From the Institut für Mikrobiologie, Westfälische Wilhelms-Universität Münster, Corrensstr. 3, D-48149 Münster, Germany
Received for publication, October 15, 2002, and in revised form, December 19, 2002
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ABSTRACT |
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Triacylglycerols (TAGs) and wax esters are
neutral lipids with considerable importance for dietetic, technical,
cosmetic, and pharmaceutical applications. Acinetobacter
calcoaceticus ADP1 accumulates wax esters and TAGs as
intracellular storage lipids. We describe here the identification of a
bifunctional enzyme from this bacterium exhibiting acyl-CoA:fatty
alcohol acyltransferase (wax ester synthase, WS) as well as
acyl-CoA:diacylglycerol acyltransferase (DGAT) activity. Experiments
with a knock-out mutant demonstrated the key role of the bifunctional
WS/DGAT for biosynthesis of both storage lipids in A. calcoaceticus. This novel type of long-chain acyl-CoA
acyltransferase is not related to known acyltransferases including the
WS from jojoba (Simmondsia chinensis), the DGAT1 or DGAT2
families present in yeast, plants, and animals, and the phospholipid:diacylglycerol acyltransferase catalyzing TAG formation in
yeast and plants. A large number of WS/DGAT-related proteins were
identified in Mycobacterium and Arabidopsis
thaliana indicating an important function of these proteins. WS
and DGAT activity was demonstrated for the translational product of one
WS/DGAT homologous gene from M. smegmatis
mc2155. The potential of WS/DGAT to establish novel
processes for biotechnological production of jojoba-like wax esters was
demonstrated by heterologous expression in recombinant
Pseudomonas citronellolis. The potential of WS/DGAT
as a selective therapeutic target of mycobacterial infections is discussed.
The capability for biosynthesis of neutral lipids is
widely distributed in nature and is found in animals and plants as well as microorganisms. In bacteria, the most abundant class of neutral lipids are polyhydroxyalkanoic acids serving as intracellular carbon
and energy storage compound (1), but also few examples of substantial
triacylglycerol (TAG)1
accumulation have been reported for species mainly belonging to the
actinomycetes genera: Mycobacterium (2),
Nocardia, and Rhodococcus (3) and
Streptomyces (4). Furthermore, biosynthesis of wax esters
(oxoesters of long-chain primary fatty alcohols and long-chain fatty
acids) has been frequently reported for members of the genus
Acinetobacter (5).
TAGs are the dominating storage lipid in animals, plants, and
eukaryotic microorganisms. TAG biosynthesis is involved in animals in
numerous processes such as regulation of plasma TAG concentration, fat
storage in adipocytes, and milk production (6). In plants, TAG
synthesis is mainly important for the generation of seed oils (7).
Using diacylglycerol (DAG) as a substrate, three different classes of
enzymes are known that mediate TAG formation (reviewed in Ref. 8).
Acyl-CoA:DAG acyltransferase (DGAT) catalyzes the acylation of DAG
using acyl-CoA as a substrate. Two DGAT families designated as DGAT1
and DGAT2 are known that exhibit no sequence homologies to each other.
Members of the DGAT1 gene family occur in animals and plants
(9-12), whereas members of the DGAT2 gene family were found
in animals (13), plants (14), and yeast (15). In human, one
DGAT1-related gene and five DGAT2-related genes
were identified (13). Recently, DGAT has attracted great interest since
it is a potential therapeutical target for obesity treatment (16).
Acyl-CoA-independent TAG synthesis is mediated by a phospholipid:DAG
acyltransferase found in yeast and plants that uses phospholipids as
acyl donors for DAG esterification (17). A third alternative mechanism
present in animals and plants is TAG synthesis by a
DAG-DAG-transacylase, which uses DAG as acyl donor and acceptor
yielding TAG and monoacylglycerol (18, 19), although no gene coding
such a transacylase could be identified as yet.
