Cooperation between a coenzyme A-independent stand-alone initiation module and an iterative type I polyketide synthase during synthesis of mycobacterial phenolic glycolipids

Article abstract of DOI:10.1021/ja904792q

Several Mycobacterium tuberculosis strains, Mycobacterium leprae, and other mycobacterial pathogens produce a group of small-molecule virulence factors called phenolic glycolipids (PGLs). PGLs play key roles in pathogenicity and host-pathogen interaction.

Full text of DOI:10.1021/ja904792q

Published on Web 10/02/2009
Cooperation between a Coenzyme A-Independent Stand-Alone
Initiation Module and an Iterative Type I Polyketide Synthase
during Synthesis of Mycobacterial Phenolic Glycolipids
Weiguo He,†,§ Clifford E. Soll,‡ Sivagami Sundaram Chavadi,† Guangtao Zhang,#
J. David Warren,# and Luis E. N. Quadri*,†
Department of Microbiology and Immunology, Department of Biochemistry, and Milstein
Chemistry Core Facility, Weill Medical College of Cornell UniVersity, 1300 York AVenue,
New York, New York 10065, and Department of Chemistry, Hunter College, 695 Park AVenue,
New York, New York 10021
Received June 11, 2009; E-mail: leq2001@med.cornell.edu
Abstract: Several Mycobacterium tuberculosis strains, Mycobacterium leprae, and other mycobacterial
pathogens produce a group of small-molecule virulence factors called phenolic glycolipids (PGLs). PGLs
play key roles in pathogenicity and host-pathogen interaction. Thus, elucidation of the PGL biosynthetic
pathway will not only expand our understanding of natural product biosynthesis, but may also illuminate
routes to novel therapeutics to afford alternative lines of defense against mycobacterial infections. In this
study, we report an investigation of the enzymatic requirements for the production of long-chain
p-hydroxyphenylalkanoate intermediates of PGL biosynthesis. We demonstrate a functional cooperation
between a coenzyme A-independent stand-alone didomain initiation module (FadD22) and a 6-domain
reducing iterative type I polyketide synthase (Pks15/1) for production of p-hydroxyphenylalkanoate
intermediates in in vitro and in vivo FadD22-Pks15/1 reconstituted systems. Our results suggest that Pks15/1
is an iterative type I polyketide synthase with a relaxed control of catalytic cycle iterations, a mechanistic
property that explains the origin of a characteristic alkyl chain length variability seen in mycobacterial PGLs.
The FadD22-Pks15/1 reconstituted systems lay an initial foundation for future efforts to unveil the mechanism
of iterative catalysis control by which the structures of the final products of Pks15/1 are defined, and to
scrutinize the functional partnerships of the FadD22-Pks15/1 system with downstream enzymes of the
PGL biosynthetic pathway.
Introduction
pathway will not only expand our understanding of natural
product biosynthesis, but may also illuminate routes to novel
Mycobacterial infections cause serious diseases. In particular,
tuberculosis, produced by Mycobacterium tuberculosis, is
responsible for nearly two million deaths per year, and leprosy,
produced by Mycobacterium leprae, accounts for over three
million people with neuropathy-derived disabilities worldwide.
Several M. tuberculosis strains (e.g., strains of the W-Beijing
family), M. leprae, and other mycobacterial pathogens produce
a group of small-molecule virulence factors called phenolic
glycolipids (PGLs) (Figure 1).1 PGLs play key roles in
pathogenicity and host-pathogen interaction by virtue of their
immunomodulatory properties or, in the case of leprosy, their
ability to cause peripheral nerve degeneration.2 Moreover, PGL
production has been suggested as a trait predisposing M.
tuberculosis strains of the W-Beijing family to their character-
istic epidemic spread and increased likelihood of developing
drug resistance.3 Thus, elucidation of the PGL biosynthetic
therapeutics to afford alternative lines of defense against
mycobacterial infections.4
We have recently demonstrated that the protein FadD22 is
required for PGL biosynthesis and reported a FadD22 inhibitor
that blocks PGL production.4b FadD22 is comprised of a
p-hydroxybenzoic acid (pHBA) adenylation (A) domain and an
aroyl carrier protein (ArCP) domain.4b This didomain protein
(2) (a) Ng, V.; Zanazzi, G.; Timpl, R.; Talts, J. F.; Salzer, J. L.; Brennan,
P. J.; Rambukkana, A. Cell 2000, 103, 511–524. (b) Rambukkana,
A.; Zanazzi, G.; Tapinos, N.; Salzer, J. L. Science 2002, 296, 927–
931. (c) Reed, M. B.; Domenech, P.; Manca, C.; Su, H.; Barczak,
A. K.; Kreiswirth, B. N.; Kaplan, G.; Barry, C. E., III. Nature 2004,
431, 84–87. (d) Ruley, K. M.; Ansede, J. H.; Pritchett, C. L.; Talaat,
A. M.; Reimschuessel, R.; Trucksis, M. FEMS Microbiol. Lett. 2004,
232, 75–81. (e) Collins, D. M.; Skou, B.; White, S.; Bassett, S.; Collins,
L.; For, R.; Hurr, K.; Hotter, G.; de Lisle, G. W. Infect. Immun. 2005,
73, 2379–2386. (f) Tsenova, L.; Ellison, E.; Harbacheuski, R.; Moreira,
A. L.; Kurepina, N.; Reed, M. B.; Mathema, B.; Barry, C. E., III;
Kaplan, G. J. Infect. Dis. 2005, 192, 98–106. (g) Robinson, N.; Kolter,
T.; Wolke, M.; Rybniker, J.; Hartmann, P.; Plum, G. Traffic 2008, 9,
1936–1947.
