Enzymatic extender unit generation for in vitro polyketide synthase reactions: Structural and functional showcasing of Streptomyces coelicolor MatB

Article abstract of DOI:10.1016/j.chembiol.2010.12.014

In vitro experiments with modular polyketide synthases (PKSs) are often limited by the availability of polyketide extender units. To determine the polyketide extender units that can be biocatalytically accessed via promiscuous malonyl-CoA ligases, structural and functional studies were conducted on Streptomyces coelicolor MatB. We demonstrate that this adenylate-forming enzyme is capable of producing most CoA-linked polyketide extender units as well as pantetheine- and N-acetylcysteamine-linked analogs useful for in vitro PKS studies. Two ternary product complex structures, one containing malonyl-CoA and AMP and the other containing (2R)-methylmalonyl-CoA and AMP, were solved to 1.45 A and 1.43 A resolution, respectively. MatB crystallized in the thioester-forming conformation, making extensive interactions with the bound extender unit products. This first structural characterization of an adenylate-forming enzyme that activates diacids reveals the molecular details for how malonate and its derivatives are accepted. The orientation of the α-methyl group of bound (2R)-methylmalonyl-CoA, indicates that it is necessary to epimerize α-substituted extender units formed by MatB before they can be accepted by PKS acyltransferase domains. We demonstrate the in vitro incorporation of methylmalonyl groups ligated by MatB to CoA, pantetheine, or N-acetylcysteamine into a triketide pyrone by the terminal module of the 6-deoxyerythronolide B synthase. Additionally, a means for quantitatively monitoring certain in vitro PKS reactions using MatB is presented.

Full text of DOI:10.1016/j.chembiol.2010.12.014

Chemistry & Biology
Article
Enzymatic Extender Unit Generation for In Vitro
Polyketide Synthase Reactions: Structural and Func-
tional Showcasing of Streptomyces coelicolor MatB
1
1,
Amanda J. Hughes and Adrian Keatinge-Clay *
1
Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, TX 78712, USA
Correspondence: adriankc@mail.utexas.edu
*
DOI 10.1016/j.chembiol.2010.12.014
SUMMARY
produced by enzymatic assembly lines known as modular poly-
ketide synthases (PKSs). Each PKS module is a collection of
In vitro experiments with modular polyketide syn- domains that catalyze the condensation and processing of
thases (PKSs) are often limited by the availability of small carbon building blocks, called extender units. Within
a module, an acyltransferase (AT) selects an extender unit, a
ketosynthase (KS) catalyzes the condensation reaction, and
polyketide extender units. To determine the poly-
ketide extender units that can be biocatalytically
accessed via promiscuous malonyl-CoA ligases,
structural and functional studies were conducted
optional b-carbon processing enzymes, such as a ketoreduc-
tase (KR), a dehydratase (DH), and an enoylreductase (ER),
set the functional groups and stereochemistries at the a- and
b-carbons. The growing polyketide chain is shuttled through
on Streptomyces coelicolor MatB. We demonstrate
that this adenylate-forming enzyme is capable of
producing most CoA-linked polyketide extender
units as well as pantetheine- and N-acetylcyste-
˚
the PKS in a hand-over-hand fashion via an ꢀ18 A phosphopan-
tetheinyl arm attached to the acyl carrier protein (ACP) of each
module. The tremendous diversity of polyketide metabolites
amine-linked analogs useful for in vitro PKS studies. produced by modular PKSs largely results from extender unit
Two ternary product complex structures, one con- selection (Figure 1A). Modular PKSs select two types of
taining malonyl-CoA and AMP and the other contain- extender units: CoA-linked and ACP-linked (Chan et al., 2009).
The most commonly selected extender units are CoA-linked,
ing (2R)-methylmalonyl-CoA and AMP, were solved
˚
˚
such as malonyl-CoA, (2S)-methylmalonyl-CoA, and (2S)-ethyl-
malonyl-CoA. The less common ACP-linked extender units,
such as (2R)-methoxymalonyl-ACP, (2R)-hydroxymalonyl-ACP,
and (2S)-aminomalonyl-ACP, are synthesized on the phospho-
pantetheinyl arm of an ACP separate from the PKS assembly
line (Chan and Thomas, 2010).
to 1.45 A and 1.43 A resolution, respectively. MatB
crystallized in the thioester-forming conformation,
making extensive interactions with the bound
extender unit products. This first structural charac-
terization of an adenylate-forming enzyme that
activates diacids reveals the molecular details for
how malonate and its derivatives are accepted. The
To study the activities of PKS modules in vitro, a supply of
polyketide extender units is necessary; however, the cost and
orientation of the a-methyl group of bound (2R)- availability of these molecules is prohibitive. If the extender units
methylmalonyl-CoA, indicates that it is necessary to are obtained from a commercial source, experiments designed
epimerize a-substituted extender units formed by to produce over a milligram of polyketide product in vitro
become impractical. An alternative is to use N-acetylcysteamine
MatB before they can be accepted by PKS acyltrans-
(SNAC-)-linked extender units, which have been employed in
ferase domains. We demonstrate the in vitro incorpo-
ration of methylmalonyl groups ligated by MatB to
CoA, pantetheine, or N-acetylcysteamine into a tri-
ketide pyrone by the terminal module of the 6-deoxy-
erythronolide B synthase. Additionally, a means for
quantitatively monitoring certain in vitro PKS
reactions using MatB is presented.
feeding studies and in vitro reactions (Carroll et al., 2002; Pohl
et al., 1998). Pohl et al. (1998) showed that on incubation of
synthetic (2RS)-methylmalonyl-SNAC with DEBS3, the third
protein of the 6-deoxyerythronolide B synthase, in the presence
of (2S, 3R)-3-hydroxy-2-methylpentanoate-SNAC and NADPH,
the anticipated triketide lactone was generated; this result
indicates that the terminal DEBS3 AT accepts methylmalonyl
units from (2S)-methylmalonyl-SNAC. To access CoA-linked
extender units enzymatically, Pohl and coworkers utilized the
malonyl-CoA synthetase MatB from Rhizobium trifolii, which
has a broad substrate tolerance, and demonstrated that it can
INTRODUCTION
Polyketides are secondary metabolites produced by diverse produce malonyl-CoA, methylmalonyl-CoA, ethylmalonyl-CoA,
microbes that have been employed as antibiotics, antifungals, as well as unnatural extender units such as cyclopropyl-
anticancer agents, and as many other types of therapeutics malonyl-CoA, cyclobutylmalonyl-CoA, benzylmalonyl-CoA, and
(Khosla et al., 2007; Walsh, 2004; Pfeifer and Khosla, 2001; cyclopropylmethylenemalonyl-CoA (Pohl et al., 2001). We have
Staunton and Wilkinson, 2001). Many of these metabolites are employed the previously uncharacterized Streptomyces
Chemistry & Biology 18, 165–176, February 25, 2011 ª2011 Elsevier Ltd All rights reserved 165

Chemistry & Biology
Enzymatic Synthesis of PKS Extender Units by MatB
Figure 1. Polyketide Extender Unit Incorporation and MatB Reaction
A) The most common CoA-linked polyketide extender units are mCoA, (2S)-mmCoA, and (2S)-emCoA; the most common ACP-linked polyketide extender units
(
are (2R)-oxACP, (2R)-hmACP, and (2S)-amACP. These extender units are incorporated into polyketides 1–3 and a polyketide-nonribosomal peptide hybrid, 4. +,
Priming unit; *, mCoA; -, mmCoA; A, emCoA; C, oxACP; z, amACP; y, hmACP.