Wax esters have diverse and important biological functions including
coating of aerial surfaces of plants as epicuticular waxes to provide
protection against desiccation, ultraviolet light, and attack of
pathogens; regulation of buoyant density as the principal component of
the spermaceti oil of sperm whales; and serving as energy storage
materials in the seeds of the jojoba plant (20). The latter is the main
natural source of wax esters for commercial applications since the
world-wide ban on whale hunting. However, the high price of jojoba oil
has limited its use. Wax esters have a multitude of important technical
applications in a variety of areas, including medicine, cosmetics, and
food industries as well as their more traditional usage as lubricants. Acinetobacter calcoaceticus accumulates wax esters
intracellularly as insoluble inclusions under growth-limiting
conditions. The chemical structure is similar to those wax esters
produced by jojoba and the sperm whale with mainly a
C32-C36 carbon length composed of saturated and
unsaturated C16 and C18 fatty acid and fatty
alcohol moieties (21). Thus, analysis of microbial wax ester
biosynthesis could provide a promising basis for the biotechnological production of low-cost jojoba-like wax esters. The key enzymatic step
in wax ester biosynthesis is the final condensation of acyl-CoA and
fatty alcohol yielding the wax ester catalyzed by the acyl-CoA:fatty alcohol acyltransferase (wax ester synthase, WS). The only WS gene
described so far has been cloned from jojoba, but this WS was not
active in Escherichia coli or yeast (22). Here we report on
the identification of a bifunctional WS/DGAT, which is the first
description of a bacterial protein mediating WS or DGAT activity. This
bifunctional WS/DGAT is a new type of long-chain acyl-CoA
acyltransferase, which also might have great importance for
Mycobacterium and Arabidopsis thaliana.
Strains, Plasmids, and Growth Conditions--
The strains used
are: A. calcoaceticus ADP1 (ATCC 33305),
Escherichia coli XL1-Blue (23), E. coli S17-1
(24), Pseudomonas citronellolis (DSM 50332),
Rhodococcus opacus PD630 (DSM 44193), and
Mycobacterium smegmatis mc2155 (25). Plasmids
used for cloning are pBluescript SK Isolation of Mutants of A. calcoaceticus ADP1 Defective in
Storage Lipid Accumulation--
miniTn10Km-induced mutants
of an isolated spontaneous nalidixic acid-resistant strain of A. calcoaceticus ADP1 were generated according to Ref. 29.
Non-auxotrophic transposon mutants were selected on MSM agar plates
containing gluconate, kanamycin, and nalidixic acid. Mutants defective
in the accumulation of storage lipids were identified by staining with
the lipophilic dye Sudan Black B as described in Ref. 30 and subjected
to TLC for further analysis.
Lipid Analysis--
Thin-layer chromatography (TLC) was
performed as described (31) using the solvent systems
hexane/diethylether/acetic acid (90:15:1, v/v/v) for wax ester analysis
or (80:20:1, v/v/v) for TAG analysis. Triolein was used as TAG
reference substance and cetylpalmitate as wax ester reference
substance. Fatty acid analysis of whole cells and purified TAGs and wax
esters was done by gas chromatography (GC) according to Ref. 31.
Genotypic Characterization of the miniTn10Km Insertion Mutant
A. calcoaceticus ACM7--
An 8.9-kbp EcoRI fragment
conferring kanamycin-resistance to E. coli due to the
presence of miniTn10Km was isolated from total DNA of the
mutant A. calcoaceticus ACM7. After subcloning into pBluescript SK Cloning of the WS/DGAT Gene and Heterologous
Expression--
The WS/DGAT gene was amplified by tailored PCR
employing the oligonucleotides
5'-AAAGAATTCAAGGAGGTATCCACGCTATGCGCCCATTAC-3' (5'-end)
introducing a ribosome binding site and
5'-TTTGGATCCAGGGCTAATTTAGCCCTTTAGTT-3' (3'-end) and was cloned
into pBluescript KS Determination of Enzyme Activities--
WS activity was measured
in a total volume of 250 µl containing 3.75 mM
1-hexadecanol, 4.63 mg ml Data Deposition--
The WS/DGAT nucleotide sequence has been
deposited in the GenBankTM data base under GenBank
Accession Number AF529086.