† Department of Microbiology and Immunology, Weill Medical College.
# Department of Biochemistry and Milstein Chemistry Core Facility,
Weill Medical College.
‡ Hunter College.
(3) Reed, M. B.; Gagneux, S.; Deriemer, K.; Small, P. M.; Barry, C. E.,
III. J. Bacteriol. 2007, 189, 2583–2589.
§ Current address: The University of Texas Health Science Center at
Houston, 1825 Pressler Street, Houston, TX 77030.
(1) Onwueme, K. C.; Vos, C. J.; Zurita, J.; Ferreras, J. A.; Quadri, L. E.
Prog. Lipid Res. 2005, 19, 259–302.
(4) (a) Quadri, L. E. Infect. Disord. Drug Targets 2007, 7, 230–237. (b)
Ferreras, J. A.; Stirrett, K. L.; Lu, X.; Ryu, J. S.; Soll, C. E.; Tan,
D. S.; Quadri, L. E. Chem. Biol. 2008, 15, 51–61.
9
16744 J. AM. CHEM. SOC. 2009, 131, 16744–16750
10.1021/ja904792q CCC: $40.75  2009 American Chemical Society

Biosynthesis of Mycobacterial Phenolic Glycolipids
A R T I C L E S
3-6, and 8). Conversely, neither FadD22(S576A) (lane 7), a
phosphopantetheinylation site-deficient mutant with Ser-576
substituted by Ala,4b nor apo-FadD22 (not shown) were capable
of autoacylation, indicating that a functional ArCP domain is
required for autoacylation. These results are in agreement with
FadD22 autoacylation studies reported previously.4b Holo-
Pks15/1 was robustly radiolabeled in the reaction containing
holo-FadD22 (lane 3). Conversely, holo-Pks15/1 radiolabeling
was not detected in reactions lacking holo-FadD22 (lane 2),
containing FadD22(S576A) in place of holo-FadD22 (lane 7),
or lacking ATP (lane 8).
Starter unit loading onto three Pks15/1 site-directed mutants
was investigated as well. These mutants had Ala substitutions
at the predicted catalytic Cys (Cys-211) of the ꢀ-ketoacyl
synthase (KS) domain and/or the phosphopantetheinylation site
(Ser-2039) of the acyl carrier protein (ACP) domain. These key
amino acid residues were identified by sequence analysis (Figure
S1, Supporting Information). Pks15/1(S2039A), the ACP do-
main mutant defective for carrier domain phosphopantethein-
ylation (Figure S2, Supporting Information), was radiolabeled
like wild-type (cf., lanes 3 and 5). In contrast, holo-Pks15/
1(C211A), the KS domain mutant, and Pks15/1(C211A/
S2039A), the double mutant, were not competent for label
incorporation (lanes 4 and 6, respectively). These observations
indicate that a functional KS domain is required for starter unit
loading, whereas a functional ACP domain is not. Overall, the
results from the starter unit loading analysis are consistent with
a model in which the p-hydroxybenzoyl unit loaded onto the
holo-ArCP domain of FadD22 is transferred to the KS domain
active-site Cys-211 residue in Pks15/1. Formation of a p-
hydroxybenzoyl-Cys-KS domain thioester intermediate would
parallel the priming mechanism of other type I PKSs with
ordinary acetyl-CoA- or propionyl-CoA-derived starter units.9
Pks15/1 was also competent for autoacylation with the predicted
extender unit, the malonyl group derived from malonyl-CoA.