(B) Two-step reaction mechanism of Streptomyces coelicolor. MatB: In the adenylate-forming reaction, the ATP a-phosphate is attacked by a malonate deriv-
ative carboxylate to form the adenylate intermediate and pyrophosphate. In the thioester-forming reaction, CoA displaces AMP to produce the extender unit
and AMP.
coelicolor MatB to synthesize CoA-linked extender units and acyl-adenylate and pyrophosphate; in the thioester-forming
derivatives thereof (Figure 1B).
conformation the thiol acceptor CoA is selected to perform the
The S. coelicolor malonyl-CoA synthetase MatB is an adeny- second half-reaction, generating acyl-CoA and AMP (Kochan
late-forming enzyme that is best classified with acyl-CoA synthe- et al., 2009).
tases. MatB is anticipated to be enzymatically and structurally
Here, we demonstrate the S. coelicolor MatB-catalyzed
homologous to such enzymes as acyl-CoA synthetases (Protein synthesis of polyketide extender units malonyl-CoA (mCoA),
Data Bank [PDB] codes: 3EQ6, 1V26, 1PG3, 1RY2)(Kochan methylmalonyl-CoA (mmCoA), ethylmalonyl-CoA (emCoA), me-
et al., 2009; Hisanaga et al., 2004; Gulick er al., 2003; Jogl and thoxymalonyl-CoA (oxCoA), and hydroxymalonyl-CoA (hmCoA),
Tong, 2004), 4-chlorobenzoyl-CoA ligase (PDB code: 1T5D) marking the first enzymatic syntheses of oxCoA and hmCoA.
(Gulick et al., 2004), the adenylation domains (A-domains) of Equivalent pantetheine- (PANT-) and SNAC-linked extender
nonribosomal peptide synthetases (NRPSs; PDB codes: 3ITE, units were also generated, demonstrating that MatB is not only
3
E7W, 1AMU) (Lee et al., 2010; Yonus et al., 2008; Conti et al., promiscuous toward malonyl derivatives, but also thiol accep-
997; Strieker et al., 2010; Mootz et al., 2002; Marahiel, 2009), tors. Crystal structures of two MatB ternary product complexes
1
and firefly luciferase (PDB code: 2D1R)(Nakatsu et al., 2006). A were obtained with malonyl-CoA and AMP (1.45 A resolution)
˚
˚
structure of a diacid-activating adenylate-forming enzyme has and with (2R)-methylmalonyl-CoA and AMP (1.43 A resolution).
not yet been reported. Reactions catalyzed by adenylate-form- These are the first structures of a diacid-activating adenylate-
ing enzymes can be represented by a medium-chain acyl-CoA forming enzyme. The strong electron density surrounding the
synthetase: in the adenylate-forming conformation a medium- protein, thioester product, and AMP provides some of the
sized fatty acid is selected to attack the a-phosphoryl moiety most complete snapshots of product formation in the enzyme
of adenosine triphosphate (ATP) via its carboxylate to yield an superfamily. The orientation of the (2R)-methylmalonyl-CoA
166 Chemistry & Biology 18, 165–176, February 25, 2011 ª2011 Elsevier Ltd All rights reserved

Chemistry & Biology
Enzymatic Synthesis of PKS Extender Units by MatB
a-methyl group reveals the stereoselectivity of MatB toward observed mass (calculated mass) of the triketide pyrone was
a-substituted malonate derivatives and also reveals the pocket 169.2(169.1). In all reactions, mmSNAC is formed as a byproduct
that enables MatB to be promiscuous toward a-substituents. generated by MatB using methylmalonate and SNAC liberated
In vitro reactions in which the methylmalonyl groups of MatB- from (2S, 3R)-2-methyl-3-oxopentanoate-SNAC, although
synthesized extender units mmCoA, mmPANT, and mmSNAC thioesterase-catalyzed hydrolysis of (2S, 3R)-2-methyl-3-oxo-
are incorporated into a triketide pyrone by the final module of pentanoate-SNAC may also contribute to mmSNAC formation
the 6-deoxyerythronolide B synthase exemplify the use of (Figure 2E) (Wang et al., 2009). Monitoring mmSNAC production
MatB. A protocol for monitoring such reactions through represents a powerful, new method of following in vitro PKS
a coupled reaction, also catalyzed by MatB, is described.
reactions, especially those producing polyketides lacking
chromophores.
RESULTS
Overall MatB Structure
MatB Synthesizes Diverse CoA-, PANT-,
and SNAC-Linked Extender Units
The conditions that yielded protein crystals contained all of the
substrates required for the synthesis of mCoA or mmCoA as
Products from incubations of CoA and a diacid (malonic acid, described in the Experimental Procedures. Briefly, purified
methylmalonic acid, ethylmalonic acid, methoxymalonic acid, protein was incubated with the substrates of the MatB reaction:
and tartronic acid) with MatB were collected during high-perfor- ATP, magnesium, CoA, and malonate or methylmalonate. Clus-
mance liquid chromatography (HPLC) runs and analyzed by ters of elongated, orthorhombic crystals, often hollowed along
mass spectrometry. The observed masses (calculated masses) their long axes, grew overnight. Diffraction data was collected
from the hmCoA, oxCoA, mCoA, mmCoA, and emCoA reactions using crystals flash-frozen in their mother liquor. Molecular
were 867.8(868.1), 881.9(882.1), 851.7(852.1), 865.9(866.1), and replacement proved unsuccessful, as is often the case with ad-
8
79.9(880.1), respectively, indicating the production of each of enylate-forming enzymes (Shah et al., 2009). The mCoA/AMP-
the five CoA-linked extender units (Figure 2A). Aminomalonyl- bound MatB structure was solved via single-wavelength anom-
CoA could not be produced. MatB was also found capable of alous dispersion using selenomethionine-labeled protein. The
synthesizing PANT- and SNAC-linked extender units; employing native malonyl-CoA/AMP-bound and methylmalonyl-CoA/
identical HPLC methanol gradients, the retention times of AMP-bound complexes were then refined at resolutions of
˚
the PANT-linked extender units were increased relative to the 1.45 A and 1.43 A, respectively (Table 1). A comparison of the
˚
˚
CoA-linked extender units, whereas the retention times of the ternary complexes yields root-mean-square deviation of 0.06 A.
SNAC-linked extender units were decreased (Figures 2B and MatB is comprised of 485 residues, possesses a molecular
C). Two product peaks were observed in each of the hmCoA, weight of 50.6 kDa, and is monomeric as determined through
2
hmPANT, and hmSNAC reactions. Both peaks from the gel filtration chromatography and through an analysis of the
hmSNAC reaction were collected and subjected to LC/MS packing within the crystals. Like other adenylate-forming
analysis. The observed masses (calculated masses) of 222.2 enzymes, MatB has a large N-terminal body (residues 1–387)
(
222.0) and 206.2(206.1), correspond to hmSNAC and mSNAC, and a small C-terminal lid (residues 388–485), yet is the smallest
respectively. The mSNAC peak may have resulted from of the adenylate-forming enzymes solved to date (Figure 3A). No
a malonic acid impurity in the tartronic acid stock. With the addi- density was observed for the N-terminal histidine tag, residues
tion of sufficient ATP, thiol, and diacid, MatB reactions can be 1–3, or residues 472–485 of the C-terminal lid. No density for
0
0
scaled up to yield multimilligram quantities that can be readily the 3 ,5 -diphosphoadenosine of CoA was observed either,
analyzed by nuclear magnetic resonance (NMR). In summary, permitting only the phosphopantetheine moiety of CoA to be
S. coelicolor MatB produces each of the CoA-, PANT-, and modeled. The overall structure for mCoA/AMP-bound MatB
SNAC-linked extender units mCoA, mmCoA, emCoA, oxCoA, contains 467 residues, 348 waters, 1 mCoA, 1 AMP molecule,
2+
hmCoA, mPANT, mmPANT, emPANT, oxPANT, hmPANT, and 1 solvated Mg in the asymmetric unit. The overall structure
mSNAC, mmSNAC, emSNAC, oxSNAC, and hmSNAC.
of mmCoA/AMP-bound MatB contains 467 residues, 407 water
2+
molecules, 1 mmCoA, 1 AMP molecule, and 1 solvated Mg in
the asymmetric unit. The electron density clearly reveals an R
stereochemistry at the a-carbon indicating that (2R)-mmCoA is
Incorporation of MatB-synthesized mmCoA, mmPANT,
and mmSNAC into a Triketide Pyrone
The final module and thioesterase from the 6-deoxyerythronolide bound. Other than the a-methyl group, the positions of the
B synthase (Mod6TE) has been used in previous experiments to bound mCoA and (2R)-mmCoA extender units are superimpos-
produce triketide lactones (Pohl et al., 1998; Gokhale et al., 1999; able between the two structures.