Identification of a Gene Locus Involved in Storage Lipid
Accumulation--
A. calcoaceticus ADP1 accumulates wax
ester and TAG intracellularly during cultivation under so-called
storage conditions from gluconate with TAGs and wax esters amounting to
up to 1.4 and 6.9% (w/v) of the cellular dry weight, respectively, as
estimated by gas chromatographic fatty acid analysis of isolated TAGs
and wax esters purified by TLC. We isolated the
miniTn10Km-induced mutant A. calcoaceticus ACM7,
which was unable to synthesize wax ester and accumulated only trace
amounts of TAG as estimated by TLC (Fig.
1B). Genotypical
characterization of the mutant revealed insertion of the transposon
with a 9-bp direct repeat (5'-CGCTATGCG-3') 4-bp upstream of
the ATG start codon (shown in bold face) of a 1377-bp open reading
frame, which we designated as wax/dgat (Fig. 1A). Since the coding region of
wax/dgat remained intact in A. calcoaceticus ACM7, we generated the knock-out mutant A. calcoaceticus ADP1wax/dgat wax/dgat Encodes a Bifunctional
WS/DGAT--
Wild-type A. calcoaceticus ADP1
exhibited a WS activity of 90.37 pmol (mg min)
Heterologous expression of wax/dgat
conferred the capability to recombinant E. coli XL1-Blue
harboring pKS::wax/dgat to catalyze the
acyl-CoA-dependent acylation of fatty alcohol as well as of diacylglycerol (Fig. 2A) at
rates similar to those of A. calcoaceticus ADP1 (Table I).
These results clearly show that both, WS and DGAT activity, arise from
wax/dgat, which therefore codes for a
bifunctional WS/DGAT enzyme. Furthermore, the experiments with the
knock-out mutant indicate that no other protein exhibiting WS or DGAT
activity contributes significantly to wax ester or TAG biosynthesis in
A. calcoaceticus ADP1. This was supported by the fact that
no other wax/dgat homologue could be identified by a BLAST (34) search in the preliminary A. calcoaceticus
ADP1 genome sequence data accessible online at www.genoscope.fr.
However, residual trace amounts of TAGs accumulated in the mutants
indicate the presence of a minor alternative pathway, which is,
however, only active at a very low rate. Furthermore, functional
heterologous expression of wax/dgat was not only
demonstrated in E. coli S17-1 but also in P. citronellolis (Table I).
Characteristics of the Bifunctional WS/DGAT--
The
bifunctional WS/DGAT comprises 458 amino acids with a theoretical
molecular mass of 51.8 kDa. The basic theoretical pI of 9.05 is
consistent with those observed for the jojoba WS (22) and other lipid
biosynthetic enzymes (35). It is a rather amphiphilic protein, and it
possesses one putative predicted membrane-spanning region (Fig.
2C). A broad range of long-chain fatty alcohols could be
utilized by the bifunctional enzyme as acyl acceptor in the WS reaction
with lower specificities toward longer chain length (Fig.
3A). Whereas the WS reaction
accepted a wide range of various chain length acyl-CoA molecules almost
equally, the DGAT reaction preferred longer chain length acyl-CoAs
(Fig. 3, B and C). Kinetic studies on the
bifunctional enzyme by monitoring the formation of radiolabeled
reaction products as a function of time revealed no obvious lag phase
and approximately constant WS and DGAT reaction rates over
a period of 40 min (Fig. 4A).
Recording the dependence of enzyme activities on palmitoyl-CoA
concentration, hyperbolic saturation curves were obtained indicating
that the bifunctional WS/DGAT does not behave as an allosteric enzyme
(Fig. 4B). Assuming substrate saturation for 1-hexadecanol
and dipalmitin under assay conditions, Lineweaver-Burk plot analysis
revealed Km values for palmitoyl-CoA of 15.6 and
21.1 µM and Vmax of 212.8 and 54.3 pmol (mg min) Heterologous Wax Ester Production in P. citronellolis--
Heterologous functional expression of the
wax/dgat gene in the alkane-degrading bacterium
P. citronellolis resulted in an active enzyme, which
maintained its bifunctionality (Table I). During cultivation of
P. citronellolis
(pBBR1MCS-2::wax/dgat) under storage conditions, no accumulation of wax esters
could be detected by TLC if 0.5% (w/v) gluconate, 0.3% (w/v)
1-hexadecane, or 0.3% (w/v) palmitate were used as carbon sources
(data not shown). However, cultivation on 0.3% (w/v) 1-hexadecanol,
which can serve as a direct substrate for the WS, resulted in
recombinant production of wax esters (Fig.