Covalently bound [14C]malonyl-CoA-derived label was readily
incorporated onto Pks15/1 (not shown). This labeling is expected
to result from AT domain-catalyzed acylation of the ACP
domain as seen in other PKSs.9
Synthesis of PHPA Products in the in Vitro and in Vivo
FadD22-Pks15/1 Reconstituted Systems. Having demonstrated
FadD22-dependent loading of the starter unit onto Pks15/1 and
the ability of Pks15/1 for autoacylation with the extender unit,
we next evaluated the ability of Pks15/1 to synthesize PHPA
products in the in vitro and in vivo FadD22-Pks15/1 reconsti-
tuted systems. In these studies, PHPA formation was assessed
by liquid chromatography-mass spectrometry (LC-MS) analy-
sis of extracted products released from the enzyme after alkaline
hydrolysis of in vitro enzymatic reactions or lysates of engi-
neered E. coli strains expressing the FadD22-Pks15/1 system.
Gratifyingly, incubation in vitro of holo-FadD22 and holo-
Pks15/1 in the presence of pHBA, malonyl-CoA, ATP, and
NADPH led to formation of a characteristic product series. The
products in this series had exact masses consistent with predicted
PHPAs differing by multiples of the 28-amu unit (-CH2CH2-)
expected from the presence of different numbers of two-carbon
extender units (Figure 3a; Table 1; Figure S3 in Supporting
Figure 1. Representative PGLs. The carbon-chain variability shown is that
of the PGLs produced by the opportunistic human pathogen Mycobacterium
marinum. The glycosyl substituent in PGLs is strain/species-specific. The
involvement of the FadD22-Pks15/1 enzyme system in the biosynthesis of
the phenolphthiocerol moiety of PGLs is illustrated according to the model
proposed herein based on the results of in vitro and in vivo studies carried
out with the proteins from M. marinum.
forms a p-hydroxybenzoyl-S-FadD22 thioester intermediate from
pHBA and ATP in a coenzyme A (CoA)-independent manner.4b
We hypothesize that this intermediate primes the biosynthesis
of the long-chain ꢀ-diol-containing moiety of PGLs [phenol-
phthiocerol (PPOL)] by presenting the pHBA starter unit for
elongation by Pks15/1 to form p-hydroxyphenylalkanoate
(PHPA) intermediates during PPOL biosynthesis (Scheme 1).
Genetic studies have demonstrated that the pks15/1 gene is
required for PGL biosynthesis,2c,5 and our sequence analysis
indicates that Pks15/1 is well-conserved among PGL producers
(2104-2118 amino acid residues, 79-100% sequence identity;
Figure S1, Supporting Information). Pks15/1 is a 6-domain
protein with homology to reducing iterative type I polyketide
synthases (PKSs),1 a poorly understood family of enzymes that
synthesize highly reduced products such as lovastatin, T-toxin,
and fumonisin.6 The proposed PHPA intermediates synthesized
by the FadD22-Pks15/1 system are likely to be further extended
by the PpsA-E noniterative type I PKS system to complete
PPOL biosynthesis.1 Subsequently, PPOL is esterified with
characteristic long-chain multimethyl-branched fatty acids
(Figure 1) by the acyltransferase PapA5.7
Herein, we report in vitro and in vivo investigations that
demonstrate the functional partnership between FadD22 and
Pks15/1 for production of PHPA intermediates from the PGL
biosynthetic pathway.
Results and Discussion
Loading of Starter and Extender Units onto the FadD22-
Pks15/1 System. We first evaluated whether Pks15/1 could be
loaded with pHBA in a FadD22-dependent manner in vitro. To
this end, incorporation of covalently bound [14C]pHBA-derived
label onto Pks15/1 and FadD22 was monitored by standard
sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) followed by autoradiography (Figure 2).8 Phos-
phopantetheinylated (holo)-FadD22 was readily autoacylated in
an ATP-dependent and CoA-independent manner (cf., lanes 1,
(5) Constant, P.; Perez, E.; Malaga, W.; Laneelle, M. A.; Saurel, O.; Daffe,
M.; Guilhot, C. J. Biol. Chem. 2002, 277, 38148–38158.
(6) Cox, R. J. Org. Biomol. Chem. 2007, 5, 2010–2026.
(7) (a) Buglino, J.; Onwueme, K. C.; Ferreras, J. A.; Quadri, L. E.; Lima,
C. D. J. Biol. Chem. 2004, 279, 30634–30642. (b) Onwueme, K. C.;
Ferreras, J. A.; Buglino, J.; Lima, C. D.; Quadri, L. E. Proc. Natl.
Acad. Sci. U.S.A. 2004, 101, 4608–4613.
(8) See the Supporting Information for additional experimental procedures
(plasmid constructions, protein purification, and PHPA standard
synthesis), sequence alignment, and Figures S1-S6.
(9) (a) Staunton, J.; Weissman, K. J. Nat. Prod. Rep. 2001, 18, 380–416.
(b) Fischbach, M. A.; Walsh, C. T. Chem. ReV. 2006, 106, 3468–
3496.
