Retrobiosynthetic approach delineates the biosynthetic pathway and the structure of the acyl chain of mycobacterial glycopeptidolipids

Article abstract of DOI:10.1074/jbc.M112.384966

Glycopeptidolipids (GPLs) are dominant cell surface molecules present in several non-tuberculous and opportunistic mycobacterial species. GPLs from Mycobacterium smegmatis are composed of a lipopeptide core unit consisting of a modified C₂₆-C₃₄ fatty acyl chain that is linked to a tetrapeptide (Phe-Thr-Ala-alaninol). The hydroxyl groups of threonine and terminal alaninol are further modified by glycosylations. Although chemical structures have been reported for 16 GPLs from diverse mycobacteria, there is still ambiguity in identifying the exact position of the hydroxyl group on the fatty acyl chain. Moreover, the enzymes involved in the biosynthesis of the fatty acyl component are unknown. In this study we show that a bimodular polyketide synthase in conjunction with a fatty acyl-AMP ligase dictates the synthesis of fatty acyl chain of GPL. Based on genetic, biochemical, and structural investigations, we determine that the hydroxyl group is present at the C-5 position of the fatty acyl component. Our retrobiosynthetic approach has provided a means to understand the biosynthesis of GPLs and also resolve the long-standing debate on the accurate structure of mycobacterial GPLs.

Full text of DOI:10.1074/jbc.M112.384966

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 36, pp. 30677–30687, August 31, 2012
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Retrobiosynthetic Approach Delineates the Biosynthetic
Pathway and the Structure of the Acyl Chain of Mycobacterial
□
S
Glycopeptidolipids*
Received for publication, May 24, 2012, and in revised form, June 27, 2012 Published, JBC Papers in Press, July 13, 2012, DOI 10.1074/jbc.M112.384966
Archana Vats‡§1,2, Anil Kumar Singh¶ʈ1,3, Raju Mukherjee**, Tarun Chopra§, Madhu Sudhan Ravindran§,
Debasisa Mohanty§, Dipankar Chatterji**, Jean-Marc Reyrat¶†, and Rajesh S. Gokhale‡§‡‡4
From the ‡CSIR-Institute of Genomics and Integrative Biology, Mall Road, Delhi 110007, India , ¶Université Paris Descartes, Faculté
de Médecine Paris Descartes, INSERM, U570, Paris Cedex 15 F-75730, France, the ʈDepartment of Biotechnology, CSIR-North East
Institute of Science and Technology, Jorhat, Assam 785006, India, the **Molecular Biophysics Unit, Indian Institute of Science,
Bangalore 560012, India, the §National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India, and the
‡‡Jawarharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore 560064, India
Background: Precise chemical structure and biosynthetic pathway of the acyl chain of mycobacterial glycopeptidolipids
(GPLs) is unknown.
Results: Polyketide synthases dictate biosynthesis and determine hydroxylation at C-5 position of the GPL acyl chain.
Conclusions: Retrobiosynthetic studies establish the role of bimodular polyketide synthase and fatty acyl-AMP-ligase in GPL
biosynthesis.
Significance: The long-standing ambiguity on the accurate structure and the biosynthetic mechanism of GPLs was resolved.
(GPLs)5 are one of the most abundant cell-surface glycolipids
synthesized by several non-tuberculous opportunistic myco-
bacteria. GPL molecules provide distinct surface properties to
these organisms, like sliding motility and biofilm development
(3, 4). GPLs are also known to play a significant role in patho-
genesis by activating the host immune response (5). Subtle
structural variability of these molecules generates phenotypic
heterogeneity between the different mycobacterial species.
Despite the characterization of first GPL structure (6) and the
genetic cluster (7), precise chemistry as well as mechanism
involved in synthesizing the acyl chain component of GPL is not
clear.
Glycopeptidolipids (GPLs) are dominant cell surface mole-
cules present in several non-tuberculous and opportunistic
mycobacterial species. GPLs from Mycobacterium smegmatis
are composed of a lipopeptide core unit consisting of a modified
C26-C34 fatty acyl chain that is linked to a tetrapeptide (Phe-
Thr-Ala-alaninol). The hydroxyl groups of threonine and termi-
nal alaninol are further modified by glycosylations. Although
chemical structures have been reported for 16 GPLs from
diverse mycobacteria, there is still ambiguity in identifying the
exact position of the hydroxyl group on the fatty acyl chain.
Moreover, the enzymes involved in the biosynthesis of the fatty
acyl component are unknown. In this study we show that a
bimodular polyketide synthase in conjunction with a fatty acyl-
AMP ligase dictates the synthesis of fatty acyl chain of GPL.
Based on genetic, biochemical, and structural investigations, we
determine that the hydroxyl group is present at the C-5 position
of the fatty acyl component. Our retrobiosynthetic approach has
provided a means to understand the biosynthesis of GPLs and
also resolve the long-standing debate on the accurate structure
of mycobacterial GPLs.
GPLs are reported to have a common fatty acyl tetrapeptide
core consisting of tetrapeptide amino alcohol (D-Phe-D-allo-
Thr-D-Ala-L-alaninol) and amide-linked long-chain fatty acid
(C26–34) (8). The fatty acyl-tetrapeptide core is glycosylated
with 6-deoxy-talose and variable O-methyl-rhamnose residues
(9, 10). The GPLs from Mycobacterium avium are further mod-
ified such that an additional Rha residue is added to 6-deoxy-
talose. Presently 31 distinct serotype-specific GPLs have been
identified, of which chemical structures for 16 have been
reported (11).
The GPL biosynthetic gene cluster has now been identified in
several mycobacterial species. In Mycobacterium smegmatis,
the cluster maps to a single locus of ϳ65 kb in the genome,
containing ϳ30 open reading frames (ORF) (Fig. 1) (12).
Genetic knock-out and complementation studies have estab-
lished an understanding of many of the biosynthetic enzymes
involved in modification of sugar moieties such as O-methyl-
Mycobacteria produce diverse lipid metabolites, many of
which are unique to this species (1, 2). Glycopeptidolipids
* This work was supported by Le Centre Indo-Franc¸ais Pour la Recherche
Avance´e (CEFIPRA)/Indo-French Centre for the Promotion of Advanced
Research (IFCPAR) Grant 3403-03 (to J.-M. R. and R. S. G.)
□
S
This article contains supplemental Figs. S1–S5.
1 Both authors contributed equally to this work.
2 Supported by a Department of Biotechnology, Government of India, post-
doctoral fellowship.
3 Supported by CEFIPRA/IFCPAR.
5 The abbreviations used are: GPL, glycopeptidolipid; dGPL, de-O-acylated
GPL; PKS, polyketide synthase; FAAL, fatty acyl-AMP; AT, acyltransferase;
MCoA, malonyl-CoA; NAC, N-acetylcysteamine; SNAC, thio-NAC; Fmoc,
N-(9-fluorenyl)methoxycarbonyl; KS, ketosynthase; ACP, acyl carrier pro-
tein; FaPhe, fatty acylphenylalanine.
† Deceased.
4 To whom correspondence should be addressed: CSIR-Institute of Genomics
and Integrative Biology, Mall Road, Delhi 110007, India. Tel.: 91-11-
27662407; Fax: 91-11-27667471; E-mail: rsg@igib.res.in.
