Chin. J. Chem. 2019, 37, XXX－XXX
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3
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Yi et al.
Concise Report
this kind of acyl transferase to determine their catalytic
mechanism (Figure S3), and only Cys/Ser-His was conserved in the
sequence alignment because their protein sequences were
obviously different with that of CerJ or FabD. Utilizing the
available proteomic information from the UniProt database, we
constructed the SSN and GNN of the KAS III-like acyl transferase
ChlB3 with the Enzyme Function Initiative-Enzyme Similarity Tool
(EFI-EST) and Genome Neighborhood Tool (Figure S4)[22]. More
than 1,500 biosynthetic gene clusters including possible KAS
III-like acyltransferases were found, and most of the secondary
and then coenzyme A (6.35 μmol, 1 eq) was dissolved in 0.5 mL of
H2O (O2 was removed by sonication) and added dropwise to the
reaction. After being stirred at room temperature for 2 hr, the
reaction mixture was directly purified by RP-HPLC (Venusil
XBP-C18 10.0 × 250 mm, 5 μm, 100 Å, 3ml/ml, 0-25 min 20%-60%
B, 25-29 min 60% B, 29-30 min 60%-20% B ; buffer A: 10 mM
NH4OAc in H2O; buffer B: CH3OH). These fractions containing
product were collected, and methanol was removed by
evaporation firstly, followed by the removal of water and NH4OAc
by lyophilization. More than 95% CoA was converted into its
metabolites from these gene clusters have not been excavated yet, derivative, and the mass of the product was confirmed by ESI-MS
form which possible molecular skeleton spanned polyketides,
non-ribosomal peptides and terpenes. Therefore, our exploration
will provide a reference for predicting the function of these
homologous proteins and discovering compounds with novel
structures.
[M-H]-or [M+H] +. For O-methylsalicyl-S-CoA, ESI-MS [M+H] +:cald.
902.2, found. 902.3; for 6-methyl-O-methylsalicyl-S-CoA, ESI-MS
[M-H]- cald. 914.2, found. 914.4.
Synthesis of CoA derivatives of salicyclic acid. Salicyclic acid
(100 μmol, 1.0 eq) in freshly distilled DMF (1 mL) was stirred with
PyBOP (1.5 eq), iPr2EtN (1.5 eq), and thiophenol (3.0 eq) under
argon. After being stirred at room temperature for 30 min, the
reaction mixture was directly purified by RP- HPLC column
(Venusil XBP-C18, 10.0 × 250 mm, 5 μm, 100 Å, 3ml/ml, 0-5 min
5%-70% B, 5-28 min 70%-100% B, 28-30 min 100%-5% B ; buffer A:
0.1% TFA in H2O; buffer B: 0.1% TFA in CH3CN). The solvent was
removed under reduced pressure to give the product of
salicyl-S-phenyl thioester. Then the salicyl-S-phenyl thioester
(60.8 μmol, 4.8 eq) and coenzyme A (12.7 μmol, 1.0 eq) were
stirred in phosphate buffer (8 mL, 50 mM, pH=8.5) under argon.
After being stirred at room temperature for 2 hr, the reaction
mixture was directly purified by reverse-phase HPLC (Venusil
XBP-C18, 10.0 × 250 mm, 5 μm, 100 Å, 3ml/ml, 0-25 min 20%-60%
B, 25-29 min 60% B, 29-30 min 60%-20% B ; buffer A: 10 mM
NH4OAc in H2O; buffer B: CH3OH). The fractions containing
product were pooled, and methanol was removed by evaporation
firstly, followed by the removal of water and NH4OAc by
lyophilization. More than 90% CoA was converted into its
derivative, and the mass of the product was confirmed by ESI-MS
[M-H]-: cald.888.1, found.888.2.
Conclusions
According to these data mentioned above, the biosynthesis
pathway of 1 should happen in the order showed in Scheme 1d.
Firstly ChlB1 synthesizes 6-methylsalicyl loaded on its ACP from
one acetyl CoA and three malonyl CoA. Secondly, ChlB3 transfers
6-methylsalicyl from 6-methylsalicyl-ChlB1 to holo-ChlB2. Thirdly,
ChlB6 immediately grafts 6-methylsalicyl from holo-ChlB2 to
DM-CHL, producing M-CHL. Finally ChlB4 and ChlB5 perform
chlorination and O-methylation on M-CHL to obtain DCM-CHL or
CHL. Manipulation of chain initiation has proven very useful for
generating novel products in vivo. However, manipulation of the
modified units is more powerful in increasing the structural
diversity of natural products at the biochemical level in vitro. And
our findings will provide a unique opportunity to understand
other similar functional modified enzymes and contribute to
generating new CHL analogs via combinatorial biosynthesis and
chemo-enzymatic synthesis.