Menzella et al., 2005). We were able to incorporate methylmalo-
nate from MatB-synthesized mmCoA, mmPANT, and mmSNAC MatB Observed in the Thioester-Forming Conformation
into a triketide pyrone by (1) incubating MatB ligation reactions Adenylate-forming enzymes can adopt at least two catalytically
ꢁ
overnight at 22 C; (2) adding Mod6TE, S. coelicolor methyl- distinct conformations (Reger et al., 2008; Kochan et al., 2009;
ꢁ
malonyl-CoA epimerase (Dayem et al., 2002), and (2S, 3R)-2- Lee et al., 2010). The C-terminal lid pivots 140 between the
methyl-3-oxopentanoate-SNAC (Keatinge-Clay, 2007); and (3) two catalytically-active conformations (the adenylate-forming
ꢁ
incubating at least 1 day at 22 C and analyzing for polyketide conformation and the thioester-forming conformation) in
production via HPLC (Figures 2D–2F). To confirm the identity a process termed ‘‘domain alternation’’ (Reger et al., 2008;
of the triketide pyrone, the fraction corresponding to the largest Shah et al., 2009) (Figure 3A). An aspartate located in a linker
peak (at 290 nm) was collected and subjected to LC/MS; the between the N-terminal body and the C-terminal lid of
Chemistry & Biology 18, 165–176, February 25, 2011 ª2011 Elsevier Ltd All rights reserved 167

Chemistry & Biology
Enzymatic Synthesis of PKS Extender Units by MatB
Figure 2. HPLC Analysis of MatB and Mod6TE Reactions
A–C) MatB synthesized 15 polyketide extender units valuable for in vitro PKS reactions. Retention times (min) of extender units formed using CoA, PANT, and
SNAC as the thiol acceptor are indicated. The observed masses (calculated masses) from the hmCoA, oxCoA, mCoA, mmCoA, and emCoA reactions were
(
168 Chemistry & Biology 18, 165–176, February 25, 2011 ª2011 Elsevier Ltd All rights reserved

Chemistry & Biology
Enzymatic Synthesis of PKS Extender Units by MatB
adenylate-forming enzymes, is usually considered to be the prin- similar to that of S. coelicolor MatB is from a medium-chain ad-
ciple pivot point about which the C-terminal lid rotates during enylate-forming enzyme (PDB code: 3EQ6) (Kochan et al.,
domain alternation and is hypothesized to interact with the 2009).
arginine that is the final residue of the N-terminal body in the
thiolation conformation (Wu et al., 2008). The thioester-forming
Bound Malonyl-CoA and Methylmalonyl-CoA
Both mCoA and (2R)-mmCoA products are bound between the
N-terminal body and C-terminal lid of MatB (Figure 3D). Although
conformation of 4-chlorobenzoate CoA ligase shows an ionic
interaction between hinge residue D402 and the final residue of
the N-terminal body (R400); the D402P and D402A mutants
a few adenylate-forming enzymes have been solved with CoA or
showed decreased catalytic activity (Reger et al., 2008; Wu
et al., 2008). The putative hinge residue in MatB is D387, which
thioester products bound (PDB codes: 2P2F, 1PG4, 3EQ6)
(
Reger et al., 2007; Gulick et al., 2003; Kochan et al., 2009), the
is four residues after the final residue of the N-terminal body
R383) and highly conserved among malonyl-CoA synthetases.
The side-chain electron density for the MatB linker (residues
84–388, KATDL) is too weak to observe interactions with R383.
MatB complex structures presented here provide the strongest
density for a phosphopantetheinyl arm to date. In both struc-
tures, the bound extender unit displays electron density that
continues past the CoA b-phosphoryl moiety and possesses
an average B-factor lower than the average B-factor of MatB.
The interactions with the extender unit products can be divided
into three categories:
(
3
Comparison to Adenylate-Forming Enzyme Core Motifs
Highly conserved regions of adenylate-forming enzymes have
been categorized into ten major core motifs (A1–A10, Table 2)
(
Marahiel et al., 1997). Motif A3 contains a serine/threonine-
1. Malonyl groups are bound primarily by residues S261 and
R283 (Figure 3E). The presence of the arginine in core
motif A5 is a hallmark of malonyl-CoA synthetases.
Indeed, R283 serves an important functional role: the
ternary complexes reveal that it forms strong ionic interac-
tions with the carboxylates of both mCoA and mmCoA
(another active site arginine forms strong ionic interactions
with this carboxylate in ATs when the extender unit is
selected for transfer to an ACP [Tang et al., 2006]). The
rich region hypothesized to interact with the b- and g-phos-
phates of ATP during the adenylation reaction, stabilizing the
release of pyrophosphate; S. coelicolor MatB contains an
equivalent region (Marahiel et al., 1997). The role of motifs A4
and A5 are to bind the carboxylate substrate and stabilize the
formation of the adenylate intermediate; ten residues, mostly
located between these motifs, have been identified as ‘‘speci-
ficity-conferring residues’’ that form the binding site for
a carboxylate substrate (Stachelhaus et al., 1999). A conserved
aspartate in motif A4 of A-domains (FDXS) ionically interacts
with the a-amino group of bound amino acids; in S. coelicolor
MatB, the corresponding residue is V188 (HVHG) (PDB codes:
ꢁ
ꢁ
observed c and f angles for S261 are ꢂ17.9 and 88.5 ,
respectively; that these values are outside of the expected
Ramachandran regions suggests the importance of S261
in the MatB reaction (Herzberg and Moult, 1991). In the
ternary complexes presented in this work, S261 partici-
pates in a hydrogen-bonding network that includes R283
and the extender unit carboxylate (Figure 3E).
1
AMU and 3E7W) (Conti et al., 1997; Yonus et al., 2008).
H189, also within S. coelicolor MatB motif A4, coordinates
with the specificity-conferring residues S261 and R238 to help
form the malonate binding pocket. Within the S. coelicolor
MatB motif A5 (ERYGMTE), R238 forms a salt bridge with the
extender unit carboxylate in each of the ternary product
complex structures; the corresponding residue in the phenylal-
anine-selective A-domain of the gramicidin synthetase is A322
2. The pantetheine arm is contacted by hydrogen bonds
from the backbone carbonyls of residues G392 and
G393 as well as hydrophobic interactions principally medi-
ated by Y384 (as observed in a medium-chain acyl-CoA
synthetase ternary complex, PDB code: 3EQ6) (Figure 3F)
(Kochan et al., 2009). The A8 core motif, which contains
these residues, is hypothesized to possess multiple
functions: in the adenylate-forming conformation, the first
arginine may help stabilize the pyrophosphate leaving
group; in the thioester-forming conformation, it may
interact with the pantetheinyl arm, as observed in the
MatB structure.
