In vitro kinetic study of the squalestatin tetraketide synthase dehydratase reveals the stereochemical course of a fungal highly reducing polyketide synthase

Article abstract of DOI:10.1039/c6cc10172k

Six potential diketide substrates for the squalestatin tetraketide synthase (SQTKS) dehydratase (DH) domain were synthesised as N-acetyl cysteamine thiolesters (SNAC) and tested in kinetic assays as substrates with an isolated DH domain. 3R-3-hydroxybutyryl SNAC 3R-16 was turned over by the enzyme, but its enantiomer was not. Of the four 2-methyl substrates only 2R,3R-2-methyl-3-hydroxybutyryl SNAC 2R,3R-8 was a substrate. Combined with stereochemical information from the isolated SQTKS enoyl reductase (ER) domain, our results provide a near complete stereochemical description of the first cycle of beta-modification reactions of a fungal highly reducing polyketide synthase (HR-PKS). The results emphasise the close relationship between fungal HR-PKS and vertebrate fatty acid synthases (vFAS).

Full text of DOI:10.1039/c6cc10172k

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Reactivity differences of Sc
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DOI: 10.1039/C6CC10172K
Journal Name
Communication
In Vitro Kinetic Study of the Squalestatin Tetraketide Synthase
Dehydratase Reveals the Stereochemical Course of a Fungal
Highly Reducing Polyketide Synthase
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
a
a
a
a
Emma Liddle, Alan Scott, Li-Chen Han, David Ivison, Thomas J. Simpson, Christine L. Willis
a,b,c *
DOI: 10.1039/x0xx00000x
and Russell J. Cox
www.rsc.org/
Six potential diketide substrates for the squalestatin extender units from CoA onto the PKS. In addition, SQTKS
tetraketide synthase (SQTKS) dehydratase (DH) domain were contains: -ketoacyl ACP ketoreductase (KR); -hydroxy acyl
synthesised as N-acetyl cysteamine thiolesters (SNAC) and ACP dehydratase (DH); and enoyl ACP reductase (ER) domains
tested in kinetic assays as substrates with an isolated DH which process the -carbonyl formed by the KS. Finally, a C-
β
β
β
domain. 3R-3-Hydroxybutyryl SNAC 3R-16 was turned over by methyl transferase (C-MeT) domain is responsible for adding a
the enzyme, but its enantiomer was not. Of the four 2-methyl methyl group derived from S-adenosyl methionine. SQTKS thus
substrates only 2R,3R-2-methyl-3-hydroxybutyryl SNAC contains a full set of active HR-PKS domains.
2R,3R-8 was a substrate. Combined with stereochemical
information from the isolated SQTKS enoyl reductase (ER)
domain, our results provide a near complete stereochemical
description of the first cycle of beta-modification reactions of
a fungal highly reducing polyketide synthase (HR-PKS). The
results emphasise the close relationship between fungal HR-
PKS and vertebrate fatty acid synthases (vFAS).
Iterative fungal polyketide synthases (PKS) are responsible
for the biosynthesis of complex and often biologically active
natural products such as squalestatin S1
1
a potent inhibitor of
1
squalene synthase, and lovastatin
,2
3
2, an inhibitor of human
HMG-CoA reductase. These PKS are Type I systems in which
several individual catalytic domains are covalently linked to
4
,5
form a mega-complex of ca >200 KDa. Understanding the
selectivity and programming of these systems is important
because reprogramming them could lead to the systematic
creation of new bioactive materials. In order to achieve this an
understanding of the individual catalytic domains and their
intrinsic selectivities is required.
Figure 1. Structures of fungal highly reduced polyketides, and the structure of
8
dimeric vFAS (PDB 2VZ8) which is homologous the squalestatin tetraketide
synthase (SQTKS). See text for abbreviations.
