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Journal of Bacteriology, May 2003, p . 3228-3231, Vol . 185, No . 10
The
Bacillus subtilis Acyl Lipid Desaturase Is a
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| ABSTRACT |
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Bacillus subtilis was recently reported to synthesize unsaturated
fatty acids (UFAs) with a double bond at positions
5,
7,
and
9
(M . H . Weber, W . Klein, L . Muller, U . M . Niess, and M . A . Marahiel,
Mol . Microbiol . 39:1321-1329, 2001) . Since this finding would have
considerable importance in the double-bond positional specificity
displayed by the B . subtilis acyl lipid desaturase, we
have attempted to confirm this observation . We report that the double
bond of UFAs synthesized by B . subtilis is located
exclusively at the
5
position, regardless of the growth temperature and the length chain
of the fatty acids .
| TEXT |
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Fatty acid desaturases are nonheme, iron-containing, oxygen-dependent
enzymes involved in regioselective introduction of double bonds in
fatty acyl aliphatic chains (12) . They are present in all
groups of organisms, i.e., bacteria, fungi, plants, and animals,
and they play a key role in the maintenance of the proper structure
and functioning of biological membranes . Two general classes of
fatty acid desaturases have been identified . (i) The acyl carrier
protein (ACP) desaturases, which uses acyl-ACPs as substrates, are
soluble plant enzymes and plastid-localized (15); (ii) the
membrane-bound desaturases are found in a wide range of taxa
and use acyl-CoA or acyl lipid substrates (13-15) .
They show different consensus motifs (15) . The
soluble class contains a consensus motif consisting of carboxylate
and histidine ligands that coordinate an active site di-iron cluster,
as revealed by the X-ray structure of the castor
9
stearoyl-ACP desaturase (11) . The integral
membrane class contains a different consensus motif for the putative
active site composed of three histidine-rich regions, which are
presumably involved in iron binding (14) .
Unfortunately, information about substrate specificities, regioselectivities,
and structure of membrane desaturases is scarce due to the technical
limitations in obtaining large quantities of active forms of
such enzymes .
In a previous work, it was reported that the Bacillus subtilis
des gene codes for a unique desaturase, Des (1) .
Transcription of the des gene is tightly controlled by a
two-component regulatory system composed of a membrane-associated
kinase, DesK, and a soluble transcriptional regulator, DesR (3) .
Activation of this pathway takes place when cells are shifted to low
growth temperatures (2, 6) . The
B . subtilis desaturase is a polytopic membrane protein
with six transmembrane domains and one membrane-associated domain,
which likely represents a substrate-binding motif (7) . This
desaturase, which contains a tripartite motif of His essential
for catalysis, is an acyl lipid desaturase, since it is able to
introduce a double bond into the acyl chain of membrane glycerolipids
(1, 6) . It was previously shown that
expression of the des gene in Escherichia coli resulted
in desaturation of the palmitic moieties of the membrane
phospholipids to give cis-5-hexadecenoic acid, indicating that
the B . subtilis desaturase is a
5
acyl lipid desaturase (1) . However, Weber et al . (17)
recently reported that four unsaturated fatty acid (UFA) species
differing in length, branching pattern, and position of the double
bond were synthesized in B . subtilis JH642 . Under the
same conditions, none of these fatty acids species was detected in a
B . subtilis isogenic strain possessing a des
null mutation, showing that their production is strictly dependent on
the B . subtilis desaturase (10) . On
the basis of these data, Weber et al . (17) questioned
whether the B . subtilis desaturase was a
5
desaturase and concluded that this enzyme was able to introduce
double bonds into different positions (5,
7,
or
9)
of fatty acids of chain lengths of 16 or 17 carbon atoms . Since these
data raised the possibility that Des could have different specificity
to introduce the double bond if it is expressed either in a B.
subtilis (17) or in an E . coli
(1) host, we reevaluated the structure of the UFAs synthesized
by B . subtilis by using two different complementary
techniques .
The B . subtilis strains used in this study were trpC2 phe-1 derivatives of JH642 . Strain LC5, containing a kanamycin resistance gene cassette in the des gene, was constructed as described previously (3) . Strain AKP20 overproduces the transcriptional activator DesR, resulting in constitutive expression of the desaturase gene (3) . This strain overproduced UFAs compared with the wild-type strain JH642 (Table 1), greatly facilitating the determination of the double bond position of these fatty acids by gas chromatography-mass spectrometry (GC-MS) analysis .