(
NAYGPTE), which helps create a large hydrophobic pocket
for the phenylalanine side chain (PDB code: 1AMU) (Conti
et al., 1997). Interestingly, the loop immediately following motif
A5, which contains two specificity-conferring residues, is one
residue shorter in S. coelicolor MatB than in most adenylate-
forming enzymes. Thus, the position of the specificity-deter-
mining residue M291 in S. coelicolor MatB is slightly different
than the most equivalent residue, C331, in the phenylalanine-
activating A-domain of the gramicidin synthetase. In terms of
length and orientation, the loop following motif A5 that is most
3. The CoA b-phosphate is ionically bound by R236 and R461
(Figure 3G). A third arginine, R459, may form ionically
˚
interact with the CoA a-phosphate as it is within 6 A.
867.8(868.1), 881.9(882.1), 851.7(852.1), 865.9(866.1), and 879.9(880.1), respectively. The two peaks observed for hmSNAC at 6.24 and 8.97 min were analyzed
by positive-ESI LC/MS and correspond to hmSNAC (222.2(222.0)) and mSNAC (206.2(206.1)); the presence of mSNAC is may be from a malonate impurity in the
tartronic acid stock. *AMP.
(
(
D) Mod6TE reaction. The selection of (2S)-mmCoA/PANT/SNAC extender unit and transthioesterification of (2S, 3R)-2-methyl-3-oxopentanoate-SNAC
diketideSNAC) releases CoA/PANT/SNAC and SNAC, respectively. The condensation reaction results in the formation of a triketide, which cyclizes to generate
a triketide pyrone.
E) HPLC analysis of Mod6TE reactions at 235 nm. The formation of mmSNAC (from the transthioesterification of diketide-SNAC) may be used as a diagnostic tool
to follow an in vitro PKS reaction.
F) HPLC analysis of Mod6TE reactions at 290 nm. Triketide pyrone was synthesized from all three different extender units, (2S)-mmCoA/PANT/SNAC. The iden-
tity of triketide pyrone was confirmed by positive-ESI LC/MS with an observed mass (calculated mass) of 169.2(169.1).
(
(
Chemistry & Biology 18, 165–176, February 25, 2011 ª2011 Elsevier Ltd All rights reserved 169

Chemistry & Biology
Enzymatic Synthesis of PKS Extender Units by MatB
Table 1. Data Collection and Refinement of S. coelicolor MatB Structures
MatB (Malonyl-CoA) 3NYR MatB (2R-Methylmalonyl-CoA) 3NYQ SeMatB (2R-Methylmalonyl-CoA)
Space group
C222(1)
C222(1)
C222(1)
Cell dimensions
˚
a, b, c (A)
73.2, 86.6, 153.1
90
73.4, 86.8, 153.4
90
73.9, 87.4, 154.4
90
ꢁ
a = b = g ( )
˚
Resolution (A)
50–1.45
50–1.43
50–1.75
R
merge
0.051 (0.439)
23.1 (2.6)
99.8 (99.4)
7.1 (4.5)
0.062 (0.292)
15.5 (3.9)
98.5 (82.4)
6.8 (3.8)
0.080 (0.202)
30.8 (7.1)
97.8 (94.7)
3.9 (4.0)
I/s(I)
Completeness (%)
Redundancy
Refinement
˚
Resolution (A)
50–1.45
50–1.43
50–1.75
Unique reflections
621,987
671,166
537,849
R
work/Rfree
0.1997/0.2247
0.1951/0.2161
99.34, 0.66
0.2026/0.2353
99.55, 0.45
Ramachandran allowed, outlier (%) 99.34, 0.66
Number of atoms
Protein
Extender unit
AMP
3378
28
3378
29
3320
29
23
23
N/A
338
Water
347
404
˚
2
B factors (A )
Protein
16.72
14.60
21.94
26.50
17.40
14.29
23.79
27.64
19.66
21.06
N/A
Extender unit
AMP
Water
27.75
Rmsd
˚
Bond lengths (A)
0.010
0.013
0.011
ꢁ
Bond angles ( )
1.80
1.77
1.45
˚
Highest Res. Bin (A)
1.49–1.45
1.47–1.43
1.78–1.75
AMP: adenosine monophosphate; rmsd: root-mean-square deviation.
Bound Adenosine Monophosphate
frequently used to generate malonyl-CoA for PKSs that utilize
AMP is bound in the MatB ternary complex structures at a site a-unsubstituted extender units (Gao et al., 2010). Here, we
equivalent to that observed in other adenylate-forming enzymes have demonstrated that S. coelicolor MatB, like R. trifolii MatB,
(
e.g., PDB codes: 3EQ6, 1MD9, 3E7W, 1AMU, 2P2F, 3FCC). ligates diverse malonate derivatives to CoA to generate a diver-
The 2F -F electron density maps (contoured at s = 1.6) do not sity of a-substituted polyketide extender units that are utilized by
completely surround AMP; however, the overall B-factors for AMP modular PKSs. We have shown that S. coelicolor MatB is also
o
c
2
˚
in the mCoA- and mmCoA-bound MatB structures are 22 A and promiscuous toward thiol acceptors, as established by the
2
3 A , respectively, which is only slightly higher than the average synthesis of PANT- and SNAC-linked extender units. These
˚
2
2
2
˚
˚
B-factors for the MatB residues (16.6 A and 17.3 A , respectively) extender units are nearly as efficient as CoA-linked extender
Figure 4B). The distance from the thioester carbon to the nearest units at transferring malonate derivatives to PKS modules, as
oxygen atom of the AMP phosphate, which had been covalently demonstrated by our experiments using Mod6TE in the produc-
(
˚
bonded to one another in the adenylate intermediate, is ꢀ3.6 A. tion of a triketide pyrone (Figure 2F).
Positively-charged K390 and K395 form ionic interactions with the
In an effort to understand the nature of the promiscuity of
AMP a-phosphate, whereas T287 also forms a hydrogen bond MatB toward malonate derivatives and thiol acceptors, as well
withit(Figure4E). Thebackbone carbonyl ofR283formsa hydrogen as the stereoselectivity imposed by MatB toward a-substituted
bond to the N7-amine of adenine, while a conserved aspartic acid, malonate derivatives, the structures of two MatB ternary
D368, interacts with both AMP hydroxyl groups (Figure 4C).
product complexes were obtained. These structures, combined
with information from analyses of more thoroughly studied ad-
enylate-forming enzymes, allow the following catalytic cycle to
be proposed for MatB-catalyzed synthesis of malonyl-CoA:
DISCUSSION
Malonyl-CoA synthetases are capable of generating many malonate and ATP bind to MatB, with the binding of ATP poten-
extender units for in vitro PKS reactions. R. trifolii MatB is tially causing the C-terminal lid to dock to the N-terminal body in
170 Chemistry & Biology 18, 165–176, February 25, 2011 ª2011 Elsevier Ltd All rights reserved

Chemistry & Biology
Enzymatic Synthesis of PKS Extender Units by MatB
Figure 3. MatB Structure and Active Site
Architecture
(
A) Overall structure of the mmCoA/AMP/MatB
ternary complex. The N-terminal body is colored
light blue, the C-terminal lid is colored dark blue,
and the N and C termini are indicated. (2R)-
mmCoA and AMP are colored green.
(
B) LIGPLOT representation of interactions
observed between (2R)-mmCoA and surrounding
MatB residues.
(
(
(
C) Simulated-annealing omit map of (2R)-mmCoA
contoured at s = 3.0).
D) Stereodiagram of (2R)-mmCoA in the pante-
theine tunnel with surrounding 2F
density map (2F -F map contoured at s = 1.6).