SQTKS shows sequence homology to vertebrate FAS (vFAS,
8
Figure 1). This similarity even extends to the position of a C-
The C10 side-chain of
1, known as squalestatin tetraketide
3
, is synthesised by a highly reducing (HR) iterative PKS called
6
,7
MeT domain which is inactive in vFAS, but which acts during
9
the first and second rounds of chain processing by SQTKS.
squalestatin tetraketide synthase (SQTKS). It consists of an
acyl carrier protein (ACP) which holds the growing polyketide
vFAS produces fully saturated linear 16-18 carbon chains,
whereas SQTKS produces the dimethylated and unsaturated 8-
chain, a β-ketoacyl ACP synthase (KS) which catalyses a Claisen
condensation between malonyl ACP and acyl-KS and an acyl
carbon chain 3. SQTKS thus displays a complex programme in
transferase (AT) which loads acetyl starter and malonyl
which the activities of the individual catalytic domains can be
varied (Scheme 1). Our approach to study the programming
mechanisms of fungal HR-PKS is to examine intrinsic
selectivities of isolated catalytic domains. For example, we
recently reported on the chemo- and stereo-selectivity of the
1
0
isolated SQTKS ER domain. Here we describe work to extend
this study to the isolated DH domain of SQTKS.
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O
O
+
O
O
O
O
O
KS
C-MeT
set up to include DH protein, substrate and buffer at 30 ˚C in
HO
SACP
SACP
SACP
SACP
SACP
1
00
ꢀ
points and quenched in CH
L assay volume, and 20
ꢀ
L aliquots were taken at time
CN (60 L). Protein was
O
3
ꢀ
precipitated by centrifugation and the supernatant was
SKS
KR
O
examined directly by LCMS.
O
OH
ER
DH
OH
O
a
OH
O
TBDMSO
O
A
b
SACP
OR
OR
OR
3R-4
2R,3R-5
2R,3R-6
2nd cycle
c
O
O
OH
O
TBDMSO
O
H
N
H
N
3rd cycle
d
release
S
S
SACP
SACP
3
O
O
O
2
R,3R-8
2R,3R-7
triketide
tetraketide
O
O
OH
O
O
TBDMSO
O
Scheme 1. The chemical reactions catalysed by SQTKS.
B
e
f
N
O
N
O
N
O
SQTKS is a megacomplex of 284.4 KDa encoded by the
phpks1 gene. We have been unable to obtain it as a single
soluble protein. However, by systematic variation of possible
start and stop positions for PCR from an intron-free phpks1
template, we were able to create an open reading frame which
reliably produces soluble SQTKS DH protein when expressed in
Bn
Bn
Bn
9
2
R,3S,4'R-10
2R,3S,4'R-11
g
OH
O
H
N
d
TBDMSO
TBDMSO
O
H
N
S
S
O
2
R,3S-13
2R,3S-12
O
O
O
6
E. coli BL21 with an N-terminal his tag. The DH protein of the
expected 38.0 kDa was purified to homogeneity by nickel
affinity and gel-filtration chromatography. Calibrated gel
filtration indicated that the DH exists primarily as a monomer
C
OH
O
b
TBDMSO
O
O
c
H
N
OR
OR
3R-14
S
3
R-4
3R-15
(see ESI). The isolated DH was unstable in unmodified buffers,
d
precipitating rapidly even at low temperatures. However, rapid
removal of imidazole used for the nickel ion chromatography
and use of a buffer containing 10% glycerol, 50 mM Tris pH
R = Me for 3S series
R = Et for 3R series
OH O
H
N
S
3R-16
8
.0, 150 mM NaCl and 100 mM L-arginine and L-glutamic acid
dramatically improved protein solubility and stability.
N-acetyl cysteamine (NAC) is a truncated form of the HSNAC, EDCI, DMAP, 0 ˚C;
Scheme 2. Synthesis of potential SNAC substrates for the SQTKS DH domain:
LDA (2eq.), -78 ˚C, then MeI; , TBDMSOTf, pyridine, 0 ˚C; , aq. LiOH, 60 ˚C, then
, THF/H O/HOAc, RT, 5 days; , Bu BOTf, CH Cl
, TBDMSCl, CH Cl , DMAP, imidazole, RT; , LiOOH,
O, RT, then HSNAC, EDCI, DMAP, 0 ˚C. TBDMS = (Si(Me )CMe ).
a,
b
c
d
2
e
2
2
2
,
Et
3
N, -78 ˚C, then CH
3
CHO;
f
2
2
g
phosphopantetheine (PP) cofactor which attaches acyl PKS
intermediates to the ACP domain, and SNAC thiolesters are
often used as PP surrogates for in vitro studies of PKS
H
2
2
3
1
1,12
13,14
In order to maximise sensitivity, single ion monitoring was
enzymes,
SNACs as targets for substrate synthesis (Scheme 2).
