Structural and stereochemical analysis of a modular polyketide synthase ketoreductase domain required for the generation of a cis-alkene

Article abstract of DOI:10.1016/j.chembiol.2013.04.014

The formation of an activated cis-3-cyclohexylpropenoic acid by Plm1, the first extension module of the phoslactomycin polyketide synthase, is proposed to occur through an L-3-hydroxyacyl-intermediate as a result of ketoreduction by an A-type ketoreductase (KR). Here, we demonstrate that the KR domain of Plm1 (PlmKR1) catalyzes the formation of an L-3-hydroxyacyl product. The crystal structure of PlmKR1 revealed a well-ordered active site with a nearby Trp residue characteristic of A-type KRs. Structural comparison of PlmKR1 with B-type KRs that produce D-3-hydroxyacyl intermediates revealed significant differences. The active site of cofactor-bound A-type KRs is in a catalysis-ready state, whereas cofactor-bound B-type KRs are in a precatalytic state. Furthermore, the closed lid loop in substrate-bound A-type KRs restricts active site access from all but one direction, which is proposed to control the stereochemistry of ketoreduction.

Full text of DOI:10.1016/j.chembiol.2013.04.014

Chemistry & Biology
Article
Structural and Stereochemical Analysis
of a Modular Polyketide Synthase Ketoreductase
Domain Required for the Generation of a cis-Alkene
Shilah A. Bonnett,1,5 Jonathan R. Whicher,2,5 Kancharla Papireddy,1,5 Galina Florova,1 Janet L. Smith,3,4
and Kevin A. Reynolds1,
*
1Department of Chemistry, Portland State University, Portland, OR 97201, USA
2Chemical Biology Graduate Program
3Life Sciences Institute
4Department of Biological Chemistry
University of Michigan, Ann Arbor, MI 48109, USA
5These authors contributed equally to this work
*Correspondence: reynoldk@pdx.edu
http://dx.doi.org/10.1016/j.chembiol.2013.04.014
SUMMARY
extender unit (typically malonyl-, methylmalonyl-, and ethylma-
lonyl-coenzyme A [CoA]) give rise to structurally diverse and
The formation of an activated cis-3-cyclohexylprope- complex polyketides, which are notable for their diverse phar-
maceutical applications.
noic acid by Plm1, the first extension module of the
The KR domain catalyzes the nicotinamide adenine dinucleo-
tide phosphate (NADPH)-dependent reduction of a b-keto group
phoslactomycin polyketide synthase, is proposed
to occur through an L-3-hydroxyacyl-intermediate
as a result of ketoreduction by an A-type ketoreduc-
tase (KR). Here, we demonstrate that the KR domain
of Plm1 (PlmKR1) catalyzes the formation of an L-3-
hydroxyacyl product. The crystal structure of
to give a hydroxyl group. KRs are classified as A- or B-type,
depending upon the stereochemistry of ketoreduction. In addi-
tion, some KRs catalyze epimerization of an a-substituent and
thus can be further subclassified. Distinct sequence motifs
have been correlated with known stereochemistry displayed by
PlmKR1 revealed a well-ordered active site with a
nearby Trp residue characteristic of A-type KRs.
Structural comparison of PlmKR1 with B-type
type I polyketide KR domains, allowing one to predict the stereo-
chemical outcome of ketoreduction by an uncharacterized KR
with the natural substrate. A-type KRs contain a conserved tryp-
KRs that produce D-3-hydroxyacyl intermediates tophan residue (‘‘W’’ motif) and generate an L-3-hydroxyacyl
intermediate, whereas B-type KRs contain a conserved Leu-
Asp-Asp (‘‘LDD’’ motif) sequence motif located near the active
site and generate D-3-hydroxyacyl intermediate (Caffrey, 2003;
revealed significant differences. The active site of
cofactor-bound A-type KRs is in a catalysis-ready
state, whereas cofactor-bound B-type KRs are in a
precatalytic state. Furthermore, the closed lid loop
in substrate-bound A-type KRs restricts active site
access from all but one direction, which is proposed
to control the stereochemistry of ketoreduction.
Keatinge-Clay, 2007; Keatinge-Clay and Stroud, 2006; Reid
et al., 2003; Valenzano et al., 2009).
Detailed studies of EryKR1 (B-type), EryKR2 (A-type), EryKR5
(A-type), and EryKR6 (B-type) from the erythromycin PKS
demonstrated that ketoreduction, which is analogous to fatty
acid biosynthesis, always involves the stereospecific transfer
of the 4-pro-S hydride from NADPH to the substrate (McPherson
et al., 1998; Yin et al., 2001). These findings suggest that cofactor
binding and hydride transfer are identical, regardless of KR type;
INTRODUCTION
Bacterial type I polyketide synthases (PKSs) are large, modular, thus, differences in the stereochemistry of ketoreduction
multifunctional proteins that catalyze the biosynthesis of various between the A- and B-type KRs must stem from the different
polyketides in a series of decarboxylative condensation reac- modes of substrate binding. One hypothesis is that the A-type
tions (Hopwood and Sherman, 1990; Khosla, 1997; Smith and and B-type KRs guide the substrate into the active site from
Tsai, 2007). Minimally, each module contains three domains opposite directions, thereby presenting different faces of the
required for the extension of the growing polyketide: an acyl car- carbonyl carbon of the b-keto to the NADPH, which would result
rier protein (ACP), an acyltransferase, and a ketosynthase. Mod- in opposite b-hydroxyl stereochemistries (Keatinge-Clay, 2007).
ules may also possess one or more b-carbon processing This is supported by the observation that the conserved A-type
domains, which include a ketoreductase (KR), a dehydratase (W) and B-type (LDD) sequence motifs are located on opposite
(DH), and an enoyl reductase that progressively modify a sides of the active site cleft (Keatinge-Clay, 2007). Additionally,
b-keto group to a hydroxyl, a double bond, and a single bond, previous studies suggest that substrate functional groups may
respectively. The number of extension modules within a PKS, help to orient the substrate to produce the desired stereochem-
the degree of b-carbon processing, the starter unit, and the istry (Zheng et al., 2010).
772 Chemistry & Biology 20, 772–783, June 20, 2013 ª2013 Elsevier Ltd All rights reserved

Chemistry & Biology
Structure and Stereochemical Analysis of PlmKR1
To date, crystal structures have been reported for five PKS hexylpropenoic acid diketide intermediate by Plm1 is thus pre-
KRs (two A-type and three B-type KRs) that catalyze ketoreduc- dicted to occur via an L-3-hydroxyacyl intermediate as a result
tion (Keatinge-Clay, 2007; Valenzano et al., 2009; Zheng et al., of ketoreduction by an A-type KR (Figure 1B).