Characterization of ChlB3 and ChlB6 activities on holo-ChlB2
and small molecule acyl-S-ChlB1-ACP. To generate the small
molecule acyl-S-ChlB1-ACP, the reaction was carried out in 75 mM
MOPS (pH 7.5), 10 mM MgCl2, 1 mM TCEP (pH 8.0), 100 μM
ChlB1-ACP, 150 μM indicated CoA derivatives, and 2 μM sfp for 1
hr at 30°C. To produce the holo-ChlB2, a similar reaction was
performed in 75 mM MOPS (pH 7.5),10 mM MgCl2, 1 mM TCEP
(pH 8.0), 300 μM ChlB2, 450 μM CoA, and 5 μM sfp for 1 hr at
30°C. For the transferring of small molecule acyl group, the
reaction harboring 75 mM MOPS (pH 7.5), 10 mM MgCl2, 1 mM
TCEP (pH 8.0), 30 μM indicated small molecule acyl-S-ChlB1-ACP
and 30 μM holo-ChlB2, was initiated by addition of 0.2 μM ChlB3
or ChlB6, and quenched with 0.25 volume 10% formic acid at
indicated time. The quenched reaction was directly analyzed by
reverse-phase HPLC (Vydac 218TP54 C18 , 250 × 4.6 mm, 5 μm,
300 Å, 1ml/min, 0-3 min 20% B, 3-5 min 20%-35% B, 5-25min
35%-55% B, 25-26 min 55%-99% B, 26-29 min 99% B, 29-30 min,
99%-20% B; buffer A: 0.1% TFA in H2O; buffer B: 0.1% TFA in
CH3CN).
Characterization of ChlB3 and ChlB6 activities on
desmethylsalicyl CHL (DM-CHL) and small molecule acyl-S-ChlB2.
To generate the small molecule acyl-S-ChlB2, the reaction was
carried out in 75 mM MOPS (pH 7.5), 10 mM MgCl2, 1 mM TCEP
(pH 8.0), 300 μM apo-ChlB2, 450 μM indicated CoA derivatives,
and 5 μM sfp for 1 hr at 30°C. The reaction containing 75 mM
MOPS (pH 7.5), 10mM MgCl2, 1 mM TCEP (pH 8.0), 1 mg/mL BSA,
5% DMSO,150 μM small molecule acyl-S-pantetheinyl ChlB2 and
150 μM DM-CHL, was initiated with 20.0 μM ChlB3 or ChlB6, and
quenched with 0.25 volume 10 % formic acid or 2 volume
methanol at indicated time. The reaction quenched with formic
acid was directly analyzed by reverse-phase HPLC (Vydac 218TP54
Experimental
Bacterial Strains, Plasmids, Biochemicals, and chemicals. E.
coli DH5α was used for general gene clone, and E. coli BL21 (DE3)
was used for protein expression (Novagen). The Chlorothricin
producer, S. antibioticus DSM40725 was purchased from DSMZ.
Cloning vectors pSP72 (Promega) and expression vectors pET28a,
pET37b (Novagen) and pMal-c2X (NEB) were originally from
commercial sources. HCoA was from Sigma, and all the other
common biochemicals and reagents came from standard
commercial sources.
DNA Isolation and Manipulation. DNA isolation and
manipulation in E. coli and Streptomyces were carried out
according to standard methods. PCR amplifications were carried
out on an Authorized Thermal Cycler (Eppendorf AG) using either
Taq DNA polymerase or PfuUltra High-Fidelity DNA polymerase.
Primer synthesis and DNA sequencing were performed at the
Shanghai Invitrogen Biotech Co., Ltd., and Chinese National
Human Genome Center.
Expression and purification of sfp, ChlB1-ACP, ChlB2, ChlB3,
and ChlB6. Sfp, ChlB1-ACP, ChlB2, ChlB3 and ChlB6 were
expressed and purified as we have reported previously.[15]
Synthesis of CoA derivatives of 5-chloro-6-methyl-O-
methylsalicyclic acid and 6-methyl-O-methylsalicyclic acid. CoA
derivatives of 5-chloro-6-methyl-O-methylsalicyclic acid and
6-methyl-O-methylsalicyclic acid were synthesized and purified as
we reported previously. [15]
Synthesis of CoA derivatives of O-methylsalicyclic acid.
6-methyl-O-methylsalicyclic acid or O-methylsalicyclic acid (10
μmol, 1.6 eq), PyBOP (10 μmol, 1.6 eq) and K2CO3 (25.4 μmol, 4.0
eq) were dissolved in 0.5 mL of freshly distilled THF under argon,
4
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Insights into the Functionalization of the Methylsalicyclic Moiety
Chin. J. Chem.
250 × 4.6mm, 5 μm, 300 Å, the gradient elution condition was as
described above). The reaction quenched with methanol was
placed at -20°C for 30min, and centrifuged at 12,000 rpm for
20min to remove proteins, followed by the supernatant analysis
by reverse-phase HPLC column (COSMOSIL 3C18-AR-II, 250 ×
4.6mm, 5 μm, 1ml/min, 0-5 min 40% B, 5-25 min 40%-85% B,
25-30 min 80%-40% B; buffer A: 0.1% TFA in H2O; buffer B: 0.1%
TFA in CH3CN).