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Polyketide Synthase Dictates Synthesis of Acyl Chain in GPL
transferase, acetyltransferase (13–15), and also of those in- Microsynth. Radioactive malonyl-CoA was purchased from
volved in the transport of these metabolites to the cell surface PerkinElmer life Sciences (1.9166 GBq/mmol), whereas radio-
(16). The disruption of genes involved in lipopeptidic core unit active methylmalonyl-CoA was purchased from American
led to the loss of metabolites. These include three large multi- Radiolabeled Chemicals (55 mCi/mmol). XLI-blue (Stratagene)
functional proteins whose biochemical function is not known. and BAP1 (29) strains of Escherichia coli were used for cloning
Two of these ORFs correspond to non-ribosomal peptide syn- and expression, respectively. DNA purification kits and nickel-
thetases, designated as mps1 and mps2. Each of these proteins nitrilotriacetic acid-agarose resin were procured from Qiagen.
possesses two sets of modules that can be postulated to be All other chemicals were of analytical grade from Sigma.
responsible for synthesizing the tetrapeptide backbone, and the
Cloning, Expression, and Purification of GPL Protein—The
novel C-terminal reductase domain present on non-ribosomal 10,959-bp pks gene was cloned in parts, which were assembled
peptide synthetase has been recently shown to reductively together by utilizing the restriction site engineered during the
release the chain as acyl-peptidyl-alaninol (17). However, the PCR (supplemental Fig. S1). PKS protein was expressed in
functional relevance of the polyketide synthase (PKS) protein BAP1 strain of E. coli. The cells were grown at 30 °C in LB
cannot be predicted.
medium with 50 ␮g/ml antibiotic (carbenicillin) to an absorb-
In recent years the involvement of PKS proteins in synthesiz- ance of ϳ0.6 at 600 nm. The cells were incubated at room tem-
ing unusual acyl chains of complex lipids in Mycobacterium perature for 10 min and induced with 0.5 mM isopropyl thio-␤-
tuberculosis has been established (18, 19). Many PKSs function D-galactoside for 12–18 h at 22 °C. The cells were harvested by
along with fatty acyl-AMP ligases (FAALs), which have been centrifugation (3500 ϫ g for 15 min), resuspended in buffer A
demonstrated to provide starter fatty acid precursors (20–22). (50 mM Tris chloride (pH 8.0) containing 10% glycerol) contain-
During polyketide biosynthesis, the final chemistry of the ing 150 mM NaCl, and lysed using a French press, and cellular
polyketide is dictated by the domain organization of the partic- debris was removed by centrifugation (22,500 rpm, 40 min; Sor-
ipating PKS and the substrate specificity of the corresponding vall evolution RC, rotor: SA300). Polyethyleneimine was added
acyltransferase (AT) domain. PKSs can catalyze chain exten- to a final percentage of 0.1%, and the DNA pellet was removed
sion by either following an iterative or a modular mechanism of by centrifugation (20,000 rpm, 30 min; Sorvall evolution RC,
ketide (-CH2CO-) unit condensation. Although the iterative rotor: SA300). Nickel nitrilotriacetic-agarose resin was added
enzymes catalyze successive condensations through repetitive to the supernatant (0.75 per ml of culture), and the protein was
utilization of the same set of active sites, modular proteins work purified using affinity chromatography with an increasing con-
in an assembly line wherein each set of active sites involved in centration of imidazole in buffer A. Anion exchange chroma-
condensation and associated reduction steps is used just once tography was performed by using a 6-ml Resource Q column
during biosynthesis. Recently, another hybrid “modularly and AKTA chromatography system (GE Healthcare). The pro-
iterative” mechanism of biosynthesis was described for the tein was purified in buffer B (100 mM phosphate buffer (pH 7.2)
bimodular PKS12 protein in M. tuberculosis (23). Despite all containing 10% glycerol) and eluted with an increasing gradient
these years and several structural reports, the position of of 1 M NaCl in buffer B with a flow rate of 2 ml/min (supple-
hydroxyl group on the acyl chain is contentious, and at least two mental Fig. S2). Protein concentration was determined by using
distinct positions of hydroxylation (␤ and ␦) have been reported BCA protein estimation kit (Pierce).
(7, 24–27).
Labeling of Enzyme by [14C]Methylmalonyl- and [14C]-
In this study we demonstrate that the PKS protein from the Malonyl-CoA—Enzyme (1–5 ␮M) was incubated with [14C]-
GPL cluster (designated GPL-PKS) is involved in biosynthesis malonyl-CoA (MCoA) and [14C]methyl malonyl-CoA (36 ␮M)
of the acyl component of GPLs. Based on genetic, biochemical, at 4 °C for 2 min. The reaction was quenched by the addition of
cell-free reconstitution, and structural analyses, we show that SDS-PAGE loading dye lacking any reducing agents like dithi-
the PKS enzyme dictates the position of the hydroxyl group on othreitol or ␤-mercaptoethanol. Samples were directly loaded
the acyl chain. Our retrobiosynthetic studies provide an inter- on a 6% SDS-PAGE gel, and electrophoresis was performed at
esting approach to establish chemical structures of complex 25 mA until the dye front ran out. The gel was dried and ana-
metabolites.
lyzed using phosphorimaging (Fuji BAS5001).
Enzymatic Assay and Product Characterization—PKS enzy-
matic assays induced 100 mM phosphate buffer (pH 7.2), 150
EXPERIMENTAL PROCEDURES
General Materials and Methods—M. smegmatis mc2155 (28) ␮M acyl-N-acetylcysteamine (NAC), 75 ␮M MCoA, 7.2 ␮M
and related mutants were grown in Middlebrook 7H9 medium [14C]MCoA, 4 mM NADPH, 10% glycerol, 2 mM Tris(2-car-
(Difco) supplemented with 2% (w/v) glucose and 0.05% (v/v) boxyethyl)phosphine hydrochloride, and 2.5–4.5 ␮M protein in
Tween-80 at 37 °C. Kanamycin and hygromycin were added to a 100-␮l reaction volume. The reaction mixture was incubated
a final concentration of 50 and 100 ␮g/ml, respectively, at 30 °C for 6–12 h. The products were released with 45% KOH
required for mutants and overexpression constructs. The followed by acidification and extraction in ethyl acetate. The
growth profile of liquid cultures was obtained by measuring the extract was spotted on a TLC plate that was developed using
A
600 at regular time intervals. The sequence analysis of mc2155 ethyl acetate:hexane:acetic acid (45:90:3.75). The radio TLC
genomic DNA was done at TIGR. The restriction and modifi- plates were analyzed by using phosphorimaging. For radio-
cation enzymes were purchased from New England Biolabs. HPLC assays, the concentration of [14C]MCoA was increased
The Ampli taq gold enzyme was obtained from Applied to 25 ␮M while keeping the total MCoA concentration as 100
Biosystems. Synthetic oligonucleotides were obtained from ␮M. The band of interest was scraped from TLC, extracted in
30678 JOURNAL OF BIOLOGICAL CHEMISTRY
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Polyketide Synthase Dictates Synthesis of Acyl Chain in GPL
ethyl acetate, concentrated, and injected on C18 reverse-phase 5% ␣-naphthol/sulfuric acid in ethanol followed by charring at
120 °C for 10 min.
column (gradient: 35% B in 5 min, 100% B in 10 min, 100% B in
25 min, 20% B in 40 min, 20% B in 50 min; A: H2O, B: 5%
methanol in ACN with 0.1% formic acid) on a HPLC
(Shimadzu). The radioactivity was monitored online by a radio-
active detector (IN/US system ␤-RAM Model 3) and also by
using a Photodiode array detector.