The anti diketide SNAC 2R,3R- was made by a route
involving Fráter-Seebach methylation
available enantiopure 3-hydroxy butyrate
give . This was O-protected with TBDMS to give
in-turn hydrolysed to its corresponding acid and coupled to
HSNAC to give the protected diketide . Acidic deprotection
then yielded 2R,3R-
The syn diketide SNAC 2R,3S-13 was made using Evans
including DH domains.
We thus selected
applied for substrate and product peaks and peak areas were
integrated. The peak integrals were calibrated vs known
concentrations of substrate and product. Initial rates were
determined by plotting product concentration vs assay times,
and variation of initial substrate concentrations allowed the
estimation of kinetic parameters (Figure 2). The diketide
8
1
5
of commercially
(Scheme 2A) to
, which was
4
5
6
2R,3R-8 was dehydrated by the DH to give exclusively the E-
7
olefin product tigloyl SNAC 17, but none of the other 2-methyl
diketides showed any turn-over. Of the non-methylated
diketides, only 3R-16 was a substrate, although much slower
8.
1
6
asymmetric aldol chemistry to give the known syn aldol
product 2R,3S,4'R-10 (Scheme 2B). This was again O-protected
with TBDMS to give 11, which was hydrolysed and processed
to the protected SNAC 2R,3S-12. Acidic deprotection then
than 2S,3S-8. The ability of the non-substrates 2S,3S-8 and the
enantiomers of 13 to act as inhibitors of the DH was
investigated. However, addition of each of these compounds
to assays containing the substrate 2R,3R-
8 showed no
yielded 2R,3S-13
The non-methylated diketide 3R-16 was made from
similar protection, thiolesterification and deprotection route
Scheme 2C). The enantiomers of all the diketides were made
.
appreciable decrease in rate when added in mM
concentrations (see ESI).
4
by a
Despite having access to soluble protein we were unable to
grow satisfactory crystals of the isolated DH domain. In lieu of
other structural information we built a model of SQTKS DH
based on the known crystal structures of DH proteins from
other Type I systems reported in the literature. These form
(
from enantiomeric starting materials using identical methods.
DH activity was assayed using LCMS (See ESI) to measure
substrate consumption and product formation. Assays were
2
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1
distinctive double hot-dog folds. In particular the DH domain reaction cavity and would presumably perform a similar role
7
1
8
from CurF, a modular type I PKS, formed an appropriate with the pantetheinyl-ACP-bound substrate in the native
template for the assembly of a model by the SwissModel complex. The two methyl groups of 2R,3R- occupy open
-hydroxyl to approach the
backbone atoms of the SQTKS DH model and CurF-DH had only conserved histidine and aspartate residues. The closest
.4 Å root mean square deviation (RMSD). Almost all of the distance between the  proton and the nearest nitrogen of
observed deviation was concentrated on the periphery of the H1034 is 3.6 Å, while the distance between the -hydroxyl and
8
1
9
threading server. Comparison of the results showed that the space and allow the α-proton and β
1
α
β
model structure, and examination of the conserved active site the oxygen of D1225 is 3.0 Å. The geometry of the interactions
aspartic acid (D1225) and histidine (H1034) residues showed is consistent with a syn abstraction of water to give the
that these amino acids are located in the same positions in the observed E-2-methylbut-2-enoyl (tigloyl) SNAC product 17
.
CurF DH and the model (see ESI). In addition a highly Attempts to dock the other stereoisomers of the diketide did
conserved Y/FP motif (Y/F1041-P1042) is also preserved in the not give satisfactory results.
model.
OH
O
O
H
DH
H
N
N
S
S
Y1041
P1042
O
O
2R,3R-8
17
H1034
D1225
A
M1083
G1043
OH
O
H
N
A1082
O
S
Me
2
R,3R-8
Figure 3. Key modelled interactions between the substrate 2R,3R-7b (cyan) and
B
the DH (green).