6B). No TAG accumulation could be observed under either condition.
WS/DGAT-related Proteins--
The A. calcoaceticus ADP1 WS/DGAT exhibits no sequence similarity to any
known acyltransferase including the WS from jojoba (Simmondsia
chinensis) (22), the DGAT1 (9-12) and DGAT2 (13-15) gene family
and the phospholipid:diacylglycerol acyltransferase catalyzing TAG
formation in yeast and plants (17). Thus, it represents a new type of
long-chain acyl-CoA acyltransferase. A BLAST search comprising 189 eubacterial and 18 archaeal finished and unfinished microbial genome
sequences (as of November 2002) publicly accessible via the National
Center of Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov)
revealed that WS/DGAT-related proteins are not widely distributed among
prokaryotes. Aside from the Gram-negative bacterium A. calcoaceticus, related proteins were found only in some
actinomycetes, and interestingly within all members of the genus
Mycobacterium (Table III). Whereas only one gene coding for WS/DGAT occurs in A. calcoaceticus ADP1, numerous genes for
related proteins were found in mycobacteria (Table III). These
WS/DGAT-related proteins constitute a remarkable large group of
conserved proteins in Mycobacterium with an up to now
unknown function. M. tuberculosis H37Rv, for instance,
possesses 13 genes coding for WS/DGAT-related proteins exhibiting up to
39.7% identity with the A. calcoaceticus enzyme (Table
II), i.e. 1.43% of the 912 genes encoding conserved hypothetical proteins and 0.86% of all 1518 genes with unknown function in this strain (36, 37). A BLAST search
with 50 eukaryotic genome sequences at NCBI identified in A. thaliana a large group of conserved putative proteins with unknown
function exhibiting some similarity to the A. calcoaceticus
WS/DGAT (Table II).
Wax Ester Biosynthesis in M. smegmatis
mc2155--
Eight WS/DGAT homologous genes could be
identified in the preliminary genome sequence of the non-pathogenic
strain M. smegmatis mc2155 (see Table
III), which is publicly accessible online
via NCBI. The gene with the highest similarity exhibits 41.0% amino
acid identity to WS/DGAT (Table II). Recombinant E. coli
expressing this gene on plasmid pSK::wdh3269
showed weak WS and DGAT activity, which was slightly but reproducible
higher than the vector control (Table I). R. opacus PD630 is
a TAG-accumulating actinomycete (3), which itself exhibits WS and DGAT
activity, but heterologous expression of wdh3269 clearly
elevated both activities in this strain (Table I).
M. smegmatis mc2155 cultivated under storage
conditions with glucose as sole carbon source exhibited both high WS as
well as DGAT activity in vitro (Table I), although in
vivo only TAGs were intracellularly accumulated (Fig.
6A). However, M. smegmatis mc2155
was also capable of substantial wax ester biosynthesis in vivo when 1-hexadecanol was provided as a sole carbon source or as
a cosubstrate (Fig. 6A).
Identification of a Putative Active Site--
The A. calcoaceticus ADP1 WS/DGAT and the related proteins in
Mycobacterium and A. thaliana exhibit in their
N-terminal region partial similarity to a conserved condensing domain
found in many multidomain enzymes synthesizing peptide antibiotics
(NCBI Conserved Domain Data Base accession pfam00668). This
condensing domain contains an active-site motif (HHXXXDG),
whose second histidine residue is strictly conserved and has been
demonstrated to be essential for catalytic activity in non-ribosomal
peptide bond formation (38). The WS/DGAT and related proteins also
contain this putative active site with the motif
(133HXXXDG138) being strictly
conserved (Fig. 2B). Thus, it is very likely that this site
is catalytically participating in the acyl-CoA acyltransferase
reactions involved in wax ester and TAG formation (Fig.
2A).