Purification and Analysis of GPLs—The crude lipids were
separated on a Florisil column (60–100 mesh) with increasing
concentration of methanol in chloroform. Fractions were mon-
itored by thin layer chromatography on silica-coated plates.
Fractions of GPLs mixture were loaded on preparative Silica
plates (20 ϫ 20 cm) F254 and developed by using 10:90:1
(CH3OH/CHCl3/H2O). Each GPL species was eluted by scrap-
ing the bands on preparative TLC and extracted in CHCl3/
CH3OH (2:1), and the purity of each GPL was monitored on
TLC by spraying plates with 5% ␣-naphthol/sulfuric acid in
ethanol followed by charring at 120 °C for 10 min (supplemen-
tal Fig. S3).
Synthesis of Alkyl NAC Thioesters—Various alkyl NAC thio-
esters were synthesized according to a modified protocol
reported earlier (20, 40).
Synthesis of 5-Hydroxydecanoic Acid—5-Hydroxydecanoic
acid was synthesized by refluxing the ␦-decanolactone in 50%
KOH solution for 8–9 h at 80 °C. The reaction mixture was
extracted with chloroform and washed with water. The organic
layer was dried with sodium sulfate (anhydrous) and concen-
trated to dryness on rotavapor (Laborota 4001; Heidolph 2).
5-Hydroxydecanoic acid was purified on preparative thin layer
chromatography and characterized by using electrospray ioni-
zation mass spectrometry (API Q- STAR pulser I, Applied
Biosystems).
To conduct structural analysis, the four tentatively assigned
de-O-acylated GPLs (dGPLs) were named dGPLI, -II, -III, and
-IV, starting from the solvent front. Analysis of the dGPLs by
MALDI-TOF mass spectrometry gave a signal corresponding
ϩ
to [MϩNa] molecular ion. The molecular ions of dGPLI, -II,
-III, and -IV were observed at m/z 1187, 1173, 1173, and 1159,
respectively, and consistent with reported GPLs from Myco-
bacterium butyricum (30).
Synthesis of Hydroxyl Alkyl Phenylalanine—Various acyl-
peptide substrates were synthesized by standard solid phase
peptide synthesis using 9-fluromethoxy carbonyl (Fmoc)
chemistry. Wang resin was preloaded with Fmoc-L-phenylala-
nine by using 1,3-diisoprylcarbodimide in the presence of 4-(di-
methylamino) pyridine in dimethylformamide. The Fmoc
group was deprotected by shaking with 20% piperidine in dim-
ethylformamide (v/v) at room temperature for 30 min and then
washed with dichloromethane followed by 3 washes of dimeth-
ylformamide. The hydroxy acyl acids were coupled with depro-
tected Fmoc-L-phenyl alanine resin using 3 eq of O-(benzotri-
azil-1-yl)-N-N-N-N-triamethyluranium-hexafluorophosphate
and 3 eq of N-N-diisopropylethylamine with shaking at room
temperature for 8 h. The resin beads were washed with dichlo-
romethane three times followed by dimethylformamide. The
alkyl peptide was cleaved from the resin by stirring with mix-
ture of TFA and water (95:5) over a period of 3 h. The cleaved
alkyl peptide was collected in chilled diethyl ether and concen-
trated on rotavapor. The hydroxylated alkyl phenylalanine was
purified by reverse phase HPLC, column C5. The chemical
identity of hydroxylated alkyl phenylalanine was confirmed by
using electrospray ionization mass spectrometry.
Acid Hydrolysis of the Glycopeptidolipids—Purified glyco-
peptidolipids (dGPLI, dGPLII, dGPLIII, and dGPLIV) were
resuspended in a minimum amount of CHCl3/CH3OH (2:1)
and refluxed at 110 °C for 24 h in a sealed tube containing 6 M
HCl solution (31). The hydrolysates thus generated were cooled
and extracted with chloroform, and the organic layer was
washed twice with water. The organic phase was concentrated,
and the hydrolyzed products were checked on a Q-TOF/MS
(API Q-STAR pulser I, Applied Biosystems).
Purification of the Fatty Acyl Acids—Hydrolyzed product,
having fatty acids, was purified by adsorption chromatography.
Concentrated mixtures of fatty acids were loaded on an open
column (silica mesh size 100–200) and washed extensively with
2% ethyl acetate in hexane and then eluted with an increment of
5–50% ethyl acetate in hexane. Fractions containing each fatty
acid were concentrated and checked by TLC and mass spectro-
metric analysis. These partially pure fractions were injected
into a preparative HPLC silica-column (Phenomenex) and
purified further. An isocratic solvent system comprising 25%
ethyl acetate in hexane at a flow of 0.2 ml/min was used, and
elution was monitored with a refractive index detector coupled
with the HPLC pumps (Waters). The acyl chain with phenyla-
lanine group were also isolated by partial hydrolysis of GPLs
and purified by column chromatography eluted in 10% chloro-
form in hexane. The alkyl-Phe-Ala moieties were also charac-
terized by mass spectrometry (Q-TOF/MS (API Q-STAR
pulser I, Applied Biosystems).
Extraction and Purification of GPLs—The M. smegmatis
mc2155 strain was grown in Middlebrook 7H9 medium till late
stationary phase, and cells were harvested (5000 rpm for 20
min). Lipids were extracted with CHCl3/CH3OH (2:1, v/v) at
room temperature for 24 h. The organic supernatant was dried
and dissolved in CHCl3/CH3OH (2:1, v/v). The crude lipids
were deacetylated by treating with an equal volume of 0.2 M
NaOH in methanol at 37 °C for 1 h and neutralized with a few
drops of glacial acetic acid. After drying, lipids were dissolved in
CHCl3/CH3OH/H2O (4:2:1) and centrifuged. The aqueous
layer was discarded, and the organic layer-containing lipids was
washed with supersaturated brine and concentrated further.
The deacylated lipids were spotted onto silica-coated TLC
plates and developed in a CHCl3:CH3OH:H2O (90:10:1) solvent
Disruption of M. smegmatis pks and Faal28 Genes—The
M. smegmatis pks mutant (MYC55) was isolated as a rough
mutant from a library of more than 19,000 transposition
mutants (16). The site of insertion of Tn611 (32) was mapped
using ligation-mediated PCR (33). Sequencing of the PCR prod-
uct has shown that Tn611 has inserted in orientation 3Ј-tnpA-
tnpR-5Ј at position 5649/10,956 of the pks gene. The GPLs
extracted from pks mutant was examined by both TLC and
system further visualized with 10% sulfuric acid in ethanol and MALDI-TOF, showing no indication of GPLs as compared
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Polyketide Synthase Dictates Synthesis of Acyl Chain in GPL
FIGURE 1. Biosynthetic pathway of GPL. A, shown is organization of GPL gene cluster in M. smegmatis. B, shown is a schematic representation of the modular
organization of PKS and non-ribosomal peptide synthetase (NRPS) of GPL cluster of M. smegmatis. Bimodular GPL-PKS contains different functional domains:
ACP, KS, AT, ketoreductase (KR) in module I along with dehydratase (DH) and enoyl reductase (ER) in module II. The PKS product from module II is then
transferred sequentially onto the non-ribosomal peptide synthetase modules (MPS1 and MPS2) of the GPL cluster, leading to the formation of peptidolipid
backbone that undergoes further modifications to form GPLs.
with wild type mc2155. The essential requirement of pks in GPL incorporation of completely reduced 2-carbon unit by the sec-
biosynthesis was confirmed by complementing the pks knock-
out with a wild type M. smegmatis pks on a mycobacterial shut-
tle vector. The complementary strain regained its smooth phe-
notype and produced GPL. faal28 gene was disrupted using the
homologous recombination method. Both pks and faal28
knock-out strains were confirmed by southern hybridizations.
ond module. Because certain PKS modules are known to con-
tain cryptic domains (36, 37), our prediction requires biochem-
ical validation. The analysis of the substrate specificity of AT
domain shows the presence of a crucial Phe residue in the active
site pocket that is known to dictate the specificity of AT
domains to utilize malonyl-CoA as the extender unit (34) (Fig.