2010, 2012) and for one KR that catalyzes epimerization of an
Here, we examine the stereochemical outcome and control of
a-methyl group but not ketoreduction (Zheng and Keatinge- ketoreduction by the KR domain from module 1 of the PLM PKS
Clay, 2011). A clear understanding of the mechanism by which (PlmKR1) at a biochemical and structural level. We successfully
A-type and B-type KRs produce opposite hydroxyl group stereo- cloned and purified PlmKR1 as an individual domain and carried
chemistries has yet to emerge from these structures.
out kinetic and stereochemical characterization studies using
Double bonds in polyketides are generally established by the SNAC thioester derivatives of the natural 3-ketocyclohexylpro-
successive action of the ketoreductase-dehydratase (KR-DH) pionic and 3-hydroxycyclohexylpropionic acid substrates. We
didomain. The DH removes the hydroxyl group generated by demonstrate that PlmKR1-catalyzed reduction yields an L-3-
the KR domain, producing an olefin with either a cis or trans hydroxyacyl product. Additionally, the crystal structure of
˚
configuration. Most unsaturated polyketides generated by PlmKR1 was solved to 1.68 A resolution and revealed structural
modular PKSs contain trans double bonds that are formed by differences between A-type and B-type KRs that may be impor-
DH dehydration of a D-3-hydroxyacyl intermediate produced tant in achieving stereochemical control.
by a B-type KR domain (Caffrey, 2003; Reid et al., 2003; Wu
et al., 2005). Double bonds of the cis configuration are less com- RESULTS AND DISCUSSION
mon but nonetheless occur in a number of polyketides and arise
through at least two mechanisms. In some cases, such as epo- Stereochemical Determination of the 3-Hydroxyl Group
thilone (Tang et al., 2004), borrelidin (Vergnolle et al., 2011), and The production of cis double bonds was hypothesized to occur
hypothemycin (Reeves et al., 2008) biosynthesis, cis double through the DH-catalyzed dehydration of the L-3-hydroxyacyl
bonds are formed by an enzyme-catalyzed isomerization of trans intermediate generated by the KR (Alhamadsheh et al., 2007),
double bonds. In other pathways, such as the phoslactomycin but this has not been experimentally verified. Experimental
(Alhamadsheh et al., 2007), rifamycin (Schupp et al., 1998), and validation came by demonstrating that the incubation of the
fostriecin (Kong et al., 2013) synthases, it has been postulated stand-alone PlmKR1 with 3-ketocyclohexylpropionyl-SNAC (2)
that cis double bonds arise from the DH-catalyzed dehydration resulted in the exclusive formation of the predicted L-3-hydrox-
of an L-3-hydroxyacyl intermediate, which is formed by an yacyl thioester product (4). as established by Mosher ester anal-
A-type KR domain (Reid et al., 2003). The correlation of an ysis. The L configuration at C-3 was deduced from the Dd values
A-type KR with DH-catalyzed cis double bond formation led to obtained by subtracting the chemical shift (dR) of each proton of
the postulate that the stereochemical outcome of the KR-cata- the (R)-methoxy(trifluoromethyl)phenylacetic acid (MTPA) ester
lyzed ketoreduction is key in establishing the configuration of (4a) from that (dS) of the (S)-MTPA ester (4b) (Figure S1A avail-
the double bond (Reid et al., 2003). Although no biochemical able online). In fact, all the protons lying on the left- and right-
evidence to date supports this hypothesis, it is consistent with hand sides of the plane containing the ester group of MTPA
the structures of DH domains, where the narrow active site is had negative and positive Dd values, respectively, as a conse-
better suited to binding an L-3-hydroxyacyl substrate in a pre- quence of the shielding effect of the Mosher’s acid phenyl ring.
cis conformation and a D-3-hydroxyacyl substrate in a pre-trans In the (R)-MTPA ester (4a), the phenyl (Ph) group and thioester
conformation (Akey et al., 2010; Keatinge-Clay, 2008).
moiety are in the same plane as shown in Figure S1B; thus Ph
Phoslactomycins (PLMs; Figure 1A) are phosphorylated poly- group shields the thioester moiety, and the resultant chemical
ketide natural products produced by Streptomyces sp. HK-803 shifts of the thioester moiety side protons appear in the upfield
and a number of other Streptomyces that exhibit antifungal, anti- region relative to (S)-MTPA ester (4b). Conversely, in the
tumor, and antiviral activity (Fushimi et al., 1989a, b). The unusual (S)-MTPA ester (4b), the Ph group and cyclohexyl moiety are in
architecture of PLMs is exemplified by the presence of three the same plane as shown in Figure S1B; thus, Ph group shields
cis (Z) (D14,15, D12,13, and D2,3) and one trans (E) (D6,7) double the cyclohexyl moiety, and the resultant chemical shifts of cyclo-
bonds (Alhamadsheh et al., 2007; Fushimi et al., 1989a, b; hexyl moiety side protons appear in upfield region relative to (4a).
Stampwala et al., 1983). Module 1 of the phoslactomycin PKS Since the natural diketide substrate of PlmKR1, 3-ketocyclohex-
(Plm1) contains the loading domain and the first extension mod- ylpropionic acid as its SNAC thioester (2), was used in this study,
ule and is responsible for the generation of the D14,15 cis double we do not expect the observed stereochemical outcome to differ
bond of PLM. In previous studies, a Dplm1 deletion mutant was if examined in a modular context or for a substrate tethered to the
constructed and shown to be unable to produce PLM (Alhamad- appropriate ACP. Thus, the PlmKR1 domain reduces the 3-keto-
sheh et al., 2007). However, when Dplm1 was grown in the pres- cyclohexylpropionyl substrate to the L-3-hydroxycyclohexyl-
ence of cis-3-cyclohexylpropenoic N-acetylcysteamine (SNAC) prionyl product, as predicted. In order to generate the 3-L
thioester, production of PLM was restored. Conversely, the trans isomer, PlmKR1 must catalyze the transfer of the 4-pro-S
isomer failed to restore PLM production to the Dplm1 mutant hydride from NADPH to the si face at C3 of the substrate
(Alhamadsheh et al., 2007). These findings demonstrate that (McPherson et al., 1998; Yin et al., 2001).
the activated cis-3-cyclohexylpropenoic acid is the diketide
intermediate passed from Plm1 to Plm2. Therefore, the stereo- Enzyme Activity
chemistry is directly established by the DH-KR didomain of The results stemming from the analysis of PlmKR1 activity with
Plm1 and not from a subsequent isomerization step (Alhamad- (1), (2), and (4) (obtained from the PlmKR1 catalyzed conver-
sheh et al., 2007). The generation of the proposed cis-3-cyclo- sion of (2) and stereoconfiguration confirmed by Mosher ester
Chemistry & Biology 20, 772–783, June 20, 2013 ª2013 Elsevier Ltd All rights reserved 773

Chemistry & Biology
Structure and Stereochemical Analysis of PlmKR1
Figure 1. General Structure of PLMs and Proposed Reaction Catalyzed by the Plm1 KR-DH Didomain
(A) General structure of PLMs.
(B) Proposed reaction catalyzed by the Plm1 KR-DH didomain.
(C) Synthetic approaches to generate substrates for PlmKR1.
See also Figures S1 and S5–S7.
analysis) are summarized in Table 1. Substrate solubility issues slower, with a calculated kcat/Km of 0.23
0.03 minꢀ1mMꢀ1
prevented the use of substrate concentrations well above Km. when using DL-3-hydroxycyclohexylpropionyl-SNAC (1) as
However, approximate kcat/Km values were obtained from the a substrate. When L-3-hydroxycyclohexylpropionyl-SNAC (4)
linear fit of the limiting case of the Michaelis-Menten model was incubated with PlmKR1, an approximate
(v = (kcat/Km)[S][E]o) for low substrate concentration (where 0.53
[S] << Km).
kcat/Km of
0.01 minꢀ1mMꢀ1 was obtained, which is ꢁ2.3-fold
higher than that for 1. When the CoA derivative of DL-3-hydrox-
Incubating PlmKR1 with 3-ketoacyl-SNAC thioester (2) pro- ycyclohexylpropionyl (3) was analyzed as a substrate, very
vided an approximate kcat/Km of 10.1 0.3 minꢀ1mMꢀ1. The little activity (<1% compared to the SNAC derivative) was
reaction was reversible, but the reverse direction proceeded observed.