TAGs and wax esters are rather uncommon storage lipids in
bacteria. Large amounts of TAG accumulation has been reported
particularly for actinomycetes (2-4), whereas wax esters occur
frequently among species of the genus Acinetobacter, which
are able to accumulate wax esters of up to 14% of the cellular dry
weight depending on the culture conditions (5). The A. calcoaceticus strain ADP1 synthesizes both classes of storage
lipids under growth-limiting conditions. In the present study we
identified a novel bifunctional enzyme from this strain, which mediates
the final reaction steps in the biosynthesis of both lipids
simultaneously. This bifunctional WS/DGAT is a new and unique type of
long-chain acyltransferase, which is not related to members of the
DGAT1 and 2 families or phospholipid:DAG acyltransferases mediating TAG
synthesis, or to the jojoba WS, or to any other known gene in the data base.
The residual trace amounts of TAGs, which are still present in the
mutants, indicate that there might be an alternative low-rate pathway
for TAG synthesis. Multiple alternative TAG biosynthesis pathways have
been reported for Saccharomyces cerevisiae, where a DGAT, a
phospholipid:DAG acyltransferase, and the DGAT side-activity of a
sterol:acyl-CoA acyltransferase contribute cumulatively at various
rates to TAG synthesis (15). In the preliminary A. calcoaceticus ADP1 genome sequence; however, no gene similar to
those mentioned could be identified by BLAST search that could be a
candidate for residual TAG synthesis.
The highly conserved motif HHXXXDG corresponding to amino
acids 132-138 of the A. calcoaceticus ADP1 WS/DGAT (Fig.
2B) may be the catalytic site responsible for ester bond
formation. In analogy to the mechanism of non-ribosomal peptide bond
formation catalyzed by condensing domains, which also contain this
putative active site motif (38), a proposed catalytic mechanism for the WS/DGAT could involve abstraction of a proton from the hydroxyl group
of the fatty alcohol or DAG, respectively, by the strictly conserved
histidine residue His133 acting as base catalyst, which
would than enable the nucleophilic attack on the thioester bond of the
fatty acyl-CoA molecule (see Fig. 2A).
Beside the genus Acinetobacter, WS/DGAT-related proteins
seem to be restricted almost exclusively to mycobacteria among
prokaryotes (Table III). Whereas only one WS/DGAT gene is present in
A. calcoaceticus ADP1, an extensive group of related
proteins occurs in mycobacteria (Table III). Wax ester synthesis and WS
activity in vitro have been reported for M. tuberculosis (39), and TAG accumulation and DGAT activity have
been shown for M. smegmatis (40, 41). In the present study,
we demonstrated in addition in vitro WS activity (Table I)
and in vivo wax ester production in M. smegmatis mc2155 (Fig. 6A). Until this study, however, no
proteins or genes had been reported to which these activities could be
attributed. By heterologous functional expression of the homologue from
M. smegmatis mc2155, which exhibited the highest
similarity to the A. calcoaceticus ADP1 WS/DGAT
(wdh3269), in recombinant E. coli and R. opacus, it was unambiguously demonstrated that wdh3269
also codes for a bifunctional WS/DGAT. However, it is not known yet to
what extent wdh3269 contributes to storage lipid
accumulation in M. smegmatis mc2155. It is
likely that the observed in vitro and in vivo WS
and DGAT activities in this strain are the cumulative result of several WS/DGAT homologues. Alternatively to storage lipid synthesis, some of
the WS/DGAT homologues could also participate in biosynthesis of other
lipids like mycolic acids.
Interestingly, only recently the first evidence for lipid accumulation
in M. tuberculosis, in vivo, has been reported (42). By
employing a combined staining method, the occurrence of substantial lipophilic intracellular inclusions in mycobacterial cells in sputum
samples from patients with clinical tuberculosis was demonstrated, which indicates that lipid accumulation could be an essential factor
participating in pathogenesis. Simply the large number of
WS/DGAT-related proteins identified in mycobacteria already supposes an
important function, and it is likely that a least some of them are
involved in lipid accumulation in M. tuberculosis. Thus,
detailed investigations on the biological function of these WS/DGAT
homologous proteins and their importance for the pathogenesis of
harmful mycobacteria seem to be worthwhile. Since they can be found
almost exclusively within mycobacteria and are not widely distributed
among other prokaryotes or eukaryotes, they could be an ideal
therapeutical target for treatment of major global health problems
caused by mycobacteria like tuberculosis.