2A). Comparative analysis of the AT domain across genomes of
several mycobacterial species of GPL-PKS showed an interest-
ing trend in their sequence conservation. The AT domains
from the two modules (AT1 and AT2) cluster independently in
different clades of the dendrogram, which indicates horizontal
transfer of this pks gene across various species (Fig. 2A).
GPL-PKS possesses another ACP domain at the N terminus
of the first module. Such a domain in mycobacterial PKS pro-
teins has been demonstrated to facilitate loading of the starter
unit from FAAL enzymes (22). Interestingly, an acyl activating
enzyme homologue from the fadD family of enzymes could be
identified in the M. smegmatis GPL cluster. Sequence analysis
indicates the presence of the FAAL specific insertion sequence
in this protein (Fig. 2B), which has been recently demonstrated
to dictate the catalytic function of this family of proteins (39).
FAAL28 is thus expected to activate starter fatty acid substrates
RESULTS
Computational Analysis of the GPL-PKS—The domain orga-
nization of the PKS protein was investigated by using the non-
ribosomal peptide synthetase-PKS database (34, 35), which
allows easy identification of domains present in the PKS
enzymes. The program predicted the GPL-PKS to encode a
bimodular protein that contains two sets of domains that could
bring about the extension of the starter chain by two ketide
units (Fig. 1). Both the modules contain the ketosynthase (KS),
AT, and acyl carrier protein (ACP) domains, which are essential
for the activity of a PKS module. Among the ␤-carbonyl modi-
fying domains, the first module contains only the ketoreductase
domain, whereas all the three auxiliary domains (dehydratase,
enoyl reductase, and ketoreductase) are present in the second
module. Thus chain extension by the first module should result
in the addition of a hydroxylated ketide group followed by
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Polyketide Synthase Dictates Synthesis of Acyl Chain in GPL
FIGURE 2. Multiple sequence alignment of the PKS-AT domain and FAAL/fatty acyl-AMP (FACL) proteins. A, shown is multiple sequence alignment and
dendrogram of PKS AT domain from module 1 and 2 for different mycobacterial species: M. avium paratuberculosis K10 (MAP), M. avium 104 (MAV), and
Mycobacterium smegmatis (MSG). B, shown is multiple sequence alignment of M. smegmatis FAAL28 with other mycobacterial FAALs and fatty acyl-AMPs,
indicating FAAL-specific insertion present in the M. smegmatis protein.
as acyl-adenylates, which are transferred to the module 1 of tein was expressed in the holo-form by phosphopantetheinyla-
GPL-PKS. As discussed above, the two rounds of condensation tion of the ACP domains by the surfactin phosphopantetheinyl
by the PKS enzyme would result in a hydroxyl group at the transferase (sfp) from Bacillus subtilis. This was achieved by
␦-position in the acyl chain of GPLs.
expressing the protein in the BAP1 strain of E. coli, which has a
Genetic Studies with pks and faal Genes—Computational single copy of the sfp gene integrated into the genome (29).
analysis suggests the possible involvement of GPL-pks and Purification by nickel-nitrilotriacetic acid chromatography fol-
faal28 genes in the synthesis of the lipopeptide core of GPLs. lowed by anion exchange chromatography showed a protein
We, therefore, predicted that knock-out of these genes would band migrating above the 212-kDa marker (Fig. 4A, lane 2). The
not be able to synthesize GPLs. faal28 gene was disrupted using size of the protein band of ϳ400 kDa was confirmed by simul-
a homologous recombination method (40, 41), the positive taneously loading PKS12 protein from M. tuberculosis on the
clones were selected on kanamycin plates, and resistant colo- same gel, which had been confirmed earlier to be of ϳ456 kDa
nies were then confirmed by southern hybridization (Fig. 3A). (supplemental Fig. S2) (23). The protein could also be detected
The pks mutant was isolated as a rough phenotype from a by an anti-histidine antibody, specific to the His6 tag. More-
library of more than 19,000 transposition mutants. The site of over, mass spectrometry sequencing of the protein showed
mutation was first mapped by using ligation-mediated PCR and peptides covering the entire protein sequence of GPL-PKS. The
then confirmed by performing a southern hybridization (Fig. specificity of the AT domains was probed by performing SDS-
3B). Both faal28 and pks mutant strains showed altered pheno- PAGE gel binding assays using MCoA and [14C]methylmalo-
typic morphology, as demonstrated by Congo Red staining, nyl-CoA, two common extenders used by majority of the PKS
sliding motility, and biofilm formation experiments (Fig. 3C). enzymes. As can be seen in Fig. 4A, lane 3 and 4, the PKS
Mass spectrometric analysis of the total lipid extract of these enzyme showed specificity only for the malonate extender, and
two strains also showed the absence of GPL (Fig. 3D). The no labeling could be detected with the methyl malonate
essential requirement of these two genes was proved unambig- extender unit. The catalytic activity of the protein was further
uously by complementing the mutant strains with cognate wild investigated by incubating various starters (C3 to C18) acyl-
type genes cloned in a mycobacterial shuttle vector. The mass NACs with radiolabeled MCoA and NADPH. Because the pro-
spectrometric analysis of these complemented strains indeed tein does not contain a chain-releasing domain, the enzyme-
confirmed the presence of GPLs (Fig. 3D). These comple- bound product was released by alkali hydrolysis (Fig. 4B) and
mented strains also substantially regained their phenotypic was detected on TLC by autoradiography. Autoradiography
GPL(ϩ) characteristics.
analysis showed a product band running at an Rf of ϳ0.2 for the
Biochemical Characterization of the GPL-PKS—The reaction primed with hexanoyl-NAC as starter (Fig. 4C, lane 2).
10,959-bp pks gene was cloned in a T7 expression vector such The Rf of this band progressively increased as the starter chain
that the protein expressed with a C-terminal His6 tag. The pro- length increased from C6 to C16 (Fig. 4C, lanes 2–6). The other
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Polyketide Synthase Dictates Synthesis of Acyl Chain in GPL
FIGURE 3. Construction and characterization of the M. smegmatis pks and faal28 mutant and complemented strains. A, shown is a schematic represen-
tation of the wild type and mutant faal28 gene and the strategy for confirming the mutants by southern hybridization. For Southern blotting, DNA was isolated
and digested with EcoNI restriction enzyme. Fragments were separated on 0.8% agarose gel, transferred, and hybridized with 32P-radiolabeled probe covering
584 bp upstream of the fadD28 gene. Restriction sites are indicated as a solid line. B, shown is a schematic representation of the wild type and mutant pks gene.