774 Chemistry & Biology 20, 772–783, June 20, 2013 ª2013 Elsevier Ltd All rights reserved

Chemistry & Biology
Structure and Stereochemical Analysis of PlmKR1
Table 1. Kinetic Parameters of PlmKR1
Table 2. Crystallographic Data and Refinement Statistics
Substrate
kcat/Km (minꢀ1mMꢀ1 a,b
)
Plm1 KR
DL-3-Hydroxycyclohexylpropionyl-SNAC (1) 0.23 0.03
Data Collection
3-Ketocyclohexylpropionyl-SNAC (2)
10.1 0.3
Space group
P21
L-3-Hydroxycyclohexylpropionyl-SNAC (4)
0.53 0.01
Cell Dimensions
˚
a,b,c (A)
a, b, g (o)
See Figure 1 for structures of (1), (2), and (4). See also Figures S2 and S3.
aSolubility issues prevented the use of substrate concentrations well
above Km. However, approximate kcat/Km values were obtained from
the linear fit of the limiting case of the Michaelis-Menten model (v =
(kcat/Km)[S][E]o) for low substrate concentration (where [S] << Km). The
approximate kcat/Km values were calculated from the average of mini-
mally three sets of triplicate.
45.2, 123.4, 79.9
90, 90.27, 90
APS 23ID-B
1.033
1.68 (1.74–1.68)a
0.077 (0.457)
23.5 (3.9)
X-ray source
˚
Wavelength (A)
˚
dmin (A)
Rsymm (%)
bData reported as mean SD.
ave I/sI
Completeness (%)
Average redundancy
Refinementb
96.9 (94.3)
4.6 (4.8)
The 3-hydroxyacyl thioester (1) was prepared as a racemic
mixture, and it is unclear if the D-isomer functions as a substrate
for PlmKR1. The greater kinetic efficiency using the L-isomer (4)
suggests the D-isomer is either not a substrate or a very poor
one. Comparing the extent of nicotinamide adenine dinucleotide
phosphate (NADP)+ conversion by PlmKR1 under limiting sub-
strate (either [4] or [1]) led to the same conclusion. If PlmKR1
only accepts the L-isomer (4) as a substrate, then approximately
twice as much NADP+ conversion should be observed with the
L-isomer as with the same concentration as the presumed
racemic mixture. In the reductive direction using limiting (2)
(0.1 mM) and a 2-fold excess of NADPH (0.2 mM), the reaction
went to completion (Figure S2A). It is possible to obtain reversal
of this reaction by using either (1) or (4) (0.25 mM) with a much
larger excess (12-fold) of NADP+ (2 mM). Under these conditions,
˚
Data range (A)
50–1.68
85,333
0.16/0.19
2
No. reflections
Rwork/Rfree
Polypeptide chains
Number of Atoms
Protein
6,479
1,008
96
Water
Ligand
Rmsds
˚
Bond length (A)
0.012
1.4
˚
Bond angle (A)
2
˚
B-Factors (A )
approximately 21.0
0.5 mM of NADPH was formed when
Protein
13.4
26
PlmKR1 was incubated with L-isomer (4), whereas only 13.1
0.5 mM NADPH was generated when the racemic mixture (1)
was utilized as a substrate (Figure S2B).
Water
Ligand
9.34
Confirmation of the PlmKR1 reaction products in the reductive
and oxidative direction was achieved by liquid chromatography-
mass spectrometry (LC-MS) analysis (Figure S3). Incubating
PlmKR1 in the presence of (2) (broad m/z peak eluting at
20.5 min) (Figures S3B and S3C) and NADPH resulted in the for-
mation of an ion that had the same m/z (274, [M+H]+ and 296,
[M+Na]+) and retention time (19.5 min) as (1) (Figures S3F and
S3G). The reaction appeared to go to completion under the con-
Ramachandran Plot
Allowed (%)
Outliers (%)
aValues in parentheses pertain to the outermost shell of data.
bThe final structure is deposited in the PDB with accession code 4HXY.
100
0
The PlmKR1 contains two domains: an N-terminal structural
ditions implemented (Figure S3E). Conversely, incubating domain (KRstruc) and a C-terminal catalytic domain (KRcat) (Fig-
PlmKR1 with (1) resulted in <20% conversion of the substrate ures 2A and 2B). Both domains have the familiar Rossmann
to a single product that had the same m/z (272, [M+H]+ and fold common to the short-chain dehydrogenase/reductase
294, [M+Na]+) and retention time as (2) (data not shown).
enzymes (SDR). However, the structural domain contains a trun-
cated Rossmann fold and other features that prevent NADPH
binding, consistent with its presumed role to stabilize the cata-
Structure of PlmKR1
The excised KR domain used for crystallization comprised resi- lytic domain. The lengths of PKS KRstruc domains vary. KRstruc
dues 1814–2248 of Plm1. The elution profile from a gel filtration of PlmKR1 and the second-module KR from the spinosyn
column indicated that the excised KR domain was monomeric. PKS (SpnKR2) are significantly shorter than the structural
˚
The crystal structure was determined to 1.68 A using molecular domains in previously reported KR crystal structures (Figure 3;
replacement with the B-type KR from the first module of the Zheng et al., 2012). Specifically, when compared with EryKR1,
erythromycin PKS (EryKR1) (Keatinge-Clay and Stroud, 2006) PlmKR1 KRstruc lacks a b strand and an a helix and contains
as a search model. The binary complex of PlmKR1 with NADP+ two truncated b strands and two truncated a helices (Figures 3
crystallized in space group P21, with two polypeptide chains in and S4).
the asymmetric unit (Table 2). The final crystallographic model
of PlmKR1 was complete, excepting a few residues: chain A res- NADP+ cofactor (Figure 2B). Clear density was observed for
idues 14–16 and 147–153 and chain B residues 147–152.
the entire NADP+ cofactor, which forms multiple contacts with
The KRcat domain contains a Rossmann fold with a bound
Chemistry & Biology 20, 772–783, June 20, 2013 ª2013 Elsevier Ltd All rights reserved 775

Chemistry & Biology
Structure and Stereochemical Analysis of PlmKR1
the protein. Specifically, Asp246 hydrogen bonds to the adenine
N6 amine. Arg218 and Gln219 hydrogen bond to the adenosine
20-phosphate. The backbone amides of Gly195 (of the
conserved GXGXXG dinucleotide binding motif), Leu196, and
Met366 form hydrogen bonds to the diphosphate. Catalytic
Lys297 and Tyr332 hydrogen bond to the nicotinamide ribose
hydroxyl groups. The backbone amide of Trp361 is hydrogen
bonded to the nicotinamide carbonyl. Finally, the nictinamide
amide forms a water-mediated hydrogen bond to the backbone
carbonyls of Trp361 and Gly364.
Active Site Structure
All features of the PlmKR1 active site are well defined by elec-
tron density and well ordered. The active site is comprised of
the NADP+ cofactor and the conserved catalytic residues
Tyr332, Ser319, and Lys297. The three amino acids are
common to all PKS KRs and to members of the larger SDR
family (Oppermann et al., 2003). According to the widely
accepted model for SDR catalysis (Oppermann et al., 2003),
NADPH transfers a hydride ion to the substrate carbonyl car-
bon atom and the catalytic Tyr hydroxy donates a proton to
the carbonyl oxygen atom. The Lys amino group is thought to
assist catalysis through a hydrogen bond donor-acceptor
network that orients the Tyr hydroxy proton toward the sub-
strate and also stabilizes the resulting Tyr hydroxylate. The
PlmKR1 active site is structured as expected for this mecha-
nism. The Lys amino group donates a proton to the nicotin-
amide ribose 20-hydroxy. The ribose 20-hydroxy donates a
proton to the Tyr hydroxy, thereby directing the Tyr hydroxy
proton toward the substrate site (Figures 2C, 4A, and 4B).
The same Lys-ribose-Tyr hydrogen-bonding network occurs
in cofactor-substrate ternary complexes of well-studied SDR
enzymes, such as UDP-galactose 4-epimerase and is thought
to be essential to catalysis (Thoden et al., 1997). The active
site Ser hydroxy may orient the substrate for catalysis (Opper-
mann et al., 2003).