Determination of the kinetic parameters of ChlB3 for the
substrates of 6-methylsalicyl-ChlB1-ACP and 5-chloro-6-methyl
-O-methylsalicyl-S-ChlB1-ACP. To determine the kinetic
parameters of ChlB3, reactions were carried out at 30 °C in total
volume of 100 μl that contained 50 mM MOPS (pH 7.5), 10 mM
MgCl2, 1 mM TCEP (pH 8.0), 5% DMSO, 150 μM holo-ChlB2, 0.1
References
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μM
ChlB3,
and
various
concentrations
of
6-methylsalicyl-S-ChlB1-ACP (10, 20, 30, 40, 50, 60, 70, and 80μM)
for 3 min. The conversion ratio of 6-methylsalicyl-S-ChlB1-ACP to
6-methylsalicyl-S-ChlB2 was analyzed by RP-HPLC as described
above. The initial rate conditions were maintained by computing
the substrate conversion at less than 10% on the assumption that
these conditions were within the linear range of enzyme
concentration and enzyme turnover. The data were fitted with
the Michaelis-Menten equation to extract the kinetic constants.
While for 5-chloro-6-methyl-O- methylsalicyl-S-ChlB1-ACP,
reactions were carried out at 30 °C in total volume of 100 μL that
contained 50mM MOPS (pH 7.5), 10 mM MgCl2, 1 mM TCEP (pH
8.0), 5% DMSO, 150 μM holo-ChlB2, 10μM ChlB3, and various
concentrations of 5-chloro-6-methyl-O-methylsalicyl-S-ChlB1-ACP
(10, 20, 30, 40, 50, 60, 70, and 80μM) for 30 min, and these
remaining steps were the same as described above.
Determination of the kinetic parameters of ChlB6 for the
substrates of 6-methylsalicyl-S-ChlB2 and 5-chloro-6-methyl-O-
methylsalicyl-S-ChlB2. To determine the kinetic parameters of
ChlB6, reactions were carried out at 30 °C in total volume of 100
μL that contained 50 mM MOPS (pH 7.5), 10 mM MgCl2, 1mg/mL
BSA, 1 mM TCEP (pH 8.0), 5% DMSO, 250μM DM-CHL, 5μM ChlB6,
and indicated concentrations of 6-methylsalicyl-S-ChlB2 (25, 50,
75, 100, 125, 150, and 200μM) for 5 min. The ratio of
6-methylsalicyl-S-ChlB2 to holo-ChlB2 conversion was analyzed by
RP-HPLC as described above. The initial rate conditions were
maintained by computing the substrate conversion at less than 10%
on the assumption that these conditions were within the linear
range of enzyme concentration and enzyme turnover. The data
were fitted with the Michaelis-Menten equation to extract the
kinetic constants. While for 5-chloro-6-methyl-O-methylsalicyl-S
-ChlB2, reactions were carried out at 30 °C in total volume of 100
μL that contained 50 mM MOPS (pH 7.5), 10 mM MgCl2, 1mg/mL
BSA, 1 mM TCEP (pH 8.0), 5% DMSO, 250μM DM-CHL, 10μM
ChlB6, and various concentrations of 5-chloro-6-methyl-O-
methylsalicyl-S-ChlB2 (25, 50, 75, 100, 125, 150, and 200μM) for
30 min, and other steps were the same as described above.
Supporting Information
The supporting information for this article is available on the
WWW under https://doi.org/10.1002/cjoc.2018xxxxx.
Acknowledgement
This work was supported in part by grants from NSFC
(21520102004, 31430005, 21750004 and 21621002), CAS
(QYZDJ-SSW-SLH037 and XDB20020200), STCSM (17JC1405100,
15JC1400400
and
18401933500),
SMEC
(2019-01-07-00-10-E00072) and the Drug Innovation Major
Project (2018ZX091711001-006-010).
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© 2019 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
5
This article is protected by copyright. All rights reserved.

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(The following will be filled in by the editorial staff)
Manuscript received: XXXX, 2019
Manuscript revised: XXXX, 2019
Manuscript accepted: XXXX, 2019
Accepted manuscript online: XXXX, 2019
Version of record online: XXXX, 2019
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6
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© 2019 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2019, 37, XXX－XXX
This article is protected by copyright. All rights reserved.

Insights into the Functionalization of the Methylsalicyclic Moiety
Chin. J. Chem.
Entry for the Table of Contents
Page No.
Insights into the Functionalization of the
Methylsalicyclic Moiety during the Biosynthesis
of Chlorothricin by Comparative Kinetic Assays
of the Activities of Two KAS III-like
Acyltransferases
a,c,†
b,†
,
Zhenhua Tianc,
Speculating the Reaction Timing of 5-chloro-6-methyl-O-methylsalicyclic Acid by Kinetic
Parameters of Two KAS III-like Acyltransferases for Different Substrates in Chlorothricin
Biosynthesis
Xuan Yi
,
Qunfei Zhao
Xinying Jiac, Weiguo Caoa , Wen Liu*c,d, and
Qing-Li He*b
.
Chin. J. Chem. 2019, 37, XXX－XXX
© 2019 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
7
This article is protected by copyright. All rights reserved.