The position where Tn611 inserted in the pks gene is indicated with a black triangle (at nucleotide 5649/10,959). Transposition of Tn611 results in the insertion
of two copies of IS6100. For Southern blotting, DNA was isolated and digested with PstI restriction enzyme. Fragments were separated on 0.8% agarose gel,
transferred, and hybridized with 32P-radiolabeled probe covering the 450-bp internal fragment of IS6100 plus 409 bp corresponding to the coding sequence
of pks gene. Restriction endonuclease sites are shown as a solid line. C, shown is phenotypic analysis of M. smegmatis mc2155 (wt), pks-mutant, faal28 mutant,
and complemented strains. Phenotype on Congo Red plates, sliding motility, and biofilm formation of wild type, mutant, and complemented strains is
compared. D, shown is MALDI-TOF analysis of crude lipid fractions of the M. smegmatis mc2155 (wt), pks-mutant, and the pks complemented strains.
bands observed in the autoradiogram varied from assay to assay panel). To obtain 5-hydroxydecanoic acid, we performed alkali
and could be a result of degradation of acyl-CoAs used in the hydrolysis of the standard (Ϯ)␦-decanolactone and subjected it
reaction. According to PKS enzymology, the bimodular PKS to HPLC analysis. Surprisingly, we observed four peaks in chro-
protein would be expected to add four carbons to the starter matogram (Fig. 4D, bottom panel). One of the peaks eluted with
chain, leading to the formation of a ␦-hydroxy fatty acid a retention time identical to ␦-decanolactone, and another
that can also spontaneously cyclize to form ␦-decanolactone showed identical retention time to the GPL-PKS product. To
(Fig. 4B).
ascertain the chemical nature of this peak, we resorted to
To unambiguously establish the identity of the products, MS/MS analysis using electrospray tandem mass spectrometry.
HPLC-based assays were performed with appropriate stand- MS/MS fragmentation revealed a dehydration peak at 169
ards. Enzyme assays included purified PKS protein and a radio- atomic mass unit and a peak at 125 atomic mass unit, which is
labeled extender unit, and hexanoyl-NAC was used as a starter characteristic of loss of water and a CO2 molecule from
unit. The assay yielded one major peak at retention time of 15.7 hydroxyl fatty acids (Fig. 4E). Some other fragments observed in
min (Fig. 4D, upper panel). This peak could either correspond this spectrum could not be explained based on predicted frag-
to 5-hydroxydecanoic acid or its corresponding ␦-decanolac- mentation of 5-hydroxydecanoic acid and were shifted by 2
tone. The standard ␦-decanolactone showed a retention time of mass units. Interestingly, many of these peaks could be
18.5 min under identical conditions at 220 nm (Fig. 4D, middle observed in the MS/MS fragmentation of the ␦-decanolactone
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Polyketide Synthase Dictates Synthesis of Acyl Chain in GPL
FIGURE4. BiochemicalcharacterizationofM. smegmatisGPL-PKS. A, gelbindingassaysusingpurifiedPKSproteinwereperformedwithradiolabeledMCoA
and[14C]methylmalonyl-CoA(MMCoA). Coomassie-stainedproteinbandsinlanes1and2correspondtothepurifiedGPL-PKSthatmigratesabovethe212-kDa
marker protein (lane 3). The PKS protein was radiolabeled in the presence of [14C]MCoA (lane 4) and not in presence of 14C- methyl malonyl-CoA (lane 5). B, a
schematic representation of the extension of hexanoyl diketide from PKS protein is shown. The substrate is transferred from KS domain of module 1 to module
2. Furthermore, two-carbon chain extension and chemical hydrolysis leads to cyclization to form ␦-decanolactone as the final product. C, an PKS enzymatic
assay with [14C]malonyl-CoA as the extender unit and different starter units as their NAC thioesters (mimics of fatty acyl-CoA; hexanoyl-SNAC (lane 2),
octanoyl-SNAC (lane 3), decanoyl-SNAC (lane 4), dodecanoyl-SNAC (lane 5), and palmitoyl-SNAC (lane 6)) indicates an increase in the Rf of product formed from
C6 (0.23), C8 (0.26), C10 (0.27), and C12 (0.28) to C16 (0.29) with increasing chain lengths. PKS protein did not show any activity with propinoyl-SNAC (lane 1) and
stearoyl-SNAC (lane 7) as starters. Lane 8 indicates negative control where no starter unit was added. SF, solvent front. D, the radioactive product synthesized
by PKS protein was analyzed on reverse phase HPLC, and chromatograms were recorded at 220 nm by simultaneously using photomultiplier and radioactive
detector. To verify the PKS product formed, chemical standards were also injected. The 18.5-min peak corresponds to standard ␦-decanolactone (Sigma)
(middle panel). The 15.7-min HPLC peak corresponded to the retention time of hydrolyzed ␦-decanolactone or 5-hydroxydecanoic acid (arrow, lower panel). *,
corresponds to contamination. E, shown is electrospray ionization-MS/MS characterization of a HPLC peak with a retention time of 15.7 min. A molecular ion
peak at m/z 187.13 indicates the eluate to be 5-hydroxydecanoic acid. The fragmentation pattern obtained by tandem MS of [M-H]Ϫ1 187.13 peak shows
fragments at m/z 169 corresponding to the loss of water molecule and at m/z 125 corresponding to the loss of water and CO2 molecules from the hydrolyzed
fatty acid. Further putative cleavage fragments are indicated for three peaks with a mass shift of 2 Da (*). The possible explanation for this is described under
“Results.” F, shown is electrospray ionization-MS/MS characterization of the HPLC peak with a retention time of 18.5 min. A molecular ion peak at m/z 169
indicates the eluate to be ␦-decanolactone. Tandem MS of [M-H]Ϫ1 169 produced peaks at m/z 99 (-C5H8O2) and 113 (-C3H5O2), in concordance with the
fragmentation of hydrolyzed ␦-decanolactone.
(Fig. 4f). It is possible that the hydroxyl fatty acid partially lac- that the major product observed in the GPL-PKS enzymatic
tonizes in the gas phase during mass spectrometric analysis, assay corresponds to 5-hydroxydecanoic acid.
and thus we observe this pattern. Such a fragmentation pattern
Structural Elucidation of Acyl Chain of GPL—Because in
could also be explained through mechanisms involving charge vitro analysis clearly demonstrated that the GPL-PKS would
remote fragmentation, which results in the formation of result in the generation of the hydroxyl group at ␦-position in
terminally unsaturated product ions. The charge remote frag- GPLs, we decided to confirm this in the GPL extracted from
mentation mechanism has been demonstrated earlier for colli- M. smegmatis cultures. Mass spectrometric analysis of intact
sion-induced ionization of fatty acids in the negative ion frag- GPLs did not reveal any information about the nature of the
mentation to produce terminally unsaturated anions that show acyl subsistent as the majority of the peaks in the MS/MS spec-
a mass difference of 2 Da (42, 43). Our studies thus demonstrate tra corresponded to fragments obtained by cleavage across the
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Polyketide Synthase Dictates Synthesis of Acyl Chain in GPL
FIGURE 5. ESI-MS/MS analysis of the fatty acyl chain of purified GPL. Tandem MS/MS spectra of saturated hydroxy fatty acid of mass 468 Da together with
the observed cleavage pattern is indicated.