In PKS KRs, a mobile helix and loop adjacent to the cofactor
nicotinamide, the ‘‘lid helix’’ and ‘‘lid loop,’’ move upon cofactor
and/or substrate binding (Keatinge-Clay, 2007; Keatinge-Clay
and Stroud, 2006; Zheng et al., 2010). In PlmKR1, the lid helix
(residues 369–378) and lid loop (residues 361–368) are well
ordered and adjacent to the nicotinamide ring (Figures 2A, 5A,
and 5B).
Figure 2. Structure of PlmKR1
Substrate Binding Site
(A) Trace of the polypeptide chain from N (blue) to C terminus (red). The mobile
lid helix and lid loop of the catalytic domain are labeled in the stereo image. The
NADP+ cofactor is shown as sticks with atomic coloring (blue N, red O, yellow
S, and orange P) and green C.
(B) PlmKR1 active site and NADP+ density. Side chains of the catalytic resi-
dues (Tyr 332, Ser319, and Lys297) are shown as sticks with magenta C. The
NADP+ cofactor is shown as in (A) with the 2Fo-Fc electron density contoured
at 1 s. The residues that interact with NADP+ are shown as sticks with gray C.
The conserved Trp332 of A-type KRs is shown with light red C. The ribbon
diagram of the polypeptide has an orange structural domain and cyan catalytic
domain.
Electron density between the nicotinamide ring and catalytic
Tyr332 in one of the PlmKR1 molecules in the asymmetric unit
was interpreted as a 6-aminohexanoic acid additive to the crys-
tallization solution (Figure 2C). The carboxylate carbon of 6-ami-
nohexanoic acid is positioned above C4 of the nicotinamide ring,
and a carboxylate oxygen is hydrogen bonded to active site
Tyr332 and Ser319. Presumably 6-aminohexanoate is not a
PlmKR1 substrate, because hydride transfer to the carboxylate
anion is disfavored. The active site of AmpKR2, another A-type
KR, also contains a carboxylic acid ligand, malic acid (Zheng
et al., 2010). In both PlmKR1 and AmpKR2, the ligand carbox-
ylate is bound between the NADPH nicotinamide and the cata-
lytic Tyr, with hydrogen bonds to Tyr and Ser side chains as
expected for KR substrates.
(C) Density for 6-aminohexanoic acid. The 2Fo-Fc electron density for the
6-aminohexanoic acid is shown contoured at 1 s. The 6-aminohexanoic acid is
shown as sticks with gray C. Hydrogen bonds between active site residues,
NADP+ nicotinamide ribose, and 6-aminohexanoic acid are shown in red. The
catalytic residues are shown and colored as in (B). The NADPH cofactor is
shown as in (A).
776 Chemistry & Biology 20, 772–783, June 20, 2013 ª2013 Elsevier Ltd All rights reserved

Chemistry & Biology
Structure and Stereochemical Analysis of PlmKR1
Stereochemical Control in A-type and B-type KRs
hydrogen bonded to the lid loop through the Trp361 amide. A
Conserved sequence motifs in each type of PKS KR, the W motif well-ordered water molecule further organizes the lid loop
for A-type and the LDD motif for B-type, are strongly correlated through hydrogen bonds with the Trp361 carbonyl, the nicotin-
with stereochemical outcome of the KR reaction (Caffrey, 2003; amide amide, and the Gly364 carbonyl (Figures 4A and 4B).
Reid et al., 2003). These motifs are proposed to control, by an Finally, the Met366 side chain extends into the active site toward
unknown mechanism, the direction of entry of the phosphopan- the nicotinamide ring and catalytic Tyr332 (Figures 5A and 5B).
tetheinylated substrate into the active site and thereby control These protein-cofactor contacts were observed in PlmKR1,
which face of the b-ketone is accessible to the NADPH cofactor AmpKR2, and AmpKR2 mutants (3MJT and 3MJV), in which sub-
for hydride transfer (Keatinge-Clay, 2007). The W motif (Trp324) strate mimics or unmodeled electron density were in the pre-
in the PlmKR1 sequence (Figure 3) and the exclusive formation of sumed substrate site.
the L-3-hydroxyacyl product demonstrate that PlmKR1 is an
For A-type KR structures with no evidence of binding in the
A-type KR. However, the position of Trp324 in the PlmKR1 struc- substrate site (PlmKR1 molecule B, AmpKR2 [PDB 3MJE], and
ture provides no insights into its role in substrate binding or the AmpKR11 [PDB 4DI7]), the lid loop and helix have weaker den-
stereospecificity of ketoreduction. Furthermore, mutagenesis sity or are disordered. Nevertheless, even with an unoccupied
of the A- and B-type sequence motifs in AmpKR2 (Zheng substrate site, A-type KR active sites have a catalysis-ready
et al., 2010) and in a full module from the erythromycin PKS Lys–20-O-ribose-Tyr hydrogen-bonding network.
(Kwan et al., 2011) demonstrated that the motifs are not the
Based on our detailed structure comparison, the active site of
sole determinants of hydroxy group stereochemistry. Therefore, A-type KRs bind cofactor with a Lys–20-O-ribose-Tyr hydrogen-
to identify other structural features that control stereochemical bonding network that is ready for catalysis. In most of these
outcome, we compared PlmKR1 to the four previously reported structures, the lid loop is closed, extending Met366 into the
Type 1 PKS KR structures. The comparison included three A- active site. The Met side chain blocks access to the nicotinamide
type KRs, PlmKR1, AmpKR2, (Zheng et al., 2010), and AmpKR11 and catalytic Tyr from one direction, which we designate ‘‘north-
(Protein Data Bank [PDB] 4DI7), and three B-type KRs, EryKR1 west,’’ while a ‘‘southeast’’ entry is accessible (Figure 5A). Previ-
(Valenzano et al., 2009), TylKR1 (Keatinge-Clay, 2007), and ous studies of A-type KRs demonstrated that this methionine
SpnKR2 (Zheng et al., 2012). Structures with bound NADP(H) is essential for catalysis, as a mutation to alanine abolishes
cofactor are available for all but TylKR1, and views of the KR activity (Zheng et al., 2010). We modeled the PlmKR1 substrate
both with and without cofactor are reported for AmpKR2 and Er- in the active site based on the positions of 6-aminohexanoate in
yKR1. In addition, structures of A-type KRs were reported with a the PlmKR1 crystal structure (Figure 2C) and malate in an
substrate mimic bound (AmpKR2 and PlmKR1) and without a AmpKR2 structure (Zheng et al., 2010; Figure 5B). A carboxyl
substrate mimic bound (AmpKR2 and AmpKR11).
oxygen of these ligands is hydrogen bonded to the catalytic
The most striking difference between A-type and B-type KR Tyr and the carboxyl carbon is near the NADPH hydride position,
structures is the ordering of the active site. The nicotinamide as expected for KR substrates. Substrate entry from the
ribose in A-type KRs is bound in a well ordered and catalysis- southeast places the appropriate face of the b-ketone toward
ready conformation with
a
Lys–20-O-ribose-Tyr hydrogen- the nicotinamide for A-type ketoreduction (Figure 5B). The Trp
bonding network and a ribose C20-endo pucker (Figures 2C, side-chain nitrogen of the W motif in A-type KRs points into
4A, and 4B). The hydrogen bond network does not exist in the southeast entry channel, where it may help orient the
cofactor-bound B-type KRs, where the ribose is poorly ordered substrate phosphopantetheine by hydrogen bonding.