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A R T I C L E S
He et al.
Scheme 1. CoA-Independent Loading of the pHBA Starter Unit and Chain Elongation for Biosynthesis of PHPA Intermediates by the
FadD22-Pks15/1 System during PGL Assemblya
a Pks15/1 is shown loaded with the malonyl extender unit. The indicated range of iterative cycles and consequent carbon-chain variability in the
p-hydroxyphenylalkanoyl-S-Pks15/1 thioester intermediate is that expected during biosynthesis of PGLs from M. marinum. Domain abbreviations: A, adenylation;
ACP, acyl carrier protein; ArCP, aroyl carrier protein; AT, acyltransferase; DH dehydratase; ER, enoylreductase; KR, ketoreductase; KS, ketosynthase. The
thiols of the phosphopantetheinyl group in the carrier domains and the catalytic cysteine in the KS domain are shown.
petent for PHPA formation either (not shown). These results
are consistent with the fact that Pks15/1(C211A) and Pks15/
1(C211A/S2039A) cannot be loaded with the starter unit (Figure
2) and Pks15/1(S2039A) is defective for ACP domain phos-
phopantetheinylation (Figure S2, Supporting Information). The
results obtained with the mutant proteins are in agreement with
the hypothesized functions of Cys-211 and Ser-2039.
Encouragingly, FadD22-Pks15/1-dependent biosynthesis of
the PHPA product series was achieved also in an E. coli strain
engineered to express FadD22 and Pks15/1 from plasmids
pCDF-FadD22 and pETDuet-Pks15/1 (Figure 3b; Table 1;
Figure S3 in Supporting Information). This strain also contained
the plasmid pSU20-Sfp. pSU20-Sfp expresses Bacillus subtilis
phosphopantetheinyl transferase Sfp,11 which was used herein
to obtain FadD22 and Pks15/1 in their holo forms. Conversely,
the PHPA product series was not detected in control strains in
which pETDuet-Pks15/1 was replaced with the pETDuet-1
vector, pCDF-FadD22 was replaced with the pCDF-1 vector,
pSU20-Sfp was omitted, or the culture medium was not
supplemented with the starter unit, pHBA (not shown).
Figure 2. FadD22-dependent loading of pHBA onto Pks15/1. (a) Image
of a coomassie blue-stained tris-glycine polyacrylamide gel run under
denaturing conditions (7.5%, SDS-PAGE). Lanes 1-8 were loaded with
acylation reaction mixtures with the indicated composition of key compo-
nents. Lane M, molecular weight marker. (b) Autoradiograph image of the
gel showing incorporation of [14C]pHBA-derived covalent label onto the
proteins.
The results of the PHPA production studies support the idea
that the FadD22-Pks15/1 reconstituted systems produce PHPAs
with alkyl chain length variability consistent with a 5-to-14 range
of iterative extension cycles with the malonyl-CoA-derived
extender unit. This suggests that Pks15/1 is a reducing iterative
type I PKS with a relaxed control of catalytic cycle iterations.
Importantly, such a mechanistic property would explain the
origin of the characteristic alkyl chain length variability seen
in the PPOL moiety of PGLs.1 Notably, one of the most
prominent PHPA peaks observed in the product series (peak 7,
Figure 3) corresponds to 23-(4-hydroxyphenyl)tricosanoic acid
(C29H50O3; calculated exact mass, 446.3760; experimental
neutral mass in the in vitro and in vivo reconstituted systems,
446.3760 and 446.3763, respectively; Table 1). This PHPA
product is predicted to arise from elongation of the starter unit
with 11 extender units, and its generation will require a total of
68 catalytic operations by the FadD22-Pks15/1 system [68 ) 1
(pHBA adenylation) + 1 (pHB-S-ArCP formation) + 1 (pHB-
S-KS formation) + 11 × 4 (condensation, reduction, dehydra-
tion, enoyl reduction) + 11 (AT-catalyzed malonyl unit loading
onto the ACP at each of 11 cycles) + 10 (transfer of
intermediate from ACP to KS, except in the last cycle)].
Coincidently, 23-(4-hydroxyphenyl)tricosanoic acid would be
the hypothesized PHPA intermediate extended by the PpsA-E
PKS system to form the PPOL with the largest carbon chain
reported in M. marinum (Figure 1).