FIGURE 6. NMR spectra of methoxy GPL fragment. 13C DEPT-135 spectra of saturated methoxy phenyl fatty acyl in CDCl3 are shown. The inset shows the
heteronuclear spin-quantum correlation (HSQC) experiment spectra of the methine carbon. *, contaminating peak.
more labile sugar and peptide moieties on GPLs. To perform a lactone product. Further fragmentation gives 407.46 and
structural analysis of the GPL acyl chain, four main GPL vari- 393.43 ions that correspond to subsequent loss of one and two
ants produced by M. smegmatis were purified using combina- methylene group from 421.45 fragments (Fig. 5). These frag-
tion of silica chromatography and preparative TLC (supple- mentations clearly suggest that the position of the hydroxyl
mental Fig. S3). These compounds were confirmed by using group has to be after C-␤ carbon atom. Another fragment at
MALDI-TOF/MS analysis. To perform structural analysis of 117.06 could also be observed in the spectrum, and this could
the acyl chain, the purified GPLs were subjected to extensive correspond to the C-terminal fragment resulting from the
hydrolysis with 6 M HCl at 110 °C. Purified C30 fatty acyl chain cleavage between the C5 and C6. This would indicate that the
Ϫ1
on mass spectrometry analysis showed the expected [M-H]
hydroxyl group could be either at C4 or C5.
at m/z 467 Da. MS/MS analysis of fatty acid first showed a loss
Structural Analysis of GPL Fragment by Using NMR—The
Ϫ
of the [M-46] peak. As discussed in the earlier section, this partial acid hydrolysis of GPLs generates another fragment that
fragment could be ascribed to the loss of carboxylate anion and could be assigned to fatty acylphenylalanine (FaPhe). In some of
the formation of terminal unsaturation through charge remote the GPLs, hydroxyl group is modified to methoxy. We per-
fragmentation mechanism or through the fragmentation of the formed NMR studies with the methoxy-form of this GPL frag-
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Polyketide Synthase Dictates Synthesis of Acyl Chain in GPL
FIGURE 7. ESI-MS/MS analysis of chemically synthesized fatty acylphenyl analogs and fatty acyl chain of GPL molecule in negative ion mode. A, a–h
indicate the fragmentation ion profile on tandem MS analysis of [M-H]Ϫ1 306 characteristic of 2-hydroxyoctanoyl phenylalanine. B, a–h indicate the fragmen-
tation ion profile on tandem MS analysis of [M-H]Ϫ1 390 characteristic of 3-hydroxymyristoyl phenylalanine. C, a–i indicate the fragmentation ion profile on
tandem MS analysis of [M-H]Ϫ1 334 characteristic of 5-hydroxy decanoyl phenylalanine. D and E, fragmentation patterns were obtained by tandem MS/MS for
C27 and C29 fatty acyl chain of GPL molecules. respectively. amu, atomic mass units.
ment (FaPhe) that facilitated spectroscopic characterization. 78.04 1H/13C). Finally, the correlations obtained from the
The chemical shift of the methine proton is deterministic of a COSY spectrum (supplemental Fig. S5) helped to build connec-
C5 methoxylation, which however cannot be distinguished tivity between the protons of the consecutive carbons. A cross-
from others in a 1H spectrum. To perform unambiguous peak due to scalar coupling between methine protons of C5 and
assignments, we first acquired the edited 13C subspectra to the diastereotopic methylene protons of C4 at ␦ppm 3.46/2.35
know the number of protons attached to each carbon. The was observed. The C3 protons at ␦ppm 1.65 were further cou-
DEPT-135 (distortionless nhancement by polarization trans- pled to the C4 protons at ␦ppm 2.35 and C2 protons at ␦ppm
fer) edited 13C subspectra, which distinguish carbon bearing 1.32. These NMR studies provided proof that the position of
odd and even protons, identified two methine carbons at ␦ppm hydroxyl/methoxy group is present at the C-5 position.
52.95 and 78.04 (labeled a and e in Fig. 6). This also identified
Confirmation of the C-5 Position of the Hydroxyl Group on the
aromatic and methoxy carbons. Furthermore, the correspond- Fatty Acyl Chain of GPL—The FaPhe fragment of GPL ionizes
ing single-bonded protons coupling to the respective carbons much better than the fatty acyl fragment in the electrospray
were determined by using a heteronuclear spin-quantum cor- ionization. To determine the position of hydroxyl group, we
relation experiment (supplemental Fig. S4). A heteronuclear synthesized several FaPhe analogs that possess hydroxylation at
spin-quantum correlation experiment spectrum of the the 2, 3, and 5 position of the fatty acyl chain. The fragmenta-
methoxy peptidolipid confirmed the protons of the Phe tion patterns of these synthetic analogs were systematically
together with that of the methoxy group (␦ppm 3.26/56.42 investigated by using ESI-MS/MS. All these three compounds
1H/13C) (Fig. 6). Moreover, it also helped to unambiguously showed several common fragments as shown in Fig. 7. At the
assign the two methines of the phenylalanine C␣ (␦ppm 4.88/ same time, few characteristic fragments were also detected dur-
52.95 1H/13C) and that on the fatty acyl chain (␦ppm 3.46–3.5/ ing these studies, which correlate with the position of hydroxy
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Polyketide Synthase Dictates Synthesis of Acyl Chain in GPL
substituent (marked in red in Fig. 7, A–C). This signature of ity or for remodeling of mycobacterial cell wall during specific
fragmentation was then used to unambiguously determine the conditions. Although the spatiotemporal mechanisms involved
position of hydroxyl group in GPLs. The two GPL FaPhe frag- in modifying lipids during various physiological niches are
ments corresponding to m/z 572.5 (C27) and 600.5 (C29) were unclear, the ability of microbes to reprogram the outer coat
purified and subjected to MS/MS analysis (Fig. 7, D and E). must be an important mechanism of survival or pathogenesis.
Clearly, the mass differences observed in the two FaPhe GPL For example, Azotobacter during formation of dormant cysts
fragments in MS/MS spectra correspond to the mass differ- has been shown to replace its outer cell envelope by resorcinolic
ences observed for the standard 5-hydroxy-analog. Together, and pyrone lipids (38).
our studies thus establish that hydroxylation is located at the 5
position of the acyl chain.
In conclusion, our studies resolve long-standing ambiguity in
terms of chemical structure of GPL and also provide under-
standing of the involvement of FAAL28 and GPL-PKS in bio-
synthesis of GPLs. By understanding exact mechanism of bio-
DISCUSSION
The cell envelope of mycobacteria contains several unique synthesis, it is now feasible to generate variants of GPLs at the
complex lipid metabolites. Other than defining unique mor- cell surface by suitably engineering PKSs.
phological features, many of these molecules possess a variety
Acknowledgment—We thank Dr. S. Raghothama from the Indian
Institute of Science, Bangalore, India, for useful discussions and help
in NMR experiments.
of biological activities. The fatty acyl component of these lipid
molecules is unusually long, and carbon chain length of C72 has
been reported for mycolic acids. Some of these metabolites also
contain methyl-branching that arise from the incorporation of
methyl malonyl-CoA instead of commonly used malonyl-CoA
(30). Although chemical structures of many of these molecules
have been known for years, the molecular mechanisms under-
lying their biosynthesis were elucidated more recently after
the availability of complete genome sequences. Studies with
M. tuberculosis has led to the identification of a new paradigm,
wherein the complex acyl chains of lipids are produced by the
biochemical cross-talk between PKSs and FAALs. We demon-
strate here that the same theme is also relevant in the biosyn-
thesis of GPLs, an abundant glycolipid found on non-tubercu-
lous and opportunistic mycobacterial species. Cell-free
constitution studies of GPL-PKS have also led to the precise
identification of the position of the hydroxylation on the acyl
chain of GPL.