(EryKR1) or is in a position with the 20 hydroxy directed away
The available structural data indicate that cofactor binds
from the catalytic Tyr (SpnKR2). We examined electron densities loosely to B-type KRs. For example, the TylKR1 crystal structure
for ribose in the B-type KR crystal structures to determine has no electron density for the cofactor that was present in crys-
whether the ribose could be rebuilt to form the hydrogen-bonding tallization solutions (Keatinge-Clay, 2007). The nicotinamide and
network, but this was not compatible with the electron density. ribose are poorly ordered in the EryKR1 structure (Keatinge-
Additionally, in B-type KRs with bound cofactor, the catalytic Clay and Stroud, 2006), and the lid loop is open so that substrates
˚
Tyr is shifted ꢁ2 A away from the catalysis-ready position could access the active site from both the northwest and south-
in A-type KRs (Figure 4A). The precatalytic position of the Tyr in east(Figure5A). InSpnKR2, thelidloopandhelixopen completely
cofactor-bound B-type KRs is identical to the Tyr position in to form a ‘‘domain swap’’ with another molecule in the crystal lat-
cofactor-free structures of both A-type and B-type KRs (AmpKR2 tice (Zhenget al., 2012). As in the A-type KRs, substrate binding to
3MJC, TylKR1). Thus, cofactor-bound A-type KRs have a fully B-type KRs is expected to assist in active-site formation by
formed active site, whereas cofactor-bound B-type KRs do not. ordering the lid loop and helix. However, in contrast to A-type
In A-type KRs with a substrate mimic in the active site, PlmKR1 KRs, the catalytic Lys-20-O-ribose-Tyr hydrogen-bonding
and AmpKR2 (PDB 4HXY and 3MJS), the lid helix is well ordered network in B-type KRs appears to form only after substrate bind-
and the lid loop closes over the bound cofactor and forms an ing. We conclude that productive binding and substrate-assisted
intricate set of hydrogen bonds with the b-phosphate and nico- active-site formation occur only when the substrate enters from
tinamide base (Figures 4A and 4B). These specific contacts the northwest direction, placing the appropriate face of the
position the lid loop close to the phosphate-binding loop (amino b-ketone toward the nicotinamide for B-type ketoreduction. The
˚
acids 194–196 in PlmKR1), with conserved Gly365 only 4 A from LDD motif of B-type KRs is near the catalytic Tyr (Figure 5A) and
Gly195. The nicotinamide amide forms an intramolecular may contribute to substrate-assisted assembly of the active site.
hydrogen bond with b-phosphate, which is also hydrogen Our analysis of the structures provides an explanation of how
bonded with the Met366 amide. The nicotinamide carbonyl is the A-type KRs restrict the direction of access to the active site
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Chemistry & Biology
Structure and Stereochemical Analysis of PlmKR1
Figure 3. Structure-Based Sequence Alignment
Structure-based multiple sequence alignment of PlmKR1 (accession AAQ84156), AmpKR2 (37% sequence identity to PlmKR1, accession AAK73513), SpnKR2
(32% sequence identity to PlmKR1, accession AAG23265), EryKR1 (35% identity to PlmKR1, accession AAB84070), and TylKR1 (41% identity to PlmKR1,
accession AAB66504). The red box indicates the location of b strand and a helix in EryKR1, which PlmKR1 lacks. Additionally, dotted lines in secondary structure
(legend continued on next page)
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Chemistry & Biology
Structure and Stereochemical Analysis of PlmKR1
Figure 4. Comparison of A-type and
B-type KRs
(A) Stereo view of PlmKR1 (cyan) and SpnKR2
superposition (white, PDB 3SLK) (root-mean-square
˚
deviation [rmsd] = 1.187 A). The active site tyrosine
and the NADP(H) are in different positions in the
A-type (PlmKR1, cyan) and B-type (SpnKR2, white)
KRs. In PlmKR1, the hydrogen bond network of
Lys297, ribose 20-hydroxy, and Tyr332 is shown as
red dashes, and the hydrogen bond network of
nicotinamide amide, lid loop, and NADP+ b-phos-
phate as yellow dashes. The PlmKR1 catalytic resi-
dues are drawn in stick form with magenta C and the
NADPH cofactor with green C, and the EryKR1
NADPH was omitted because of disorder.
(B) Schematic of the hydrogen bond network of
NADP+ bound to the A-type PlmKR1. In cofactor
complexes of B-type KRs, only the hydrogen bonds
of Lys to ribose 20-hydroxy and of nicotinamide amide
to NADP(H) b-phosphate have been observed.
Our interpretation of the structures is in
agreement with the direction-of-access hy-
pothesis proposed by Keatinge-Clay
(2007). As different conformations of the
enzyme control the direction of access, the
mechanism of stereochemical control must
also include aspects of protein dynamics.
The lid loop likely populates several confor-
mations with or without bound cofactor. As
each crystal structure captures one or a
few well-populated states, we conclude
that the loop-closed
conformations
captured in the crystal structures of PlmKR1
and AmpKR2 predominate in cofactor-
bound A-type KRs. However, experimental
factors, such as introduction of nonnatural
substrates or site-directed mutagenesis,
through progressive ordering of the lid loop and helix upon can alter the distribution of conformations within the population
cofactor and substrate binding, thereby controlling the stereo- and, thus, the stereochemical outcome (Zheng et al., 2010).
chemical outcome of ketoreduction. The W motif appears to
play no role in facilitating formation of an ordered lid loop, nor SIGNIFICANCE
the LDD motif in preventing its formation. The W motif in
˚
A-type KRs is too far (10 A) from the active site to have a direct Collectively, the results presented here demonstrate that
effect (Figure 5). The LDD motif of B-type KRs is near enough, PlmKR1 is an A-type KR that catalyzes the stereospecific
but its position does not directly block a closed-lid loop. In reduction of the b-keto intermediate to generate an
fact, the side chains of the lid-loop Met and the LDD Leu could L-configured alcohol. Such findings are consistent with the
interact if the lid loop were to close in B-type KRs. The analogous hypothesis that cis double bonds arise from the syn dehy-
hydrophobic contact exists in the A-type PlmKR1 between dration of L-3-hydroxyacyl intermediates by the DH domain.
Met366 and Val277 (analogous to the LDD Leu) (Figure 5B). Furthermore, detailed structural analysis of A-type and
Thus, like other authors (Kwan et al., 2011; Zheng et al., 2010), B-type KRs demonstrates that formation of a catalysis-
we conclude that KR stereochemical control does not arise ready active site is cofactor-assisted in the A-type KRs
solely from the W and LDD motifs.
and apparently substrate-assisted in the B-type KRs.
elements indicate truncations of these elements in PlmKR1 when compared to the other KRs. The orange line indicates the end of the structural domain and start
of the catalytic domain. Green boxes indicate the location of the W motif for A-type KRs (PlmKR1 and AmpKR2) and LDD motif for (B-type KRs). Red stars indicate
the location of the active site residues. The sequence alignment was generated with MUSCLE (Edgar, 2004), and ESPript (Gouet et al., 1999) was used to generate
secondary structure annotations.
See also Figure S4.
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Chemistry & Biology
Structure and Stereochemical Analysis of PlmKR1
Figure 5. Substrate Entry into A-type and
B-type KRs
(A) Active site channel of A-type PlmKR1 (left) and
B-type EryKR1 (right). The surfaces of the catalytic
domains are cyan, with blue lid helices, light blue lid
loops, and magenta active site residues. The
cofactors are shown in stick form with white C. With
NADP+ and substrate bound, the A-type PlmKR1
active site is ordered and is accessible from the
southeast direction, where the W motif (light red
surface) is located. The Met366 side chain (light red
surface) blocks access to the active site from the
northwest direction. With NADP+ bound, the B-type
EryKR1 active site is open, with access from either
direction. The surface of the LDD motif is shown in
yellow.
(B) Model of substrate binding to PlmKR1. A 3-
ketocyclohexylpropionyl thioester (white C) was
modeled by hand into the A-type PlmKR1 active
site based on the position of 6-aminohexanoic acid
in the crystal structure. The thioester sulfur
indicates the phosphopantetheinyl-ACP attach-
ment site. The Met 366 side chain (light red C)
blocks access from the northwest direction.