E) H189, S261, and R283, in the N-terminal body,
o c
-F electron
o
c
(
interact with methylmalonyl moiety of (2R)-
mmCoA. A hydrogen-bonding network is formed
between S261, R283, and the (2R)-mmCoA
˚
carboxylate (distances in A, 2F
o
-F
c
map contoured
at s = 1.6).
(
F) Carbonyls from G392 and G393 hydrogen bond
with the pantetheine amide groups. Hydrophobic
interactions are also formed between Y394 and
˚
o c
the pantetheine (distances in A, 2F -F map con-
toured at s = 1.6).
(
G) R236 and R461 form salt-bridges with the (2R)-
mmCoA b-phosphate. No density was observed
0
0
for the 3 ,5 -diphosphoadenosine; however, the
positively charged pantetheine tunnel entrance
likely interacts with both a- and b-phosphate
˚
groups (distances in A, 2F
o
-F
c
map contoured at
s = 1.6).
(
H) V188 and M291 form a hydrophobic pocket
near the a-methyl substituent of (2R)-mmCoA
˚
o c
distances in A, 2F -F map contoured at s = 1.6).
(
tunnel’’ is created. The malonyl-adeny-
late remains bound during this domain
alternation process. CoA then binds
through hydrogen bonds with the G392
and G393 carbonyls, hydrophobic inter-
actions with Y394, and ionic interactions
with R236 and R461 (Figures 3F and
3G). Inspection of the MatB structures
does not reveal a site that recognizes
0
0
the 3 ,5 - diphosphoadenosine of CoA
as is observed other adenylate-forming
enzymes (e.g., PDB codes: 1PG3 and
the adenylate-forming conformation (Kochan et al., 2009). Mal- 2P2F). The entry of CoA into the MatB active site is halted
onate binds largely via a salt-bridge between one of its carbox- when the diphosphate moiety forms salt-bridges with positively
ylates and R283 and S261 (Figure 3E). By analogy with other ad- charged residues at the tunnel opening (Figure 3G), although
enylate-forming enzymes, MatB interacts with ATP by bonding these ionic interactions are not required for catalysis because
to its phosphates with the serine/threonine-rich A3 core motif, PANT and SNAC can serve as thiol acceptors. Because the
its ribose with the conserved aspartate D368, and its adenine pantetheinyl arm makes as many contacts to the C-terminal lid
through van der Waals interactions with loop residues S261– as it does with the N-terminal body, CoA likely enters the pante-
A263 (Figures 4C and 4E). The free carboxylate of the malonate theine tunnel after tunnel formation. After an attack of the CoA
is positioned to attack the ATP a-phosphate, releasing pyro- thiolate on the malonyl-adenylate carbonyl, the leaving group
phosphate, which may be stabilized as a leaving group by AMP, stabilized by K395, is generated. Finally, AMP and the
K476 (Kochan et al., 2009). After pyrophosphate diffuses from malonyl-CoA extender unit diffuse out of the active site, aided
the active site, the C-terminal lid pivots about D387 to adopt by the pivoting motion of the C-terminal lid during domain
the thioester-forming conformation in which the ‘‘pantetheine alternation.
Chemistry & Biology 18, 165–176, February 25, 2011 ª2011 Elsevier Ltd All rights reserved 171

Chemistry & Biology
Enzymatic Synthesis of PKS Extender Units by MatB
Table 2. Core Motifs Conserved in Adenylate-Forming Enzymes
Adenylate-Forming Enzyme
Core
A1
Function
Consensus Sequence
L(TS)YxEL
Corresponding MatB Sequence
(29)LTYAEL(34)
Structural
A2
Structural
LKAGxAYL(VL)P(LI)D
LAYxxYTSG(ST)TGxPKG
FDxS
(69)LLAGVAAVPLN(79)
(137)PALVVYTSGTTGPPKG(152)
(187)HVHG(190)
A3
Stabilize pyrophosphate leaving group
Asp interacts with a-amino groups of peptides
Stabilize product
A4
A5
NxYGPTE
(282)ERYGMTE(288)
A6
Unknown
GELxIxGxG(VL)ARGYL
Y(RK)TGDL
(334)GEIQVRGPNLFTEYL(348)
(364)FRTGDM(369)
A7
Stabilize ATP/AMP ribose
Essential for adenylation
Unknown
A8
GRxDxQVKIRGxRIELGEIE
LPxYM(IV)P
(382)GRKATDLIKSGGYKIGAGEIE(402)
(454)LAPHKRP(460)
A9
A10
Stabilize ATP/AMP ribose
NGK(VL)DR
(474)MGKIMK(479)
AMP: adenosine monophosphate; ATP: adenosine triphosphate. Ten major conserved core motifs have been identified for the adenylate-forming
enzymes (A1–A10) (Marahiel et al., 1997). Corresponding MatB sequences are listed for comparison.
o c
The 2F -F electron density maps from the mmCoA/AMP- et al., 2007, 2008; Conti et al., 1997; Yonus et al., 2008; Hisanaga
bound MatB ternary complex suggest that MatB forms (2R)- et al., 2004; Kochan et al., 2009). Due to the proximity of the
˚
mmCoA and not (2S)-mmCoA. In related structures, the position a-substituent and the AMP phosphoryl moiety (ꢀ4 A in the
of a bound AMP is similar to its position within the adenylate ternary product complex and likely closer within the adenylate
intermediate, thus this intermediate can be approximately recon- intermediate), the stereochemistry of the a-carbon is limited to
structed from the positions of the malonyl group and AMP (Reger the R-configuration (Figure 5C). The formation of (2R)-mmCoA
Figure
Bound in the Ternary Complex
A) LIGPLOT diagram of AMP and MatB interac-
4. Adenosine
Monophosphate
(
˚
tions (distances in A).
B) Simulated-annealing omit map of AMP (con-
toured at s = 3.0).
C) Interaction between conserved D368 and
(
(
o c
hydroxyl groups of AMP (2F -F map contoured
at s = 1.6).
(
(
(
o c
D) 2F -F electron density map surrounding AMP
contoured at s = 1.6).
E) Ionic interactions between the AMP a-phos-
phate and both K390 and K395. The backbone
amide and hydroxyl group of T287 form hydrogen
˚
bonds with the AMP a-phosphate (distances in A,
o c
2F -F map contoured at s = 1.6).
(
F) Stereodiagram of (2R)-mmCoA and AMP within
˚
the active site (distances in A, 2F
o
-F
c
map con-
toured at s = 1.6).
172 Chemistry & Biology 18, 165–176, February 25, 2011 ª2011 Elsevier Ltd All rights reserved

Chemistry & Biology
Enzymatic Synthesis of PKS Extender Units by MatB
Figure 5. Engineering Substrate Specificity and Product Stereochemistry
(
A) Structural alignment of MatB (blues) and DltA (greys, PDB code: 3E7W). DltA is an A-domain that ligates D-alanine onto the phosphopantheinyl arm of a pep-
tidyl carrier protein.
B) D196 (gray) of the FDxS motif in DltA ionically interacts with the of D-alanine amino group. The MatB mutant V188D could catalyze the formation of amino-
malonyl-CoA.
C) The distance between the AMP leaving group and the a-carbon is 3.91 A. Re-engineering MatB to form (2S)-a-substituted extender units may not be possible
because the a-substituent of a (2S)-a-substitued-malonyl adenylate intermediate would clash with the AMP phosphoryl moiety. MatB is hypothesized to synthe-
size (2R)-mmCoA, (2R)-emCoA, (2R)-hmCoA, and (2R)-oxCoA (2F -F map contoured at s = 1.6).