2
R,3R-8
S,3S-8
2
Our experiments report the first in vitro studies of the
2R,3S-13
stereoselectivity of an isolated DH domain from an iterative
2S,3R-13
Type I PKS. Using a kinetic assay we measured the K
M
(4.5 mM)
-1
and kcat values (0.063
⋅min ). While these values have little
absolute meaning, they are comparable with values measured
for other DH proteins. For example Aldrich, Smith and
M
coworkers reported K values in the same range for a KR-DH
didomain from module 2 of the pikromycin modular PKS
(
pikKR2-DH2) acting on triketide mimics, although their kcat
1
3
values are ten-fold higher.
Only one 2-methylated diketide, 2R,3R-
substrate for SQTKS DH, with no dehydation activity observed
for its enantiomer 2S,3S- or either of the syn diastereomers
8, is accepted as a
8
Figure 2. Kinetic data for SQTKS DH reactions. A, raw time course data showing 13. Since these stereoisomers show no measurable substrate
increase in tigloyl SNAC 17 concentration; B, comparison of DH reaction rate for the 4 or inhibition activity it seems unlikely that they can be bound
diketide stereoisomers.
at the DH active site, also supported by the failure to generate
satisfactory docked models of these isomers. However the
The diketide substrate 2R,3R-8 was then docked into the
non-methylated diketide 3R-16 is a substrate.
DH active site using a combination of manual positioning
In the active SQTKS the ACP-bound 2R,3R-diketide 20 is
created by reduction of a 3-oxo diketide 19 by the KR domain
using NADPH as the cofactor (Scheme 3). Our results strongly
suggest that the SQTKS KR releases 3R substrates, and thus it
must reduce the 3-oxo group of its substrate 19 by 3-Si
addition of hydride. Since racemisation at the 2-position of the
diketide is strongly disfavoured after reduction of the 3-oxo
group, this observation also suggests that the KR accepts and
releases 2R-methylated diketides (e.g. 2R-19, Scheme 3).
However, because facile epimerisation of 2-methyl-3-oxo
2
PyMol) and optimisation and energy minimisation using the
0
(
2
YASARA force field. For 2R,3R-8, the docked model shows
1
that the thiolester carbonyl oxygen is within hydrogen bonding
distance of the backbone NH of G1043 (2.9 Å), and aligns with
the dipole of the helix between G1043 and M1055 of the DH
model. The SNAC NH is positioned within hydrogen bonding
distance of the backbone carbonyl of conserved Y1041 (2.0 Å);
while the SNAC carbonyl is 2.8 Å from backbone NH of M1083.
These interactions locate the SNAC in the entrance to the
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substrates such as 19 is likely, it is not yet possible to infer the vFAS: its fundamental mechanisms for substate reaction and
stereochemical preference of the C-MeT without further stereoselectivity are also preserved and reinforce the idea that
experiments. Our previous results have shown that the SQTKS fungal hr-PKS and vFAS evolved from a common ancestor. Our
ER domain can process both Z 22 and E 23 substrates. current work focusses on determining the stereochemical
However since the DH can only provide E-diketides it appears preference of the CMeT domain and attempts at engineering
1
0
that the ER's ability to accept Z-olefins is merely adventitious.
SQTKS to rationaly change its selectivity.
In our earlier study of the stereoselectivity of the ER
domain we showed that the stereochemical preferences at the
Acknowledgements
β-carbon are identical for SQTKS ER and vFAS ER, in terms of
both the cofactor itself (transfer of 4'-pro-R Hydrogen) and the
We thank EPSRC (EP/F066104/1) for LCMS equipment, BBSRC
(BB/I003355/1) for funding AS and BrisSynBio Synthetic
Biology Research Centre, (BB/L01386X/1) for funding L-CH. EL
and DI were funded by the School of Chemistry, University of
Bristol. We thank Dr J. E. Nettleship at the Oxford Protein
Production Facility (OPPF) for assistance with protein
production. Hao Yao and Oliver Piech (LUH) are thanked for
technical assistance.
1
0
substrate (addition of hydride to to the 3-Re face). The
results of this study also show that the SQTKS DH has exactly
the same stereochemical selectivity as the vFAS DH which
dehydrates 2R,3R substrates 20 to give E-products 23 by syn
2
2
elimination. Even though the SQTKS substrate is methylated
at the 2-position, the 2R stereochemistry ensures that the 2-
pro-S proton is removed during reaction. Our model structure
shows that the 2R,3R substrate aligns with the active site
residues such that syn elimination gives the observed E-
product. The active site residues involved, H1034, D1225,
Y1041 and P1042 are conserved between the SQTKS and vFAS
sequences.