Hydrophobic amino acids (Val 277 and Leu 367)
in the proposed cyclohexane site are shown in
cyan. The hydrogen-bonding network between
active site residues (Lys, Tyr, and magenta C) are
shown as red dashes. NADP+ is shown with green
C and the W motif Trp324 with light red C. Other
coloring as in (A).
Synthesis of 3-Hydroxy- and 3-Ketocyclohexylpropionyl SNAC
and CoA Thioesters
Closing of the lid loop upon cofactor and substrate binding
in the A-type KRs restricts substrate access to the active
site to a direction that is compatible with the observed ste-
reochemical outcome of ketoreduction. These results pro-
vide additional information to the understanding of stereo-
chemical control in PKS KRs.
General
The synthetic strategies used in the generation of SNAC thioesters of 3-keto-
cyclohexylpropionic acid (2), DL-3-hydroxycyclohexylpropionic acid (1), and
DL-3-hydroxycyclohexylpropionl-CoA (3) are based on methods described
elsewhere (Bonnett et al., 2011; Eusta´ quio et al., 2009; Figure 1C). Reagents
were purchased from Aldrich and used without further purification. All reac-
tions were carried out under argon/nitrogen atmosphere using dry solvents
under anhydrous conditions unless otherwise noted. Reactions were moni-
tored by thin layer chromatography (TLC) carried out on 0.25 mm silica gel
(60, F-254) plates using the following visualization techniques: UV light, iodine
vapor and/or coated with 10% H2SO4 in MeOH, and subsequent charring on
hot plate. Chromatography was executed with silica gel (230–400 mesh) using
mixtures of ethyl acetate and n-hexane as eluents. 1H NMR spectra were
recorded on Bruker AMX-400 MHz spectrometer in CDCl3; 13C NMR spectra
were recorded at 100 MHz in CDCl3. Chemical shifts are reported as values
in ppm relative to CHCl3 (7.26) in CDCl3, and trimethylsilane was used as inter-
nal standard.
EXPERIMENTAL PROCEDURES
Protein Expression and Purification
The generation of the expression construct for the stand-alone KR domain,
pPlm1KR, is discussed in the Supplemental Information. Plasmid pPlm1KR
was used to transform E. coli BL21(DE3) cells and the transformant was
cultured at 37ꢂC to A600 = 0.8–1 in 0.5 l of Terrific Broth media containing
0.1 mg/ml ampicillin. Cultures were incubated for 1 hr at 20ꢂC, induced with
0.2 mM isopropyl b-D-1-thiogalactopyranoside, and allowed to express for
approximately 18 hr. Cells were harvested by centrifugation at 5,670 3 g for
25 min at 4ꢂC and stored at ꢀ80ꢂC.
The recombinant His6-tagged PlmKR1 fusion protein was purified by His
trap column outlined in the manufacturer’s manual (GE Healthcare). To
remove the His6-tag, fractions containing the recombinant protein were
pooled and incubated for 4 hr at room temperature with TEV protease
(30:1 molar ratio of protein to protease) and 2 mM dithiothreitol. After
dialyzing overnight in buffer A, the cleavage reaction was loaded onto a
His trap column to separate untagged PlmKR1 from His6-tagged TEV prote-
ase. The untagged PlmKR1 was collected in the flow-through and was puri-
fied further by size exclusion chromatography (HiPrep 16/60 Sephacryl S200
HR equilibrated with 50 mM Tris pH 7.5, 300 mM NaCl, 10% glycerol). The
resulting protein was >90% pure, based on SDS-PAGE analysis (data not
shown), and concentrated to 10 mg/ml, flash frozen in liquid nitrogen, and
stored at ꢀ80ꢂC.
Synthesis of Methyl-3-Ketocyclohexylpropionate
Methyl acetate (10.1 g, 136.9 mmol) was added to a stirred solution of lithium
diisopropylamide (76 ml of 1.8
M solution in tetrahydrofuran [THF],
136.9 mmol) in THF (60 ml) at ꢀ78ꢂC. The solution was stirred at ꢀ78ꢂC for
15 min, after which cyclohexanecarbonyl chloride (I, 10.0 g, 68.5 mmol) was
added. The reaction was allowed to warm to room temperature, diluted with
1N HCl, and extracted with ethyl acetate (3 3 100 ml). The combined
organic extracts were dried over anhydrous Na2SO4, and the solvents were
removed under reduced pressure. The crude product was purified by
silica gel column chromatography (10% EtOAc/n-hexane) to give methyl-3-
ketocyclohexylproprionate (II) (10.5 g, 83%). 1H NMR (CDCl3, 400 MHz)
d 3.72 (s, 3H), 3.50 (s, 2H), 2.46 (m, 1H), 1.89–1.65 (m, 5H), 1.39–1.18 (m, 5H).
780 Chemistry & Biology 20, 772–783, June 20, 2013 ª2013 Elsevier Ltd All rights reserved

Chemistry & Biology
Structure and Stereochemical Analysis of PlmKR1
Synthesis of Methyl-3-Hydroxycyclohexylpropionate
stirred at the same temperature for an additional 45 min. The stirring was
then stopped to allow the resulting solid to precipitate. The clear supernatant
was added slowly to a solution of hydrated coenzyme A (18 mg, 0.024 mmol)
and NaHCO3 (12 mg, 0.145 mmol) in dd H2O (2 ml). The reaction mixture was
stirred at room temperature for 4 hr, after which the THF was removed under
reduced pressure. The aqueous solution was acidified to pH 3 using 1N HCl
and then extracted with ethyl acetate (3 3 5 ml) to remove excess compound
IV. The aqueous solution was then lyophilized, and the resulting solid was
washed with MeOH to recover the 3-hydroxycyclohexylpropionoyl CoA thio-
ester (3) (11 mg) as a white solid. 1H NMR (CD3OD, 400 MHz) d 8.67 (s, 1H),
8.39 (s, 1H), 6.10 (br s, 1H), 4.75 (s, 1H), 4.49 (s, 1H), 4.29 (m, 2H), 4.03
(m, 3H), 3.82 (br s, 1H), 3.65 (m, 3H), 3.47 (m, 4H), 2.98 (m, 2H), 2.72
(m, 1H), 2.63 (m, 1H), 2.44 (m, 2H), 1.89–1.65 (m, 5H), 1.45–0.99 (m, 6H),
1.01 (s, 3H), 0.90 (s. 3H).
To a stirred solution of II (5.0 g, 27.2 mmol) in anhydrous methanol (15 ml) at
0ꢂC was added slowly NaBH4 (1.0 g, 27.2 mmol), and the reaction mixture
was stirred at 0ꢂC for 30 min. The resulting solution was quenched with 1N
HCl, extracted with ethyl acetate (3 3 100 ml), and the combined extracts
were dried over anhydrous Na2SO4 and concentrated under reduced pres-
sure. The crude product was separated by silica gel column chromatography
(30% EtOAc/n-hexane) to give methyl-3-hydroxycyclohexylpropionate
(III) (3.5 g, 69%). 1H NMR (CDCl3, 400 MHz) d 3.77 (m, 1H), 3.71 (s, 3H), 2.88
(d, J = 4.2 Hz, 1H), 2.53 (dd, J = 3.0, 16.2 Hz, 1H), 2.43 (dd, J = 9.5, 16.2 Hz,
1H), 1.89–1.64 (m, 5H), 1.37 (m, 1H), 1.26–0.99 (m, 5H).
Synthesis of 3-Hydroxycyclohexylpropionic Acid
To a stirred suspension of III (1.0 g, 5.37 mmol) in a mixture of THF (15 ml)
and water (20 ml) was added LiOH.H2O (0.86 mg, 21.5 mmol). The reaction
mixture was stirred at 60ꢂC for 6 hr, after which it was cooled to 0ꢂC and
carefully acidified to pH 2 to 3 with 1N HCl. The reaction mixture was
extracted with ethyl acetate (3 3 50 ml), and the combined extracts were
dried over anhydrous Na2SO4 and concentrated under reduced pressure.