REFERENCES
1. Neyrolles, O., and Guilhot, C. (2011) Recent advances in deciphering the
contribution of Mycobacterium tuberculosis lipids to pathogenesis. Tu-
berculosis 91, 187–195
2. Gokhale, R. S., Sankaranarayanan, R., and Mohanty, D. (2007) Versatility
of polyketide synthases in generating metabolic diversity. Curr. Opin.
Struct. Biol. 17, 736–743
3. Recht, J., Martínez, A., Torello, S., and Kolter, R. (2001) Sliding motility
and biofilm formation in mycobacteria. Acta Cient. Venez. 52, 45–49
4. Deshayes, C., Perrodou, E., Gallien, S., Euphrasie, D., Schaeffer, C., Van-
Dorsselaer, A., Poch, O., Lecompte, O., and Reyrat, J. M. (2007) Inter-
rupted coding sequences in Mycobacterium smegmatis. Authentic muta-
tions or sequencing errors? Genome Biol. 8, R20
5. Kaufmann, S. H. (2001) How can immunology contribute to the control of
tuberculosis? Nat. Rev. Immunol. 1, 20–30
6. Smith, D. W., Randall, H. M., Maclennan, A. P., and Lederer, E. (1960)
Mycosides. A new class of type-specific glycolipids of mycobacteria. Na-
ture 186, 887–888
The catalytic and mechanistic versatility polyketide biosyn-
thetic machinery has been utilized by variety of microorgan-
isms to produce tremendous chemical diversity of secondary
metabolites. As of now mycolactones produced by Mycobacte-
rium ulcerans is the only example where PKSs produce mac-
rolide-like molecules in mycobacteria (44). In this study we also
show that a FAAL28 protein is essential for GPL biosynthesis.
FAAL28 activates fatty acids as acyladenylates and transfers
them on to the N terminus ACP domain of PKSs. The bimodu-
lar PKS extends the acyl chain by four carbons, and the position
of the hydroxyl group is dictated by the absence of dehydratase
and enoyl reductase domains in the module 1 of this PKS. The
second module of GPL-PKS contains all reductive domains and
thus results in a saturated fatty acyl chain. Previously, we
showed that diols in the phthiocerol chain of phthiocerol dimy-
cocerosate are produced by PpsA and PpsB proteins, where
again the dehydratase and enoyl reductase domains are absent
from these modules. Mycobacterial biosynthetic machinery has
cleverly used PKSs to generate functional acyl chains that may
be crucial to the functional requirement of these lipids. The
hydroxyl groups in phthiocerol dimycocerosates are esterified
by methyl-branched fatty acyl chain, whereas in GPL the
hydroxyl group is sometimes modified to methoxy analog. Such
a modification can generate chemical diversity to the cell enve-
lope that may be crucial in generating phenotypic heterogene-
7. Billman-Jacobe, H., McConville, M. J., Haites, R. E., Kovacevic, S., and
Coppel, R. L. (1999) Identification of a peptide synthetase involved in the
biosynthesis of glycopeptidolipids of Mycobacterium smegmatis. Mol. Mi-
crobiol. 33, 1244–1253
8. Brennan, P. J., and Goren, M. B. (1979) Structural studies on the type-
specific antigens and lipids of the Mycobacterium avium. Mycobacterium
intracellulare, Mycobacterium scrofulaceum serocomplex, Mycobacte-
rium intracellulare serotype 9. J. Biol. Chem. 254, 4205–4211
9. Aspinall, G. O., Chatterjee, D., and Brennan, P. J. (1995) The variable
surface glycolipids of mycobacteria. Structures, synthesis of epitopes, and
biological properties. Adv. Carbohydr. Chem. Biochem. 51, 169–242
10. Belisle, J. T., and Brennan, P. J. (1989) Chemical basis of rough and smooth
variation in mycobacteria. J. Bacteriol. 171, 3465–3470
11. Chatterjee, D., and Khoo, K. H. (2001) The surface glycopeptidolipids of
mycobacteria. Structures and biological properties. Cell. Mol. Life Sci. 58,
2018–2042
12. Ripoll, F., Deshayes, C., Pasek, S., Laval, F., Beretti, J. L., Biet, F., Risler, J. L.,
Daffé, M., Etienne, G., Gaillard, J. L., and Reyrat, J. M. (2007) Genomics of
glycopeptidolipid biosynthesis in Mycobacterium abscessus and M. che-
lonae. BMC Genomics 8, 114
13. Jeevarajah, D., Patterson, J. H., McConville, M. J., and Billman-Jacobe, H.
(2002) Modification of glycopeptidolipids by an O-methyltransferase of
Mycobacterium smegmatis. Microbiology 148, 3079–3087
14. Patterson, J. H., McConville, M. J., Haites, R. E., Coppel, R. L., and Billman-
Jacobe, H. (2000) Identification of a methyltransferase from Mycobacte-
rium smegmatis involved in glycopeptidolipid synthesis. J. Biol. Chem.
275, 24900–24906
30686 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 287•NUMBER 36•AUGUST 31, 2012

Polyketide Synthase Dictates Synthesis of Acyl Chain in GPL
15. Recht, J., and Kolter, R. (2001) Glycopeptidolipid acetylation affects sliding
(1990) Isolation and characterization of efficient plasmid transformation
mutants of Mycobacterium smegmatis. Mol. Microbiol. 4, 1911–1919
29. Pfeifer, B. A., Admiraal, S. J., Gramajo, H., Cane, D. E., and Khosla, C.
(2001) Biosynthesis of complex polyketides in a metabolically engineered
strain of E. coli. Science 291, 1790–1792
motility and biofilm formation in Mycobacterium smegmatis. J. Bacteriol.
183, 5718–5724
16. Sonden, B., Kocincova, D., Deshayes, C., Euphrasie, D., Rhayat, L., Laval,
F., Frehel, C., Daffe, M., Etienne, G., and Reyrat, J. M. (2005) Gap, a my-
cobacterial specific integral membrane protein, is required for glycolipid
transport to the cell surface, Mol. Microbiol. 58, 426–440
30. Khoo, K. H., Suzuki, R., Morris, H. R., Dell, A., Brennan, P. J., and Besra,
G. S. (1995) Structural definition of the glycopeptidolipids and the pyru-
vylated, glycosylated acyltrehalose from Mycobacterium butyricum. Car-
bohydr. Res. 276, 449–455
17. Chhabra, A., Haque, A. S., Pal, R. K., Goyal, A., Rai, R., Joshi, S., Panjikar, S.,
Pasha, S., Sankaranarayanan, R., and Gokhale, R. S. (2012) Nonprocessive
[2 ϩ 2]e- off-loading reductase domains from mycobacterial nonribo- 31. López Marin, L. M., Lanéelle, M. A., Promé, D., Daffé, M., Lanéelle, G., and
somal peptide synthetases. Proc. Natl. Acad. Sci. U.S.A. 109, 5681–5686
Promé, J. C. (1991) Glycopeptidolipids from Mycobacterium fortuitum, A
variant in the structure of C-mycoside. Biochemistry 30, 10536–10542
18. Gokhale, R. S., Saxena, P., Chopra, T., and Mohanty, D. (2007) Versatile
polyketide enzymatic machinery for the biosynthesis of complex myco- 32. Guilhot, C., Otal, I., Van Rompaey, I., Martìn, C., and Gicquel, B. (1994)
bacterial lipids. Nat. Prod. Rep. 24, 267–277
Efficient transposition in mycobacteria. Construction of Mycobacterium
smegmatis insertional mutant libraries. J. Bacteriol. 176, 535–539
19. Jackson, M., Stadthagen, G., and Gicquel, B. (2007) Long-chain multiple
methyl-branched fatty acid-containing lipids of Mycobacterium tubercu- 33. Prod’hom, G., Lagier, B., Pelicic, V., Hance, A. J., Gicquel, B., and Guilhot,
losis. Biosynthesis, transport, regulation, and biological activities. Tuber-
culosis 87, 78–86
C. (1998) A reliable amplification technique for the characterization of
genomic DNA sequences flanking insertion sequences. FEMS Microbiol.