Notes and References
1
.
B. Bonsch, V. Belt, C. Bartel, N. Duensing, M. Koziol, C. M. Lazarus, A. M.
Bailey, T. J. Simpson and R. J. Cox, Chem. Commun., 2016, 52, 6777–
6
780.
OH
O
2
3
.
.
P. J. Sidebottom, R. M. Highcock, S. J. Lane, P. A. Procopiou and N. S.
Watson, J. Antibiot., 1992, 45, 648–658.
L. Hendrickson, C. R. Davis, C. Roach, D. K. Nguyen, T. Aldrich, P. C.
McAda and C. D. Reeves, Chem. Biol., 1999, 6, 429–439.
R. J. Cox, Org. Biomol. Chem., 2007, 5, 2010–2026.
SACP
18
Re
CMeT
Si
O
4
5
.
.
Y.-H. Chooi and Y. Tang, J. Org. Chem., 2012, 77, 9933–9953.
O
O
O
?
6. R. J. Cox, F. Glod, D. Hurley, C. M. Lazarus, T. P. Nicholson, B. A. M.
Rudd, T. J. Simpson, B. Wilkinson and Y. Zhang, Chem. Commun., 2004,
2260–2261.
SACP
SACP
H
Me
Me H
2R-19
2S-19
7
.
E. J. Skellam, D. Hurley, J. Davison, C. M. Lazarus, T. J. Simpson and R. J.
Cox, Mol. BioSyst., 2010, 6, 680–682.
M. Leibundgut, T. Maier, S. Jenni and N. Ban, Curr. Opin. Struct. Biol.,
KR NADPH (unknown hydride)
KR NADPH
Si
Re
Si
O
Re
8
.
2
008, 18, 714–725.
OH
O
OH
H
O
OH
Me
O
HO
H
9. K. M. Fisch, A. M. Bailey, W. Bakeer, A. A. Yakasai, Z. Song, J. Pedrick, Z.
Wasil, C. M. Lazarus, T. J. Simpson and R. J. Cox, J. Am. Chem. Soc., 2011,
133, 16635–16641.
0. D. M. Roberts, C. Bartel, A. Scott, D. Ivison, T. J. Simpson and R. J. Cox,
Chem. Sci., 2017, in press. DOI 10.1039 /c6sc03496a
SACP
SACP
SACP
SACP
Me
H
Me
H
H
Me
R,3R-20
2
2R,3S-21
DH syn
2S,3R-21
2S,3S-20
1
1
1. R. C. Harris, A. L. Cutter, K. J. Weissman, U. Hanefeld, M. I. C. Timoney
and J. Staunton, J. Chem. Res., 1998, 283–283; I. E. Holzbaur, A.
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O
H
SACP
22
DH syn
H
DH syn
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31–142.
1
SACP 23
1
3. Y. Li, G. J. Dodge, W. D. Fiers, R. A. Fecik, J. L. Smith and C. C. Aldrich, J.
Am. Chem. Soc., 2015, 137, 7003–7006.
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1
4. N. Kandziora, J. N. Andexer, S. J. Moss, B. Wilkinson, P. F. Leadlay and F.
Hahn, Chem. Sci., 2014, 5, 3563–3567; G. Berkhan and F. Hahn, Angew.
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H
H
OH
SACP
H
H
O
2
H O
1
1
1
SACP
H
Me
24
25
Scheme 3. Stereochemical course of KR, DH and ER domains of SQTKS.
18. D. L. Akey, J. R. Razelun, J. Tehranisa, D. H. Sherman, W. H. Gerwick and
J. L. Smith, Structure, 2010, 18, 94–105.
1
9. M. Biasini, S. Bienert, A. Waterhouse, K. Arnold, G. Studer, T. Schmidt, F.
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Finally, the SQTKS KR domain also operates with the same
2
stereochemical preference as the vFAS KR. Although we have
2
not yet been able to show which of the cofactor 4'-hydrides is 20. The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC.
2
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transferred by KR, the reduction does occur at the 3-Si face of
the substrate. Thus our studies show that SQTKS shares more
than just sequence homology and domain organisation with
2
4
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