The crude product was purified by silica gel column chromatography (60%
EtOAc/n-hexane) to give 3-hydroxycyclohexylpropionic acid (IV) (0.87 g,
94%). 1H NMR (CDCl3, 400 MHz) d 7.45 (br s, 1H), 3.81 (m, 1H), 2.55 (dd,
J = 3.0, 16.2 Hz, 1H), 2.45 (dd, J = 9.5, 16.2 Hz, 1H), 1.87–1.38 (m, 5H),
1.24 (m, 1H), 1.21–0.98 (m, 5H); 13C NMR (CDCl3, 100 MHz) d 177.9, 72.4,
43.0, 38.5, 28.7, 28.2, 26.3, 26.1, 25.9.
PlmKR1 Activity Assay
Activity assays for recombinant PlmKR1 were conducted spectrophotometri-
cally by monitoring the change in absorbance at 340 nm, due to the formation
or consumption of NADPH (ε340 = 6220 Mꢀ1cmꢀ1). Assays were performed in
the presence of 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10% glycerol, 1 mM
tris(2-carboxyethyl)phosphine hydrochloride, variable substrate (0–2 mM sus-
pended in DMSO to give a final concentration of 1%), and either 0.2 mM
NADPH or 1 mM NADP+ in a total volume of 0.5 ml. Reactions were initiated
by the addition of protein (1.5 or 7 mM) and allowed to proceed at 25ꢂC for
10 min, taking readings every 15 s. The approximation of kcat/Km values
were calculated, from the average of minimally three sets of triplicate, using
the limiting case of the Michaelis-Menten equation (v = (kcat/Km)[S][E]o) for
low substrate concentration (where [S] << Km).
Synthesis of 3-Hydroxycyclohexylpropionoyl N-Acetylcysteamine
Thioester
The extent of NADP(H) conversion was also monitored over the course of
2 hr. PlmKR1 (10 mM) was incubated in the presence of 0.25 mM (1) or (4)
and 2 mM NADP+ in the oxidative direction and 0.1 mM (2) and 0.2 mM NADPH
in the reductive direction.
A solution of carboxylic acid IV (650 mg, 3.8 mmol) in anhydrous CH2Cl2 (10 ml)
was cooled to 0ꢂC for 15 min. To this solution was added N-acetyl cysteamine
(540 mg, 4.53 mmol) followed by 4-(N,N-dimethylamino)pyridine (DMAP,
115 mg, 0.25 mmol), and N-(3-dimethylamino- propyl)-N0-ethylcarbodiimide
hydrochloride (866 mg, 4.53 mmol). The mixture was allowed to warm
to room temperature and stirred overnight. Saturated aqueous NH4Cl
solution (30 ml) was added, and the organic phase was separated. The
aqueous phase was extracted with diethyl ether (3 3 30 ml), and the combined
organic phases were dried over anhydrous Na2SO4 and concentrated under
reduced pressure. The residue was purified by silica gel column chromatog-
raphy (60% EtOAc/n-hexane) to give 3-hydroxycyclohexylpropionoyl N-ace-
tylcysteamine (SNAC) thioester (1) (940 mg, 91%). 1H NMR (CDCl3,
400 MHz) d 5.85 (br s, 1H), 3.83 (m, 1H), 3.44 (m, 2H), 3.05 (m, 2H), 2.75
(dd, J = 3.0, 15.4 Hz, 1H), 2.68 (dd, J = 9.4, 15.4 Hz, 1H), 2.56 (d, J = 4.2 Hz,
1H), 1.97 (s, 3H), 1.78–1.67 (m, 5H), 1.42 (m, 1H), 1.25–0.99 (m, 5H);
13C NMR (CDCl3, 100 MHz) d 200.1, 170.4, 72.9, 48.4, 43.3, 39.3, 28.9 (2C),
28.1, 26.3, 26.1, 26.0, 23.2.
LC-MS Analysis
Reactions were performed at 25ꢂC under the conditions described above in
the presence of 0.5 mM NADP(H), 0.1 mM of either (2) or (1), and 5 mM protein
in a total volume of 100 ml. Aliquots of 20 ml were taken at specific time points
and analyzed by LC-MS in the positive mode on a Bruker MicroTOF-Q mass
spectrometer equipped with an Agilent 1200 Series LC system using a Discov-
ery HS C18 (250 3 2.1, 5 micron, Supelco) column at a flow rate of 0.3 ml/min.
The following solvent gradient between buffer A (0.05% formic acid in water)
and buffer B (0.05% formic acid in acetonitrile) was used in the analysis:
(time, % buffer B): 0 min, 20%; 30 min, 80%; 35 min, 80%; 38 min, 20%;
and 45 min, 20%.
Stereochemical Determination of the 3-Hydroxyl Group
Synthesis of 3-Ketocyclohexylpropionoyl N-Acetylcysteamine
Thioester
PlmKR1 (0.5 mM) was incubated in the presence of 10 mM NADPH and 3 mM
(2) (ꢁ400 mg) in a final reaction volume of 500 ml. The reaction was monitored
at 235 nm by high-performance liquid chromatography (HPLC) on Discovery
HS C18 (250 3 4.6, 5 micron, Supelco) column at a flow rate of 1 ml/min using
the solvent system described above. After incubating for 6 hr, the reaction
appeared to have gone to completion, as determined by HPLC and LC-MS
analysis (data not shown). The resulting reaction product (4) was isolated by
ethyl acetate extraction (2 3 500 ml). Organic extracts containing the reaction
product were combined and dried under vacuo to obtain the desired product
(4) (375 mg, 94%). The absolute configuration at C-3 of (4) was elucidated by
applying the modified Mosher’s method (Dale and Mosher, 1973; Ohtani et al.,
1989).
To a stirred solution of (1) (200 mg, 0.73 mmol) in dry CH2Cl2 (15 ml) was added
pyridinium chlorochromate (236 mg, 1.1 mmol), and the mixture was stirred at
room temperature under argon atmosphere for 1 hr. Then, the reaction was
worked up by the addition of ether (20 ml), and the resulting mixture was
filtered through a silica gel pad and washed with ether (2 3 30 ml). Combined
organic phases were concentrated under reduced pressure, and the residue
was purified by silica gel column chromatography (50% EtOAc/n-hexanes)
to give 3-ketocyclohexylpropionoyl N-acetylcysteamine (SNAC) thioester (2)
(185 mg, 93%). Note: Compound (2) exists in keto-enol tautomerism. 1H
NMR (CDCl3, 400 MHz) d 12.65 (s, 0.35H-enol form), 6.24 (br s, 1H), 5.44 (s,
0.35H-enol form), 3.74 (s, 1.3H), 3.47 (m, 2H), 3.08 (m, 2H), 2.45 (m, 0.63H),
2.07 (m, 0.51H-enol form), 1.98 (s, 3H), 1.89–1.66 (m, 5H), 1.35 (m, 1H);
13C NMR (CDCl3, 100 MHz) d 205.4, 194.5, 192.6, 181.2, 170.6, 170.4, 97.3,
55.1, 51.3, 43.4, 39.9, 39.2, 29.9, 29.2, 28.1 (2C), 27.8, 27.7, 25.8, 25.7, 25.6
(3C), 25.4, 25.3, 23.2.