A large group of conserved hypothetical proteins similar to WS/DGAT was
also found in A. thaliana (Table II). Members of the DGAT1
and 2 families were already described for this plant (11, 14), thus the
presence of WS/DGAT-related proteins could possibly indicate that
acyl-CoA-dependent TAG synthesis is mediated in A. thaliana by three different non-related enzyme groups. It would also be conceivable that WS/DGAT homologues are involved in the biosynthesis of epicuticular wax esters.
A strong demand exists for large-scale production of cheap jojoba-like
wax esters, which have multiple commercial uses. Jojoba oil is the only
alternative natural source of wax esters to sperm whale oil, which is
used at a commercial scale, but the high production costs restrict
its use currently on cosmetic applications. The jojoba WS could not be
functionally expressed in microorganisms like E. coli and
S. cerevisiae (22). In contrast, we have demonstrated that
the A. calcoaceticus ADP1 WS/DGAT was active in different bacterial hosts. In P. citronellolis, the heterologous
expression of WS/DGAT led to production of wax esters if a long-chain
fatty alcohol was provided as a carbon source that also delivers fatty acyl-CoA during catabolism by the alkane degradation pathway. By
variation of the fatty alcohol used as carbon source it should be
possible to vary the composition of the produced wax esters, because
the bifunctional enzyme can utilize a broad range of substrates (Fig.
3). This study provides the basis for a potential microbial biotechnological production of cheap jojoba-like wax esters.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and KS
(Stratagene, Heidelberg, Germany), pBBR1MCS-2 (26), and the E. coli-Rhodococcus shuttle vector
pBBRKmNC903.2 Cells of
A. calcoaceticus were cultivated aerobically in LB medium (27) in Erlenmeyer flasks at 30 °C. For the induction of wax ester
and TAG formation and for determination of enzyme activities, cells
were cultivated 24 h in mineral salts medium (MSM) (28) with
0.1 g liter
1 NH4Cl and with 1% (w/v)
sodium gluconate as carbon source. These conditions are referred to as
"storage conditions." Cells of E. coli and P. citronellolis were cultivated in LB medium at 37 and 30 °C,
respectively. For recombinant wax ester production, P. citronellolis was cultivated under storage conditions for 48 h at 30 °C with 0.3% (w/v) 1-hexadecanol as sole carbon source. For
determination of enzyme activities in recombinant R. opacus PD630 cells were cultivated for 24 h under storage conditions at
30 °C with 1% (w/v) gluconate. M. smegmatis
mc2155 was cultivated for 48 h under storage
conditions at 30 °C with 1% (w/v) glucose. Antibiotics were added
at the following concentrations if appropriate: ampicillin, 75 µg
ml
1; nalidixic acid, 10 µg ml
1;
kanamycin, 50 µg ml
1; tetracycline, 12.5 µg
ml
1.
, DNA sequence was determined, and data
were analyzed with the online program pack Biology WorkBench 3.2 at
workbench.sdsc.edu.
collinear to the lacZ
promoter, resulting in pKS::wax/dgat,
and into pBBR1MCS-2 collinear to the lacZ promoter,
resulting in pBBR1MCS-2::wax/dgat. The
WS/DGAT homologue from M. smegmatis mc2155
exhibiting the highest similarity to the A. calcoaceticus ADP1 gene (designated as wdh3269 because of its
localization on contig 3269) was amplified by tailored PCR using the
following oligonucleotides:
5'-AAAGAATTCAAGGAGGTCAGCGTTGAATGAACCGGATGCA-3' (5'-end)