Information). The retention time and the observed ion [M -
H+]- m/z of a synthetic 21-(4-hydroxyphenyl)henicosanoic acid
PHPA standard (C27H46O3, calculated exact mass: 418.3447;
experimental neutral mass 418.3453, where the experimental
neutral mass is the observed ion [M - H+]- m/z plus H+,
1.00728 Da) matched the retention time and the ion [M - H+]-
m/z of the corresponding PHPA product (C27H46O3) (peak 6 in
Figure 3; Table 1; Figure S3 in Supporting Information). This
PHPA was one of the most abundant in the PHPA product
series. The compounds in the PHPA product series also had
the same MS/MS fragmentation pattern of the synthetic PHPA
standard. This pattern was consistent with loss of H2O (m/z [M
- H+ - 18, M - H+ - H2O]-) or CO2 (m/z [M - H+ - 44,
M - H+ - CO2]-) by the [M - H+]- parent ion (Figure S3,
Supporting Information).
Conversely, the PHPA product series was not detected in
control reactions lacking a single enzyme or the substrates
malonyl-CoA or NADPH (not shown). Traces of the most
prominent product peaks in the series were seen in controls
lacking pHBA or ATP (not shown). This may result from
purified FadD22 preparations containing traces of the p-
hydroxybenzoyl-S-FadD22 covalent intermediate or the stable
p-hydroxybenzoyl-AMP · FadD22 noncovalent intermediate.4b
These intermediates could form in E. coli using endogenous
pHBA, an intermediate in ubiquinone synthesis.10 Reactions in
which holo-Pks15/1 was replaced with holo-Pks15/1(C211A),
Pks15/1(S2039A), or Pks15/1(C211A/S2039A) were not com-
(11) Quadri, L. E.; Weinreb, P. H.; Lei, M.; Nakano, M. M.; Zuber, P.;
(10) Meganathan, R. FEMS Microbiol. Lett. 2001, 203, 131–139.
Walsh, C. T. Biochemistry 1998, 37, 1585–1595.
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Biosynthesis of Mycobacterial Phenolic Glycolipids
A R T I C L E S
Figure 3. Analysis of PHPA products synthesized by FadD22-Pks15/1 reconstituted systems. Extracted ion chromatograms for the [M - H+]- ion exact
masses (listed above) of the expected PHPA products from in vitro (a) and in vivo (b) reconstituted systems are shown. The PHPA structures are illustrated.
Data representative of three experiments are shown.
The 5-to-14 range of extension cycles observed in the
reconstituted systems is wider than the 9-to-11 range deduced
from reported M. marinum PPOL structures.1 In connection with
this observation, it is worth noting that, in nonreducing iterative
type I PKS systems, a product template (PT) domain and a
thioesterase (TE) domain are proposed to be involved in the
control of product chain length, cyclization, and release.6,12 In
contrast, reducing iterative type I PKSs, such as Pks15/1, lack
the PT and TE domains, implying a distinct mechanism for the
control of the structure of the final products. In fact, Du and
co-workers recently suggested that the final size of highly
reduced polyketide products of iterative type I PKSs is not
determined by the PKSs alone.13 It is possible that, in the case
of Pks15/1, the complete information for final product size is
“programmed” via the interaction of Pks15/1 with other enzymes
of the PGL pathway. In this regard, our FadD22-Pks15/1
reconstituted systems lay a first foundation for future efforts to
unveil the mechanism of iterative catalysis control by which
the structures of the final products of Pks15/1 are defined, and
to scrutinize the functional partnerships of Pks15/1 with
downstream enzymes of the PGL pathway to achieve PPOL
biosynthesis.
of our knowledge, this is the first demonstration of aroyl-S-
ArCP-dependent priming of an iterative PKS. Our results are
consistent with a model in which (1) pHBA is transferred from
the ArCP domain in p-hydroxybenzoyl-FadD22 to the KS
domain active-site Cys residue of Pks15/1 and (2) the formed
p-hydroxybenzoyl-KS transient intermediate serves as a substrate
for Pks15/1-dependent acyl chain elongation using the malonyl-
CoA-derived extender unit for biosynthesis of PHPA intermedi-
ates during PGL production (Scheme 1). The presented data
suggest that Pks15/1 has a relaxed control of catalytic cycle
iterations, a mechanistic property that explains the origin of a
characteristic alkyl chain length variability seen in mycobacterial
PGLs. Overall, this study provides novel insight into the
biosynthesis of PGLs, an important group of mycobacterial
virulence factors.
Experimental Section
Reagents. Unless otherwise stated, all commercially available
materials were purchased from Aldrich, Acros Organics, Fisher
Scientific, TCI, or Alfa Aesar and were used without any further
purification. When necessary, solvents and reagents were dried prior
to use, using standard protocols. Radiolabeled [carboxy-14C]p-
hydroxybenzoic acid and [2-14C]malonyl-CoA were purchased from
American Radiolabeled Chemicals, Inc. 12-N-Biotinyl-(aminodode-
canoyl)-Coenzyme A (biotinyl-CoA) was purchased from Avanti
Polar Lipids, Inc. Monoclonal antibiotin-alkaline phosphatase
conjugated antibody was obtained from Sigma-Aldrich. All other
molecular biology reagents were from Sigma, Invitrogen, New
England Biolabs, Novagen, QIAGEN, or Stratagene. Oligonucle-
otide primers were purchased from Integrated DNA Technologies,
Inc. The synthetic PHPA standard [21-(4-hydroxyphenyl)hen-
icosanoic acid; C27H46O3] was synthesized as described in the
Supporting Information and outlined in Figure S4.