Synthesis of MTPA Esters of 4
The NMR spectra of (4) and synthetic scheme in the generation of MTPA esters
of (4) are shown in Figures S5–S7. To a stirred solution of (4) (4.0 mg) in CH2Cl2
(1 ml) was added DMAP (a spatula tip), and (S)-MTPA chloride (15 ml) and then
maintained at room temperature under stirring overnight. After removal of the
solvent, the reaction mixture was purified by preparative TLC (50% EtOAc/
3-Hydroxycyclohexylpropionoyl CoA Thioester
To a stirred solution of compound IV (25 mg, 0.145 mmol) in anhydrous THF
(5 ml) was added triethylamine (25 ml, 0.145 mmol) followed by ethyl chlorofor-
mate (15 ml, 0.145 mmol) under nitrogen at 0ꢂC. The reaction mixture was
n-hexane) to obtain the (R)-MTPA ester (4a, 3.0 mg) in a pure state.
Using (R)-MTPA chloride, the same procedure afforded the (S)-MTPA ester
(4b) in the same yield. (R)-MTPA ester (4a): 1H NMR (CDCl3, 400 MHz)
Chemistry & Biology 20, 772–783, June 20, 2013 ª2013 Elsevier Ltd All rights reserved 781

Chemistry & Biology
Structure and Stereochemical Analysis of PlmKR1
d 7.44 (m, 2H), 7.33 (m, 3H), 5.85 (br s, 1H), 5.29 (m, 1H), 3.42 (s, 3H), 3.3198
(m, 2H), 2.9242 (m, 2H), 2.8010 (dd, J = 8.5, 15.7 Hz, 1H), 2.7026 (dd, J = 3.6,
15.7 Hz, 1H), 1.87 (s, 3H), 1.6273 (m, 6H-overlaped), 1.0410 (m, 5H-overlaped);
(S)-MTPA ester (4b): 1H NMR (CDCl3, 400 MHz) d 7.50 (m, 2H), 7.33 (m, 3H),
5.86 (br s, 1H), 5.30 (m, 1H), 3.48 (s, 3H), 3.3401 (m, 2H), 2.9661 (m, 2H),
2.8363 (dd, J = 6.5, 15.5 Hz, 1H), 2.7502 (dd, J = 3.6, 15.5 Hz, 1H), 1.87
(s, 3H), 1.5534 (m, 6H-overlaped), 0.9585 (m, 5H-overlaped).
Bonnett, S.A., Papireddy, K., Higgins, S., del Cardayre, S., and Reynolds, K.A.
(2011). Functional characterization of an NADPH dependent 2-alkyl-3-ketoal-
kanoic acid reductase involved in olefin biosynthesis in Stenotrophomonas
maltophilia. Biochemistry 50, 9633–9640.
Caffrey, P. (2003). Conserved amino acid residues correlating with ketoreduc-
tase stereospecificity in modular polyketide synthases. ChemBioChem 4,
654–657.
Collaborative Computational Project, Number 4. (1994). The CCP4 suite: pro-
grams for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50,
760–763.
Crystallization
PlmKR1 was crystallized by vapor diffusion in 4 ml drops containing equal vol-
umes of protein stock (10 mg/ml in 20 mM Tris (pH 7.5), 100 mM NaCl, and
1 mM NADP+) and well solution (200 mM calcium acetate, 39% polyethylene
glycol 3350, 100 mM bis-tris propane (pH 6.5), and 4% 6-aminohexanoic
acid). Crystals grew within 48 hr at 4ꢂC and were harvested in loops, cryopro-
tected with the corresponding well solution containing 20% glycerol, and
flash-cooled in liquid nitrogen.
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95, 512–519.
Davis, I.W., Murray, L.W., Richardson, J.S., and Richardson, D.C. (2004).
MOLPROBITY: structure validation and all-atom contact analysis for nucleic
acids and their complexes. Nucleic Acids Res. 32(Web Server issue), W615–
W619.
Data Collection and Structure Determination
Data collected at GM/CA beamline 23ID-B at the Advanced Photon Source
(APS), Argonne National Laboratory (Argonne, IL) were processed using
HKL2000 (Otwinowski and Minor, 1997). Phases for PlmKR1 were determined
by molecular replacement using Phenix (Adams et al., 2010) with the KR from
module 1 of the erythromycin biosynthetic gene cluster (EryKR1, PDB code:
2FR0) (Keatinge-Clay and Stroud, 2006) as the search model. COOT (Emsley
and Cowtan, 2004) was used for model building and REFMAC5 (Murshudov
et al., 1997) of the CCP4 (Collaborative Computational Project, 1994) suite
for refinement. Structures were validated by MOLPROBITY (Davis et al.,
2004). Sequence alignments were done with the MUSCLE alignment tool
(Edgar, 2004) and molecular figures were prepared with PyMOL (http://
www.pymol.org/).
Edgar, R.C. (2004). MUSCLE: multiple sequence alignment with high accuracy
and high throughput. Nucleic Acids Res. 32, 1792–1797.
Emsley, P., and Cowtan, K. (2004). Coot: model-building tools for molecular
graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132.
´
Eustaquio, A.S., McGlinchey, R.P., Liu, Y., Hazzard, C., Beer, L.L., Florova, G.,
Alhamadsheh, M.M., Lechner, A., Kale, A.J., Kobayashi, Y., et al. (2009).
Biosynthesis of the salinosporamide A polyketide synthase substrate chloroe-
thylmalonyl-coenzyme A from S-adenosyl-L-methionine. Proc. Natl. Acad.
Sci. USA 106, 12295–12300.
Fushimi, S., Furihata, K., and Seto, H. (1989a). Studies on new phosphate ester
antifungal antibiotics phoslactomycins. II. Structure elucidation of phoslacto-
mycins A to F. J. Antibiot. 42, 1026–1036.
ACCESSION NUMBERS
The final structure is deposited in the PDB with accession code 4HXY.
Fushimi, S., Nishikawa, S., Shimazu, A., and Seto, H. (1989b). Studies on new
phosphate ester antifungal antibiotics phoslactomycins. I. Taxonomy, fermen-
tation, purification and biological activities. J. Antibiot. 42, 1019–1025.
SUPPLEMENTAL INFORMATION
Gouet, P., Courcelle, E., Stuart, D.I., and Me´ toz, F. (1999). ESPript: analysis of
Supplemental Information includes Supplemental Experimental Procedures
and seven figures and can be found with this article online at http://dx.doi.
org/10.1016/j.chembiol.2013.04.014.
multiple sequence alignments in PostScript. Bioinformatics 15, 305–308.
Hopwood, D.A., and Sherman, D.H. (1990). Molecular genetics of polyketides
and its comparison to fatty acid biosynthesis. Annu. Rev. Genet. 24, 37–66.
Keatinge-Clay, A.T. (2007). A tylosin ketoreductase reveals how chirality is
ACKNOWLEDGMENTS
determined in polyketides. Chem. Biol. 14, 898–908.
This work was supported, in whole or part, by National Institutes of Health
grants R01 AI051629 (to K.A.R.) and DK42303 (to J.L.S.). Mass spectrometric
analysis was carried out at the Portland State Bioanalytical Mass Spectrom-
eter Facility, which was supported in part by a grant from the NSF (Grant Num-
ber 0741993).
Keatinge-Clay, A. (2008). Crystal structure of the erythromycin polyketide syn-
thase dehydratase. J. Mol. Biol. 384, 941–953.
Keatinge-Clay, A.T., and Stroud, R.M. (2006). The structure of a ketoreductase
determines the organization of the beta-carbon processing enzymes of
modular polyketide synthases. Structure 14, 737–748.
Khosla, C. (1997). Harnessing the biosynthetic potential of modular polyketide
Received: January 18, 2013
Revised: March 24, 2013
Accepted: April 16, 2013
Published: June 20, 2013
synthases. Chem. Rev. 97, 2577–2590.
Kong, R., Liu, X., Su, C., Ma, C., Qiu, R., and Tang, L. (2013). Elucidation of the
biosynthetic gene cluster and the post-PKS modification mechanism for fos-
triecin in Streptomyces pulveraceus. Chem. Biol. 20, 45–54.
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