introducing a ribosome binding site and
5'-TTTAAGCTTTCAGGCGCCTGTGGCCGTCTCGA-3' (3'-end). The obtained
1403-bp PCR product was cloned into pBluescript SK
collinear to the lacZ promoter, resulting in
pSK::wdh3269. For heterologous expression in
R. opacus PD630, HindIII-restricted pSK::wdh3269 was fused with
HindIII-restricted pBBRKmNC903 resulting in
pBBRKmNC903-pSK::wdh3269. Transfer of the fusion
plasmid to R. opacus was done according to Ref. 32. For
heterologous expression, recombinant E. coli were cultivated
for 6 h in the presence of 1 mM IPTG and recombinant
P. citronellolis for 6 h and recombinant R. opacus PD630 for 24 h without IPTG, respectively.
1 bovine serum albumin, 10 mM MgCl2, 4.72 µM
[1-14C]palmitoyl-CoA (specific activity 1.961 Bq
pmol
1), and 125 mM sodium phosphate buffer
(pH 7.4). Hexadecanol and bovine serum albumin were emulsified by
ultrasonification. The assays were incubated at 35 °C for 30 min,
and the reactions were stopped by extraction with 500 µl of
chloroform/methanol (1:1, v/v). After centrifugation, the chloroform
phase was withdrawn, evaporated to dryness, and 40 µg of
chloroform-dissolved unlabeled reference wax ester (cetylpalmitate)
were added. After separation of lipids by TLC and staining of TLC
plates with iodine vapor, spots corresponding to waxes were scraped
from the plates, and radioactivity was measured by scintillation
counting. The DGAT activity assay was identical to the WS assay except
that the reaction mixture contained 3.75 mM
1,2-dipalmitoyl-rac-glycerol instead of 1-hexadecanol. Here
triolein was used as the unlabeled TAG reference substance. Acyl-CoA
specificity of the WS and DGAT reactions was assayed in a total volume
of 250 µl containing 0.19 mM
[1-14C]hexadecanol (specific activity 1.924 Bq
pmol
1) or 0.09 mM oleic
[1-14C]diolein (specific activity 2.035 Bq
pmol
1), respectively, 4.63 mg ml
1 bovine
serum albumin, 10 mM MgCl2, 50 µM
acyl-CoA, and 125 mM sodium phosphate buffer (pH 7.4) under
the conditions described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Km by
disrupting wax/dgat by insertion of a
Km gene cassette (33) (Fig. 1A). The knock-out mutant also exhibited the wax ester-negative and TAG-leaky phenotype, thus demonstrating that
the strong impact on biosynthesis of both storage lipids was really
attributed to wax/dgat (Fig.
1B).
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Fig. 1.
Influence of wax/dgat on wax
ester and TAG accumulation in A. calcoaceticus
ADP1. A, molecular organization of the 6.9-kbp
EcoRI fragment harboring wax/dgat. The
triangle indicates the miniTn10Km insertion locus
in A. calcoaceticus ACM7. The NruI site was used
for gene disruption by inserting a Km gene cassette (33). The
bar shows the DNA region whose sequence has been deposited
in GenBankTM. The remaining part was obtained from
preliminary sequence data of the A. calcoaceticus ADP1
genome project accessible online at www.genoscope.fr. Designations
of putative genes were based on homologies found in a BLAST search:
mreC, rod-shape determining protein; maf,
putative inhibitor of septum formation; cysH,
3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase;
thrH, homoserine kinase. B, TLC of whole cell
extracts of A. calcoaceticus strains grown under storage
conditions. Lanes: TAG, TAG standard;
Wax, wax ester standard; 1, A. calcoaceticus ADP1; 2, A. calcoaceticus
ACM7; 3, A. calcoaceticus
ADP1wax/dgat
Km.
1 and a
~10-fold lower DGAT activity (Table I),
which corresponded approximately with the amounts of wax esters and
TAGs accumulated under storage conditions as estimated by TLC (Fig.
1B). Inactivation of wax/dgat not only
caused the loss of the ability for wax ester and TAG biosynthesis; it
also abolished WS and DGAT activity in the transposon-induced as well
as in the knock-out mutant (Table I).
WS and DGAT activities in crude cell extracts of different strains
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Fig. 2.
Properties of the bifunctional WS/DGAT.
A, reactions catalyzed by the bifunctional enzyme.