Conclusion
The studies reported herein demonstrate the functional
partnership between FadD22, a pHBA-specific CoA-independent
stand-alone didomain initiation module, and Pks15/1, a 6-do-
main reducing iterative type I PKS, for production of PHPA
intermediates from the PGL biosynthetic pathway. To the best
(12) Crawford, J. M.; Thomas, P. M.; Scheerer, J. R.; Vagstad, A. L.;
Kelleher, N. L.; Townsend, C. A. Science 2008, 320, 243–246.
(13) Zhu, X.; Vogeler, C.; Du, L. J. Nat. Prod. 2008, 71, 957–960.
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He et al.
Table 1. Mass Spectrometry Data for p-Hydroxyphenylalkanoate Products
a Peak numbers correspond to those shown in Figure 3. b The calculated exact masses for the molecular formulas shown were determined using
Agilent’s MassHunter Qualitative Analysis Software package version B.02.00 (Agilent Technologies, Inc.). c The experimental exact masses were
determined by processing the data using Agilent’s MassHunter Qualitative Analysis Software’s “Find Compounds by Formula” feature. The software
searches the data for ions based on a given molecular formula and, in this case, extracts the ions for the [M - H+]- ion for the molecular formulas of
interest. The experimental exact mass is calculated by the addition of a proton (H+; 1.00728 Da) to the measured m/z value. The experimental exact
mass is used to calculate the ppm difference from the calculated exact mass for a given molecular formula. d The ppm error was determined by
Agilent’s MassHunter Qualitative Analysis Software. PHPA, p-hydroxyphenylalkanoate.
Incorporation of Covalently Bound [14C]pHBA-Derived Label
onto Pks15/1 and FadD22 in Vitro. For these and all other
experiments with FadD22 and Pks15/1 described in this study,
we used the proteins from the opportunistic human pathogen
Mycobacterium marinum.14 The proteins utilized in the experi-
ments were recombinantly produced and purified as described
in the Supporting Information (Figure S5). The starter unit
loading experiments were conducted using reported approaches
for monitoring [14C]/[3H]acyl-enzyme thioester intermediate
formation.4b,15 The standard reaction mixture (20 µL) contained
12 µM [14C]p-hydroxybenzoic acid (pHBA), 100 mM sodium
phosphate buffer (pH 7.2), 10% glycerol, 1 mM tris(2-carboxy-
ethyl)phosphine hydrochloride (TCEP), 0.5 mM MgCl2, 1 mM
ATP, 4 µM FadD22 (wild-type or mutant), and 8 µM Pks15/1
(wild-type or mutant). In control reactions, selected components
were omitted as indicated in Figure 2. After incubation (30 °C,
2 h), the reactions were quenched by the addition of SDS-PAGE
loading buffer16 (10 µL, 2× concentrated, lacking reducing
reagent) and analyzed by standard SDS-PAGE (7.5%).16 For
detection of 14C-labeled proteins, the polyacrylamide gels were
treated with Amplify Fluorographic Reagent (GE Healthcare)
as recommended in the manufacturer’s manual. The treated
(15) (a) Quadri, L. E.; Keating, T. A.; Patel, H. M.; Walsh, C. T.
Biochemistry 1999, 38, 14941–14954. (b) Quadri, L. E.; Sello, J.;
Keating, T. A.; Weinreb, P. H.; Walsh, C. T. Chem. Biol. 1998, 5,
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(14) (a) Katoch, V. M. J. Med. Res. 2004, 120, 290–304. (b) Jarzembowski,
J. A.; Young, M. B. Arch. Pathol. Lab. Med. 2008, 132, 1333–1341.
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A R T I C L E S
polyacrylamide gels were exposed to storage phosphor screens
(GE Healthcare), which were subsequently scanned using a
Typhoon Trio Imager (Amersham Biosciences).
of 2 M HCl, the products were extracted with ethyl acetate (1.4
mL × 5), and the recovered organic layer was evaporated to
dryness. The residual material was dissolved in 50 µL of dichlo-
romethane/methanol (3:1, vol/vol) and analyzed by LC-MS as
described below.