(
˚
(
o
c
is consistent with observations from previous experiments that side chain with a charged amino group may preclude the forma-
failed to synthesize polyketides from mmCoA produced by tion of aminomalonyl-CoA. A structural alignment of MatB with
MatB in the absence of an epimerase (Murli et al., 2003). Thus, a D-alanine:D-alanyl carrier protein ligase (PDB code: 3E7W)
we hypothesize that MatB also synthesizes (2R)-emCoA, (2R)- suggests that the MatB mutant V144D would bind aminomalo-
hmCoA, and (2R)-oxCoA in a stereocontrolled manner. As the nate and catalyze the formation of aminomalonyl-CoA (Figures
AT domains of PKSs are very stereoselective, only accepting 5A and 5B). We have not tested for the formation of the less-
(
2S)-mmCoA and (2S)-emCoA, epimerization of mmCoA and commonly utilized extender units propylmalonyl-CoA or chloro-
emCoA is necessary for the incorporation of these MatB-synthe- ethylmalonyl-CoA; however, if MatB is not capable of selecting
sized extender units into PKSs and can be accomplished via the corresponding diacids, appropriate mutagenesis of V188
spontaneous epimerization under acidic conditions or enzymat- and M291 may enable the formation of these extender units.
ically-catalyzed epimerization through the addition of an epi-
merase (e.g., mmCoA epimerase) (Murli et al., 2003).
Thus, an enzymatic means to produce CoA-, PANT-, and
SNAC-linked polyketide extender units is provided. This reaction
Organic syntheses of mSNAC, mmSNAC, oxSNAC, and may be utilized by many PKS laboratories to produce significant
hmSNAC have been reported (Pohl et al., 1998; Carroll et al., quantities of extender units, especially when they are economi-
2
002); however, the syntheses of hmSNAC and oxSNAC involve cally unviable or commercially unavailable. The utility of MatB
multistep syntheses with 13% and 29% yields, respectively for in vitro PKS reactions was demonstrated not only through
Carroll et al., 2002). Using MatB, super-stoichiometric quantities the incorporation of the methylmalonyl groups of mmCoA,
(
of extender units can be enzymatically produced in situ to drive mmPANT, and mmSNAC by a PKS module to form a polyketide
the synthesis of milligram quantities of polyketides from in vitro product but also through the ability to observe reaction progress
reactions. MatB also serves as an extender unit regeneration by monitoring the production of the coupled product, mmSNAC.
system, as supported by the formation of mmSNAC in the The MatB ternary product complexes have provided chemical
Mod6TE reactions. With the addition of excess diacid and insights into the malonyl-CoA synthetase reaction as well as
ATP, MatB will continually regenerate product by recycling the structural insights into substrate promiscuity. Efforts to alter
appropriate thiol acceptor (e.g., CoA, PANT, or SNAC). The the stereochemistry of a-substituted extender unit products
MatB-catalyzed formation of a-substituted extender units will most likely not be successful due to the hypothesized geo-
enables the generation of otherwise unavailable isotopically- or metry of the adenylate intermediate; however, the range of the
radioactively-labeled extender units for more detailed in vitro sizes and chemistries of a-substituents of malonate derivatives
PKS studies. The generation of hmCoA and oxCoA also ligated by MatB is quite large, and apparently only limited by
suggests the potential of genetically engineering PKSs to the nearby residues V188 and M291.
produce sugar derivatives.
The hydrophobic residues V188 and M291 adjacent to the SIGNIFICANCE
a-methyl group of (2R)-mmCoA, as observed in the mmCoA/
AMP/MatB ternary complex, likely influence the promiscuity of We have demonstrated the use of Streptomyces coelicolor
MatB for a-substituted malonate derivatives and may explain MatB to enzymatically synthesize five polyketide extender
why S. coelicolor MatB was unable to synthesize aminoma- units (malonyl-, methylmalonyl-, ethylmalonyl-, hydroxy-
lonyl-CoA (Figure 3H). Within A-domains, an aspartate is usually malonyl-, and methoxymalonyl-CoA), of which some are
positioned in the A4 core motif to ionically interact with the amino commercially-unavailable or economically-prohibitive. This
group of a selected amino acid; within MatB, this position is is the first report of enzymatic synthesis of hydroxy-
occupied by V188. The chemical incompatibility of a valine malonyl-CoA and methoxymalonyl-CoA. In addition to the
Chemistry & Biology 18, 165–176, February 25, 2011 ª2011 Elsevier Ltd All rights reserved 173

Chemistry & Biology
Enzymatic Synthesis of PKS Extender Units by MatB
synthesis of these five natural extender units, we have enzy-
matically synthesized the corresponding D-pantetheine
(3 ml). When the OD600 = 0.4, each liter of media was supplemented with lysine,
phenylalanine, and threonine (each 100 mg), isoleucine, leucine, and valine
(
each 50 mg), and selenomethionine (60 mg). After 15 min protein expression
ꢁ
(PANT) and N-acetylcysteamine (SNAC) extender units.
was induced with 0.5 mM IPTG. The cultures were then cooled (15 C) and after
Furthermore, we demonstrated the use of (2S)-methyl-
malonyl-CoA, (2S)-methylmalonyl-PANT, and (2S)-methyl-
malonyl-SNAC to form a triketide pyrone in vitro using the
final module and thioesterase of 6-deoxyerythronolide B
synthase. We have also developed a coupled assay to
monitor the progress of in vitro PKS reactions via HPLC,
which proves faster and more economical than methods
previously employed, such as radio-TLC or LC/MS. The
structures of two ternary product complexes were deter-
1
6 hr cells were harvested. Protein purification, gel filtration, and concentration
of Se-MatB protein followed the methods described above.
Protein Crystallization
The following conditions were used for the protein solution of mmCoA/AMP-
bound selenomethionine-labeled MatB: 15.8 mg/ml Se-MatB, 1 mM CoA,
5
mM methylmalonate pH 7.5, 1 mM ATP, 2 mM DTT, and 5 mM MgCl
2
.
Optimal mmCoA/AMP-bound selenomethionine-labeled MatB crystals grew
2
overnight in 31% PEG 4000, 100 mM MgCl , and 100 mM Tris-HCl pH 7.5
ꢁ
via sitting drop vapor diffusion at 22 C with a protein solution to crystallization
buffer ratio of 1:1. The following conditions were used for the protein solution of
mCoA/AMP-bound MatB: 10 mg/ml MatB, 20 mM CoA, 24 mM sodium malo-
˚
mined: one with malonyl-CoA and AMP bound (1.45 A) and
another with (2R)-methylmalonyl-CoA and AMP bound
˚
2
nate pH 7.7, 24 mM ATP, and 48 mM MgCl . Optimal mCoA/AMP-bound MatB
(
1.43 A). Both ternary structures have provided insights
2
crystals grew overnight in 35% PEG 4000, 100 mM MgCl , and 100 mM Tris-
into substrate interactions, increasing the knowledge for
this subfamily of the adenylate-forming enzyme superfamily.
This research marks great progress toward preparative,
economical, in vitro, enzymatic synthesis of polyketides.
ꢁ
HCl pH 8.2 via sitting drop vapor diffusion at 22 C with a protein solution to
crystallization buffer ratio of 1:1. The following conditions were used for the
protein solution of mmCoA/AMP-bound MatB: 9.6 mg/ml MatB, 20 mM
CoA, 24 mM sodium methylmalonate pH 7.5, 24 mM ATP, and 48 mM
MgCl
2
. Optimal mmCoA/AMP-bound MatB crystals were grown overnight in
, and 100 mM Tris-HCl pH 9.5 via sitting
3
3% PEG 4000, 100 mM MgCl
2
EXPERIMENTAL PROCEDURES
ꢁ
drop vapor diffusion at 22 C with a protein solution to crystallization buffer ratio
of 1:1.