B, multiple sequence alignment of WS/DGAT with some related
proteins from M. tuberculosis H37Rv and A. thaliana using the ClustalW program (43). Only the region
corresponding to the A. calcoaceticus ADP1 WS/DGAT amino
acid residues 75-151 are shown. Residues identical in seven or more
sequences are shaded in gray. A putative active site is
boxed. M. tuberculosis H37Rv:
a, Rv3740c; b, Rv3734c; c,
Rv1425; d, Rv3480c; e, Rv2285; A. thaliana: f, At5g53380; g, At5g16350;
h, At5g12420; i, At5g22490; j,
At1g72110; k, A. calcoaceticus ADP1 WS/DGAT (for
accession numbers see Table II). C, hydrophobicity plot (44)
of WS/DGAT (window size 9). A putative transmembrane domain predicted
by the TMAP program (45) is indicated by the gray bar. The
black bar region exhibits some homology to a conserved
condensing domain containing a putative active site.
1 for WS and DGAT reactions, respectively.
In direct competition experiments, in which dipalmitin and hexadecanol
were provided together at various concentrations, the WS/DGAT exhibited
a considerably higher substrate specificity toward 1-hexadecanol in
comparison to dipalmitin, which explains the rather low DGAT activity
compared with the WS activity (Fig.
5).
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Fig. 3.
Substrate specificities of the bifunctional
WS/DGAT. Measurements were done using insoluble fraction of crude
extract of E. coli XL1-Blue
(pKS::wax/dgat) obtained after a 30-min
centrifugation at 35,000 × g. Assay conditions were as
described under "Experimental Procedures." Values are averages of
two independent experiments. A, fatty alcohol specificity of
the WS reaction. B, acyl-CoA specificity of the WS reaction.
C, acyl-CoA specificity of the DGAT reaction.
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Fig. 4.
Enzyme kinetics of the bifunctional
WS/DGAT. Measurements were done using insoluble fraction of crude
extract of E. coli XL1-Blue
(pKS::wax/dgat) obtained after a 30-min
centrifugation at 35,000 × g. Assay conditions were as
described under "Experimental Procedures." Values are averages of
two independent experiments. A, time course of wax ester and
TAG formation. B, palmitoyl-CoA dependence of WS and DGAT
reactions. , WS;
, DGAT.
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Fig. 5.
Competition between 1-hexadecanol and
dipalmitin as substrates for WS/DGAT. Measurements were done using
the insoluble fraction of crude extract of E. coli XL1-Blue
(pKS::wax/dgat) obtained after a 30-min
centrifugation at 35,000 × g. Assay conditions were as
described under "Experimental Procedures" but providing
1-hexadecanol and dipalmitin together at various ratios in the same
assay with a constant total concentration of 1-hexadecanol and
dipalmitin as 3.75 mM. Values are averages of two
independent experiments. , WS;
, DGAT.
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Fig. 6.
Wax ester biosynthesis in M. smegmatis mc2155 and recombinant P. citronellolis. A TLC of whole cell extracts
of M. smegmatis mc2155 grown under storage
conditions with 1% (w/v) glucose (lane 1), 0.3% (w/v)
1-hexadecanol (lane 2), or 1% (w/v) glucose + 0.3%
(w/v) 1-hexadecanol (lane 3). TAG, TAG standard;
Wax, wax ester standard. The arrow indicates
residual 1-hexadecanol used as carbon source. B, TLC of
whole cell extracts of P. citronellolis strains grown under
storage conditions with 0.3% (w/v) 1-hexadecanol. Lanes:
Wax, wax ester standard; lane 1, P. citronellolis (pBBR1MCS-2); lane 2, P. citronellolis
(pBBR1MCS-2::wax/dgat).
WS/DGAT-related proteins in M. tuberculosis and A. thaliana
Distribution of WS/DGAT-related proteins in bacteria
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Toni Voelker and Kathryn D. Lardizabal for helpful discussions.
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FOOTNOTES |
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* This study was supported by a grant from Monsanto Co. (St. Louis, MO).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF529086.
To whom correspondence should be addressed. Tel.: 49-251-8339821;
Fax: 49-251-8338388; E-mail: steinbu@uni-muenster.de.
Published, JBC Papers in Press, December 26, 2002, DOI 10.1074/jbc.M210533200
2 R. Kalscheuer, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are:
TAG, triacylglycerol;
WS, wax ester synthase;
DGAT, acyl-CoA:diacylglycerol
acyltransferase;
IPTG, isopropyl-1-thio--D-galactopyranoside;
DAG, diacylglycerol;
MSM, mineral salts medium.
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