Incorporation of Covalently Bound [14C]Malonyl-CoA-Derived
Label onto Pks15/1. The extender unit loading experiments were
conducted using reported approaches for monitoring [14C]/[3H]acyl-
enzyme thioester intermediate formation.4b,15 The proteins utilized
were recombinantly produced and purified as described in the
Supporting Information. The standard reaction mixture (20 µL)
contained 50 µM [14C]malonyl-CoA, 100 mM sodium phosphate
buffer (pH 7.2), 10% glycerol, 1 mM TCEP, 0.5 mM MgCl2, 1
mM ATP, and 8 µM Pks15/1 (wild-type or mutant). After
incubation (30 °C, 3 min), the reactions were quenched by the
addition of SDS-PAGE loading buffer16 (7 µL, 3× concentrated,
lacking reducing reagent) and analyzed by standard SDS-PAGE
(7.5%).16 For detection of 14C-labeled proteins, the polyacrylamide
gels were treated and exposed to storage phosphor screens as
described in the preceding paragraph. The screens were then
scanned using a Typhoon Trio Imager.
Protein Phosphopantetheinylation Analysis. Phosphopanteth-
einylation was assessed as incorporation of the biotinylated phos-
phopantetheinyl group derived from the CoA analog biotinyl-CoA
(Avanti Polar Lipids) onto the proteins. The promiscuous Bacillus
subtilis phosphopantetheinyl transferase Sfp11 was utilized to
phosphopantetheinylate Pks15/1 proteins in vitro. Sfp was recom-
binantly produced and purified as previously reported.11 The
Pks15/1 proteins were produced recombinantly and purified as
described in the Supporting Information. The standard reaction
mixture (20 µL) contained 4 µM Pks15/1 (wild-type or mutant),
100 mM sodium phosphate buffer (pH 7.2), 10% glycerol, 2 mM
TCEP, 0.5 mM MgCl2, 50 nM Sfp, and 50 µM biotinyl-CoA. After
incubation (30 °C, 15 min), the reactions were quenched by addition
of SDS-PAGE loading buffer16 (7 µL, 3× concentrated, lacking
reducing reagent). The samples were analyzed by standard SDS-
PAGE16 (5%) and by Western blot to detect Sfp-dependent
biotinylation of Pks15/1 proteins. Western blot was carried out using
standard methodologies.16 Briefly, proteins were transferred from
the polyacrylamide gel to a hydrophobic polyvinylidene difluoride
(PVDF) membrane (Hybond-P, GE Healthcare) using a Mini Trans-
Blot electrophoretic transfer cell as recommended by the manu-
facturer (Bio-Rad Laboratories). After the electrophoretic transfer
(3 h, 200 mA), the membrane was washed with TBS-T buffer (50
mM Tris, pH 7.6; 150 mM NaCl; 0.1% Tween-20) and blocked in
5% skim milk powder in TBS-T. Covalently incorporated bioti-
nylated phosphopantetheinyl group onto the proteins was detected
using a commercial mouse monoclonal antibiotin-alkaline phos-
phatase conjugated antibody as recommended by the manufacturer
(Sigma-Aldrich). The antibody was used at a dilution of 1:10 000
relative to the original stock. The colorimetric detection of
biotinylated proteins was achieved using alkaline phosphatase BCIP/
NBT (5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt/nitro-
blue tetrazolium chloride) liquid substrate system as recommended
by the manufacturer (Sigma-Aldrich).
FadD22-Pks15/1 Reconstituted System in Vivo. The engi-
neered E. coli strains used in the in vivo reconstitution
experiments were generated by introducing selected expression
plasmids into the expression host E. coli BL21(DE3) (Strat-
agene). The plasmids were constructed as described in the
Supporting Information and outlined in Figure S6. They were
introduced into E. coli BL21(DE3) using standard electropora-
tion.16 E. coli strains were routinely cultured in Luria-Bertani
(LB) media16 containing 50 µg/mL ampicillin (pETDuet-Pks15/1
or pETDuet-1 containing strains), 20 µg/mL chloramphenicol
(pSU20-Sfp containing strains), 30 µg/mL kanamycin (pRSF-
ACC containing strains), and 50 µg/mL streptomycin (pCDF-
FadD22 or pCDF-1 containing strains). Cultures (30 mL) were
incubated with orbital shaking (220 rpm) at 37 °C. When cultures
reached an OD600nm of 0.6, recombinant protein production was
induced by addition of isopropyl-ꢀ-D-1-thiogalactopyranoside
(IPTG, 0.1 mM). pHBA (5 µM) was added to the cultures
concomitantly with IPTG, except in selected control cultures.