Cloning
The matB gene was amplified from S. coelicolor genomic DNA using primers
0
0
0
Data Collection, Processing, and Refinement
5
-TCGATTGCACATATGTCCTCTCTCTTCCCGGCCCTCT-3 and 5 -ATCGG
0
ATAGCTCGAGTCAGTCACGGTTCAGCGCCCGCTT-3 . The gene encoding
Diffraction data were collected at ALS beamline 5.0.2, then integrated and
scaled using HKL2000 (Otwinowski and Minor, 1997). The structure of
mmCoA/AMP-bound selenomethionine-labeled MatB was solved by single-
wavelength anomalous dispersion using Phenix (Adams et al., 2002). This
structure was used as a search model for molecular replacement using Phaser
(CCP4, 1994), yielding the solutions for both mCoA/AMP-bound MatB and
mmCoA/AMP-bound MatB. Water molecules were identified using Coot
methylmalonyl-CoA epimerase was amplified from S. coelicolor genomic
0
0
DNA using primers 5 -ATCCCGAATCATATGCTGACGCGAATCGACCA-3
0
0
and 5 -TTAGTCTGGCTCGAGTCAGTGCTCAGGTGACTCAA-3 . Fragments
were digested with NdeI and XhoI (italicized) and ligated into pET28b plasmid
between the corresponding restriction sites. Plasmid design allowed for the
0
incorporation of a stop codon (underlined) at the 3 terminus of the DNA encod-
ing both MatB and methylmalonyl-CoA epimerase. Synthetic DNA encoding
the N-terminal docking domain from DEBS3 fused to the sixth module and thio-
esterase from DEBS3 was a gift from Kosan Biosciences (Menzella et al., 2005).
The DNA was digested at the flanking NdeI and EcoRI restriction sites and
ligated into pET28b.
o c
(F -F map contoured at s = 3.0) (Emsley and Cowtan, 2004). Refinement
cycles using Coot (Emsley and Cowtan, 2004) and Refmac5 (CCP4, 1994)
were performed until the R-factors could no longer be improved (Table 1).
Figures were prepared using PyMol (DeLano, 2002) and LIGPLOT (Wallace
et al., 1995).
Protein Expression and Purification
Synthesis of N-Acetylcysteamine
MatB and methylmalonyl-CoA epimerase expression plasmids were trans-
formed into Escherichia coli BL21(DE3), whereas the Mod6TE expression
vector was transformed into E. coli K207-3 cells (Murli et al., 2003). Starter
A total of 1.14 g cysteamine hydrochloride (10.0 mmol, 1.0 eq.), 0.56 g KOH
3
(10.0 mmol, 1.0 eq.), and 2.52 g NaHCO (30.0 mmol, 3.0 eq) was dissolved
in a round-bottom flask containing 50 ml water. After the dropwise addition
cultures (50 ml) were grown overnight and used to inoculate prewarmed
ꢁ
of 0.95 ml acetic anhydride (10.0 mmol, 1.0 eq.), the solution was stirred at
22ꢁC for 10 min. The pH was then adjusted to 7.3 using 12.1 N HCl. The result-
(
37 C) Luria broth, supplemented with 50 mg/L kanamycin. When OD600
=
ꢁ
ing mixture was extracted with 150 ml ethyl acetate. The organic layer was
0
0
.4, the media was cooled (15 C) and protein expression was induced with
.5 mM IPTG. After 16 hr, cells were harvested by centrifugation, resuspended
dried using MgSO
4
, and after filtration a quantitative yield of N-acetylcyste-
1
amine was isolated in vacuo. H-NMR (CDCl ) 400 MHz, d (ppm): 5.97 (1H,
in lysis buffer (10% glycerol, 0.5 M NaCl, 100 mM HEPES pH 7.5), sonicated,
and centrifuged (30,000 relative centrifugal force for 45 min) to remove cellular
debris. Cell-free lysate was passed over a nickel-NTA column equilibrated with
lysis buffer. The column was washed with lysis buffer containing 15 mM imid-
azole and protein was eluted using lysis buffer containing 150 mM imidazole.
Final protein concentrations were determined using a Thermo Scientific Nano-
drop 1000.
3
bs), 3.40 (2H, q, J = 8.0), 2.65 (2H, m, J = 8.4), 1.98 (3H, s), 1.33 (1H, t, J = 8.8).
Synthesis of Methoxymalonic Acid
A total of 0.850 ml dimethyl methoxymalonate (6.2 mmol, 1.0 eq.) and 2.4 g
NaOH (60 mmol, 9.7 eq.) was dissolved in a round-bottom flask containing
ꢁ
10 ml water. The resulting solution was stirred at 65 C overnight. The solution
MatB was further purified for crystallization via gel filtration using a Superdex
was then acidified with 10 ml 12.1 N HCl and extracted with 30 ml ethyl
200 column equilibrated in gel filtration buffer (10% glycerol, 150 mM NaCl,
4
acetate. The organic layer was dried using MgSO , and after filtration a quan-
and 10 mM HEPES pH 7.5). By comparison to a gel filtration standard (Bio-
Rad), MatB eluted as a ꢀ50 kDa monomer. Fractions were collected and
concentrated to 15 mg/ml in gel filtration buffer (Millipore Centrifugal Filter
Unit, 30 kDa MWCO). The final protein concentration was determined by using
a Thermo Scientific Nanodrop 1000.
titative yield of methoxymalonic acid was isolated in vacuo.
Formation of D-Pantetheine
D-pantethine (0.021 mmol, 1.0 eq.) and DTT (0.022 mmol, 1.0 eq.) were dis-
ꢁ
solved in 100 ml 20% (w/v) glycerol (aq.). The solution was incubated at 60 C
ꢁ
The same purification protocol was followed for selenomethionine-labeled
MatB (Se-MatB); however, growth and expression conditions differed. Each
for 15 min., and the resulting D-pantetheine solution was stored at 22 C.
liter of M9 growth medium contained 6 g Na
2
HPO
4
, 3 g KH
2
PO
4
, 500 mg
HPLC Analysis of MatB Reactions
NaCl, 1 g NH
4
Cl, 0.1 mM CaCl
ꢁ
2
, 1 mM MgSO
4
, 0.4% glucose, and 20 mg kana-
Enzymatic reactions to generate extender units were set up using the following
conditions: 6 nM MatB, 0.55 mM CoA or 1.0 mM D-pantetheine or 4.2 mM
mycin. Prewarmed (37 C) media was inoculated with saturated starter culture
174 Chemistry & Biology 18, 165–176, February 25, 2011 ª2011 Elsevier Ltd All rights reserved

Chemistry & Biology
Enzymatic Synthesis of PKS Extender Units by MatB
SNAC, 1.3 mM ATP, 22 mM malonate derivative, 9 mM MgCl
2
, 15% (w/v)
(2002). PHENIX: building new software for automated crystallographic struc-
ꢁ
glycerol, and 100 mM HEPES pH 7.5. All reactions were incubated at 22 C
overnight. Samples were analyzed by HPLC (Waters) using a Varian Micro-
sorb-MV 300-5 C18 column and a Waters 2998 photodiode array detector.
The mobile phases consisted of water containing 0.1% TFA (solvent A) and
methanol containing 0.1% TFA (solvent B). A linear gradient (flow rate =
ture determination. Acta Crystallogr. D Biol. Crystallogr. 58, 1948–1954.
Carroll, B.J., Moss, S.J., Bai, L.Q., Kato, Y., Toelzer, S., Yu, T.W., and Floss,
H.G. (2002). Identification of a set of genes involved in the formation of the
substrate for the incorporation of the unusual ‘‘glycolate’’ chain extension
unit in ansamitocin biosynthesis. J. Am. Chem. Soc. 124, 4176–4177.