Lett. 158, 75–81
20. Portevin, D., de Sousa-D’Auria, C., Montrozier, H., Houssin, C., Stella, A.,
Lanéelle, M. A., Bardou, F., Guilhot, C., and Daffé, M. (2005) The acyl- 34. Yadav, G., Gokhale, R. S., and Mohanty, D. (2003) Computational ap-
AMP ligase FadD32 and AccD4-containing acyl-CoA carboxylase are re-
quired for the synthesis of mycolic acids and essential for mycobacterial
proach for prediction of domain organization and substrate specificity of
modular polyketide synthases. J. Mol. Biol. 328, 335–363
growth. Identification of the carboxylation product and determination of 35. Ansari, M. Z., Yadav, G., Gokhale, R. S., and Mohanty, D. (2004) NRPS-
the acyl-CoA carboxylase components. J. Biol. Chem. 280, 8862–8874
PKS. A knowledge-based resource for analysis of NRPS/PKS megasyn-
thases. Nucleic Acids Res. 32, W405–W413
21. Trivedi, O. A., Arora, P., Sridharan, V., Tickoo, R., Mohanty, D., and
Gokhale, R. S. (2004) Enzymic activation and transfer of fatty acids as 36. Koglin, A., and Walsh, C. T. (2009) Structural insights into nonribosomal
acyl-adenylates in mycobacteria. Nature 428, 441–445
peptide enzymatic assembly lines. Nat. Prod. Rep. 26, 987–1000
22. Mohanty, D., Sankaranarayanan, R., and Gokhale, R. S. (2011) Fatty acyl- 37. Tang, Y., Lee, H. Y., Tang, Y., Kim, C. Y., Mathews, I., and Khosla, C. (2006)
AMP ligases and polyketide synthases are unique enzymes of lipid biosyn-
thetic machinery in Mycobacterium tuberculosis. Tuberculosis 91,
448–455
Structural and functional studies on SCO1815. A ␤-ketoacyl acyl carrier
protein reductase from Streptomyces coelicolor A3(2). Biochemistry 45,
14085–14093
23. Chopra, T., Banerjee, S., Gupta, S., Yadav, G., Anand, S., Surolia, A., Roy,
R. P., Mohanty, D., and Gokhale, R. S. (2008) Novel intermolecular itera-
tive mechanism for biosynthesis of mycoketide catalyzed by a bimodular
polyketide synthase. PLoS Biol. 6, e163
38. Funa, N., Ozawa, H., Hirata, A., and Horinouchi, S. (2006) Phenolic lipid
synthesis by type III polyketide synthases is essential for cyst formation in
Azotobacter vinelandii. Proc. Natl. Acad. Sci. U.S.A. 103, 6356–6361
39. Arora, P., Goyal, A., Natarajan, V. T., Rajakumara, E., Verma, P., Gupta, R.,
Yousuf, M., Trivedi, O. A., Mohanty, D., Tyagi, A., Sankaranarayanan, R.,
and Gokhale, R. S. (2009) Mechanistic and functional insights into fatty
acid activation in Mycobacterium tuberculosis. Nat. Chem. Biol. 5,
166–173
24. Ojha, A. K., Varma, S., and Chatterji, D. (2002) Synthesis of an unusual
polar glycopeptidolipid in glucose-limited culture of Mycobacterium
smegmatis. Microbiology 148, 3039–3048
25. Villeneuve, C., Etienne, G., Abadie, V., Montrozier, H., Bordier, C., Laval,
F., Daffe, M., Maridonneau-Parini, I., and Astarie-Dequeker, C. (2003)
Surface-exposed glycopeptidolipids of Mycobacterium smegmatis specif-
40. van Kessel, J. C., and Hatfull, G. F. (2007) Recombineering in Mycobacte-
rium tuberculosis. Nat. Methods 4, 147–152
ically inhibit the phagocytosis of mycobacteria by human macrophages. 41. Medjahed, H., and Reyrat, J. M. (2009) Construction of Mycobacterium
Identification of a novel family of glycopeptidolipids. J. Biol. Chem. 278,
51291–51300
abscessus defined glycopeptidolipid mutants. Comparison of genetic
tools. Appl. Environ. Microbiol. 75, 1331–1338
26. Mukherjee, R., Gomez, M., Jayaraman, N., Smith, I., and Chatterji, D.
(2005) Hyperglycosylation of glycopeptidolipid of Mycobacterium smeg-
matis under nutrient starvation: structural studies. Microbiology 151,
2385–2392
42. Griffiths, W. J., Jonsson, A. P., Liu, S., Rai, D. K., and Wang, Y. (2001)
Electrospray and tandem mass spectrometry in biochemistry. Biochem. J.
355, 545–561
43. Griffiths, W. J. (2003) Tandem mass spectrometry in the study of fatty
acids, bile acids, and steroids. Mass Spectrom. Rev. 22, 81–152
44. Stinear, T. P., Hong, H., Frigui, W., Pryor, M. J., Brosch, R., Garnier, T.,
Leadlay, P. F., and Cole, S. T. (2005) Common evolutionary origin for the
unstable virulence plasmid pMUM found in geographically diverse strains
of Mycobacterium ulcerans. J. Bacteriol. 187, 1668–1676
27. Miyamoto, Y., Mukai, T., Nakata, N., Maeda, Y., Kai, M., Naka, T., Yano, I.,
and Makino, M. (2006) Identification and characterization of the genes
involved in glycosylation pathways of mycobacterial glycopeptidolipid
biosynthesis. J. Bacteriol. 188, 86–95
28. Snapper, S. B., Melton, R. E., Mustafa, S., Kieser, T., and Jacobs, W. R., Jr.
AUGUST 31, 2012•VOLUME 287•NUMBER 36
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Enzymology:
Retrobiosynthetic Approach Delineates the
Biosynthetic Pathway and the Structure of
the Acyl Chain of Mycobacterial
Glycopeptidolipids
Archana Vats, Anil Kumar Singh, Raju
Mukherjee, Tarun Chopra, Madhu Sudhan
Ravindran, Debasisa Mohanty, Dipankar
Chatterji, Jean-Marc Reyrat and Rajesh S.
Gokhale
J. Biol. Chem. 2012, 287:30677-30687.
doi: 10.1074/jbc.M112.384966 originally published online July 13, 2012
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http://www.jbc.org/content/287/36/30677.full.html#ref-list-1