After 24 h of additional incubation at a reduced temperature
optimized for protein expression (18 °C, 220 rpm), cells were
harvested by centrifugation (6000g, 20 min) and washed with
100 mM sodium phosphate buffer pH 7.2 (5 mL × 3). After
being washed, the cells were resuspended in 500 µL of 100 mM
sodium phosphate buffer (pH 7.2) containing 1 mM TCEP, and
cell lysates were prepared using a Mini-Bead Beater cell disrupter
(BioSpec Products, Inc.) according to the manufacturer’s instruc-
tions. The cell lysates were treated by addition of 100 µL of 1
M NaOH and incubated at 65 °C for 20 min to release covalently
bound products from the enzymes. After acidification with 100
µL of 2 M HCl, the products were extracted with ethyl acetate
(700 µL × 5), and the recovered organic layer was evaporated
to dryness. The residual material was dissolved in 50 µL of
dichloromethane/methanol (3:1, vol/vol) and analyzed by LC-MS
as described below.
LC-MS Instrumentation and Analysis. Mass spectral data
were collected at the City University of New York (CUNY) Mass
Spectrometry Facility at Hunter College (New York, NY) on an
Agilent Technologies G6520A high-resolution Q-TOF mass
spectrometer attached to an Agilent Technologies 1200 Capillary
HPLC system. Samples were ionized by electrospray ionization
in negative mode. Chromatography was performed on a Zorbax
2.1 × 30 mm SB-C18 3.5 µm column (part no. 873700-902)
using water containing 0.1% formic acid and 50 µM ammonium
formate (solvent A) and 95% acetonitrile containing 0.1% formic
acid and 50 µM ammonium formate (solvent B) at a flow rate
of 350 µL/min. The gradient program was as follows: 2% B
(0-3 min), 2-100% B (3-15 min), 100% B (15-30 min). Total
analysis time was 30 min. The HPLC flow was diverted to waste
for the first 2.5 min. The temperature of the column was held at
45 °C for the entire analysis. Instrument parameters were as
follows: fragmentor ) 140 V; drying gas temperature ) 300
°C; drying gas flow ) 12 L/min; nebulizer pressure ) 40 psi;
and capillary voltage ) 3500 V. Data were collected with the
instrument set to low mass range (100-1700 m/z) under high-
resolution conditions at 4 GHz, and data were stored as both
centroid and profile mode. The mass spectra were collected over
a range of 115-1600 m/z at 1 spectra/s, and MS/MS spectra
were collected over a range of 60-800 m/z at 1 spectra/s with
an isolation width of ∼4 m/z. The collision energy was ramped
using a slope of 6.5 V/100 Da and an offset of 5 V using nitrogen
as the collision gas. The reference masses used were purine with
(M - H+)- at 119.03632 m/z and HP-922 with (M + formate)-
ion at 966.00072 m/z. They were infused into the spray chamber
using Agilent’s calibrant delivery system. The instrument was
FadD22-Pks15/1 Reconstituted System in Vitro. The proteins
utilized in the reconstitution experiments were produced recombi-
nantly and purified as described in the Supporting Information. The
standard reaction mixture (1 mL) contained 8 µM pHBA, 100 mM
sodium phosphate buffer (pH 7.2), 1 mM TCEP, 0.5 mM MgCl2,
1 mM ATP, 0.5 mM malonyl-CoA, 1 mM NADPH, 0.8 µM
FadD22 (wild-type or mutant), and 8 µM Pks15/1 (wild-type or
mutant). In control reactions, selected components were omitted
as indicated in the Results and Discussion. After incubation (30
°C, 5 h), the reactions were quenched by the addition of 200 µL of
1 M NaOH and incubated at 65 °C for 20 min to release covalently
bound products from the enzymes. After acidification with 200 µL
(16) Sambrook, J.; Russell, D. W. Molecular Cloning: A Laboratory
Manual, 3rd ed.; Cold Spring Harbor Laboratory Press: Cold Spring
Harbor, NY, 2001.
9
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A R T I C L E S
He et al.
controlled with Agilent MassHunter Workstation Acquisition
Software, and data were analyzed using Agilent MassHunter
Workstation Qualitative Analysis Software (Agilent Technolo-
gies, Santa Clara, CA). Funding for this instrument system was
provided by NIH Shared Instrumentation Grant 1S10RR022649-
01 and the CUNY Instrumentation Fund.
Note Added after ASAP Publication. Two names were added
to the list of authors of this publication on October 30, 2009.
Supporting Information Available: Experimental procedures
for plasmid constructions, protein purification, and PHPA
standard synthesis; sequence alignment; and Figures S1-S6.
This material is available free of charge via the Internet at http://
pubs.acs.org.
Acknowledgment. This work was supported by NIH Grant
AI069209 (L.E.N.Q.) and a Milstein Program in Chemical Biology
Grant (L.E.N.Q.). We are grateful to R. Moy and G. Sadhanandan
(L.E.N.Q. laboratory) for assistance with construction of recom-
binant plasmids.
JA904792Q
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16750 J. AM. CHEM. SOC. VOL. 131, NO. 46, 2009