1
ml/min) of 0%–67% B over 20 min was used for sample analysis. Reactions
CCP4 (Collaborative Computational Project, Number 4). (1994). The CCP4
were monitored at 254 nm (adenine, CoA) or 235 nm (thioester bond, SNAC,
and PANT compounds).
suite: programs for protein crystallography. Acta Crystallogr.
D Biol.
Crystallogr. 50, 760–763.
Chan, Y.A., and Thomas, M.G. (2010). Recognition of (2S)-aminomalonyl-acyl
carrier protein (ACP) and (2R)-hydroxymalonyl-ACP by acyltransferases in
Zwittermicin A biosynthesis. Biochemistry 49, 3667–3677.
HPLC Analysis of Triketide Pyrone Reactions
MatB reactions were set up using the following conditions: 5 mM CoA or 5 mM
2
D-pantetheine or 5 mM SNAC, 100 mM HEPES pH 7.5, 100 mM MgCl , 20 mM
ATP (in 100 mM HEPES pH 7.5), 50 mM sodium methylmalonate pH 7.2, 10%
ꢁ
Chan, Y.A., Podevels, A.M., Kevany, B.M., and Thomas, M.G. (2009).
Biosynthesis of polyketide synthase extender units. Nat. Prod. Rep. 26,
90–114.
glycerol, and 8 nM MatB. Reactions were incubated at 22 C overnight and
analyzed by HPLC to ensure formation of mmCoA, mmPANT, or mmSNAC
(
see HPLC Analysis of MatB Reactions). Mod6TE reactions were set up by
Conti, E., Stachelhaus, T., Marahiel, M.A., and Brick, P. (1997). Structural basis
for the activation of phenylalanine in the non-ribosomal biosynthesis of grami-
cidin S. EMBO J. 16, 4174–4183.
adding (2S, 3R)-2-methyl-3-oxopentanoate-SNAC (diketideSNAC) (10 mM,
final), S. coelicolor methylmalonyl-CoA epimerase (12 nM, final), and Mod6TE
(
4 nM, final) to the MatB reaction, so that the MatB reaction was diluted 2-fold.
ꢁ
Dayem, L.C., Carney, J.R., Santi, D.V., Pfeifer, B.A., Khosla, C., and Kealey, J.T.
Reactions were incubated at 22 C for 1 day. Samples were analyzed by HPLC
(
2002). Metabolic engineering of a methylmalonyl-CoA mutaseꢂepimerase
(
Waters) using a Varian Microsorb-MV 300-5 C18 column and a Waters 2998
photodiode array detector. The mobile phases consisted of water containing
.1% TFA (solvent A) and methanol containing 0.1% TFA (solvent B). A linear
pathway for complex polyketide biosynthesis in Escherichia coli.
Biochemistry 41, 5193–5201.
0
gradient (flow rate = 1 ml/min) of 0%–100% B over 15 min was used. Reactions
were monitored at 254 nm (adenine, CoA), 235 nm (thioester bond, SNAC, and
PANT compounds), and 290 nm (triketide pyrone).
DeLano, W.L. (2002). The PyMOL Molecular Graphics System User’s Manual
(San Carlos, CA: DeLano Scientific).
Emsley, P., and Cowtan, K. (2004). COOT: model-building tools for molecular
graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132.
Mass Spectrometry Analysis
Jogl, G., and Tong, L. (2004). Crystal structure of yeast acetyl-coenzyme A
CoA-linked extender units were collected from HPLC runs and isolated in
vacuo. Samples were subjected to low-resolution negative-ESI mass spec-
trometry performed with a Finnigan LCQ ion trap mass spectrometer with
synthetase in complex with AMP. Biochemistry 43, 1425–1431.
Gao, X., Wang, P., and Tang, Y. (2010). Engineered polyketide biosynthesis
and biocatalysis in Escherichia coli. Appl. Microbiol. Biotechnol. 10.1007/
s00253-010-2860-4.
ꢁ
the needle voltage set to 3 kV and the capillary temperature set to 120 C
(Figure 2A).
Gokhale, R.S., Tsuji, S.Y., Cane, D.E., and Khosla, C. (1999). Dissecting and
exploiting intermodular communication in polyketide synthases. Science
284, 482–485.
LC/MS Analysis
The triketide pyrone and two products from the hmSNAC reaction were
collected, isolated in vacuo, resuspended in 25% acetonitrile and 0.1% formic
acid, and subjected to positive-ESI LC/MS (Agilent Technologies 1200 Series
HPLC with a Gemini C18 column coupled to an Agilent Technologies 6130
quadrupole mass spectrometer). Mobile phases consisted of water containing
Gulick, A.M., Starai, V.J., Horswill, A.R., Homick, K.M., and Escalante-
˚
Semerena, J.C. (2003). The 1.75 A crystal structure of acetyl-CoA synthetase
0
bound to adenosine-5 -propylphosphate and coenzyme A. Biochemistry 42,
2
866–2873.
Gulick, A.M., Lu, X., and Dunaway-Mariano, D. (2004). Crystal structure of
-chlorobenzoate:CoA Ligase/synthetase in the unliganded and aryl
0
.1% formic acid (solvent A) and acetonitrile containing 0.1% formic acid
solvent B). A linear gradient (flow rate = 0.7 ml/min) of 5%–95% B over
2 min was used (Figure 2C).
(
4
1
substrate-bound states. Biochemistry 43, 8670–8679.
Herzberg, O., and Moult, J. (1991). Analysis of the steric strain in the polypep-
ACCESSION NUMBERS
tide backbone of protein molecules. Proteins 11, 223–229.
Hisanaga, Y., Ago, H., Nakagawa, N., Hamada, K., Ida, K., Yamamoto, M.,
Hori, T., Arii, Y., Sugahara, M., Kuramitsu, S., et al. (2004). Structural basis
of the substrate-specific two-step catalysis of long chain fatty acyl-CoA
synthetase dimer. J. Biol. Chem. 279, 31717–31726.
Coordinates were deposited with PDB Codes 3NYR and 3NYQ for mCoA/
AMP-bound and mmCoA/AMP-bound MatB, respectively.
ACKNOWLEDGMENTS
Keatinge-Clay, A.T. (2007). A tylosin ketoreductase reveals how chirality is
We would like to acknowledge Darren Gay for helping optimize the MatB
ternary product complex crystals and Art Monzingo for organizing data collec-
tion at ALS Beamline 5.0.2. We would also like to thank the Macromolecular
Crystallography and Mass Specrometry Facilities at the University of Texas
at Austin. Funding was provided by the Robert A. Welch Foundation.
determined in polyketides. Chem. Biol. 14, 898–908.
Khosla, C., Tang, Y., Chen, A.Y., Schnarr, N.A., and Cane, D.E. (2007).
Structure and mechanism of the 6-deoxyerythronolide B synthase. Annu.
Rev. Biochem. 76, 195–221.
Kochan, G., Pilka, E.S., von Delft, F., Oppermann, U., and Yue, W.W. (2009).
Structural snapshots for the conformation-dependent catalysis by human
medium-chain acyl-coenzyme A synthetase ACSM2A. J. Mol. Biol. 388,
Received: October 1, 2010
Revised: November 25, 2010
Accepted: December 2, 2010
Published: February 24, 2011
9
97–1008.
Lee, T.V., Johnson, L.J., Johnson, R.D., Koulman, A., Lane, G.A., Lott, J.S.,
and Arcus, V.L. (2010). Structure of a eukaryotic nonribosomal peptide synthe-
tase adenylation domain that activates a large hydroxamate amino acid in
siderophore biosynthesis. J. Biol. Chem. 285, 2415–2427.
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