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To determine the fatty acid composition, cells were grown at 37°C in
Spizizen minimal salts (16) medium supplemented
with 0.2% glucose and 50 µg each of tryptophan and phenylalanine/ml
to exponential phase at 37°C and then shifted to 25 or 15°C . The
cultures were harvested in stationary phase . Total cellular fatty
acids were prepared by the method of Bligh and Dyer (4) .
The fatty acid methyl esters were prepared by transesterification
of glycerolipids with 0.5 M of sodium methoxide in methanol (5)
and then analyzed in a Perkin-Elmer Turbo Mass gas chromatography-mass
spectrometer on a capillary column (30 mm by 0.25 mm in diameter)
of 100% dimethylpolysiloxane (PE-1, Perkin-Elmer) . Helium at 1
ml min-1 was used as the carrier gas, and the column temperature
was programmed to rise by 4°C min-1 from 140°C to 240°C .
Branched-chain fatty acids, straight-chain fatty acids, and
UFAs used as reference compounds were obtained from Sigma Chemical
Co .
As shown in Fig . 1A and Table 1, in
addition to the major branched-chain fatty acids described in B.
subtilis (9), six different UFA products
differing in length and branching pattern were identified at all
tested temperatures in strains JH642 and AKP20 . The positions of the
double bonds in these UFAs were determined by GC-MS . Fatty acid
methyl esters were converted to dimethyl disulfide (DMDS) adducts, as
described previously (18), and then separated on a
PE-1 column ramped from 140 to 280°C at 4°C min-1 . The
spectra were recorded in the electron impact mode at 70 eV, using 1-s
scans of the m/z 40-400 . The addition of DMDS to the mixture
displaces the peak of the monounsaturated components to longer
retention times . In Fig . 2A and 2B are illustrated
examples of the electron impact mass spectrum of the DMDS adducts of
iso-C16:1 and iso-C17:1 fatty acids . The mass spectrum of
the adducts shows weak ions [M]+ at m/z 362 (Fig.
2A) and m/z 376 (Fig . 2B),
corresponding to the theoretical mass of molecular ions of the DMDS
adducts of C16 and C17 monosaturated fatty
acids, respectively . Two prominent ions are formed by cleavage
between the methylthio-substituted (CH3S) carbons located at
the original site of the unsaturation . The strong ions at m/z
161 and m/z 201 indicate the position of the double bond at
5
in the iso-C16:1 fatty acid (Fig . 2A) . The ions
observed at m/z 161 and m/z 215 indicate a
5
double bond in the iso-C17:1 fatty acid (Fig.
2B) . A major ion at m/z 129 is due to loss
of methanol (CH3OH) from ion b, as shown in Fig . 2A and
2B . The fragmentation pattern obtained for the DMDS adducts of
n-C16:1, anteiso-C17:1, n-C17:1, and
iso-C18:1 also established the location of the double bond
at the
5
position (data not shown) . These results were confirmed by analyzing
the GC-MS spectrum of the picolynil ester derivatives of UFAs . An
additional advantage of this technique is that the spectra of
3-picolinyl esters contained information about the position of the
methyl branching in these UFAs . 3-Picolinyl esters were prepared from
unesterified fatty acids as described previously (8)
and then examined by GC-MS in a PE-1 column programmed at 2.5°C min-1
from 180 to 240°C . Figure 3 shows an example of the
mass spectra of the picolinyl esters of iso-C17:1 and
anteiso-C17:1 fatty acids . These spectra show prominent
ions at m/z 92, 108, 151, and 164 characteristic of picolinyl
esters (8) and a less abundant [M]+ at
359 m/z (Fig . 3) . The cleavage adjacent to the branch
point resulted in the absence of the ions at [M-29]+ (Fig.
3A) and [M-43]+ (Fig . 3B),
indicating the loss of a substituted carbon fragment . The enhanced
abundance of the ion of 14 atomic mass units (amu) below these
diagnostic ions, [M-43]+ (Fig . 3A) and
[M-57]+ (Fig . 3B) also indicates the presence of a
methyl branching at the
-1
(iso-) or
-2
(anteiso-) positions of these fatty acids . The picolinyl derivatives
from iso-C17:1 (Fig . 3A) and anteiso-C17:1
(Fig . 3B) fatty acids showed a gap of 26 amu (204
to 178), a doublet with a prominent m/z 232, and a less
prominent m/z 218, indicating that these UFAs have the double
bond at a
5
position . Similar results were obtained with picolinyl derivatives of
the other four UFAs present in B . subtilis (data not
shown) . Our assignment of the double bond position of the UFAs
synthesized by B . subtilis JH642 does not agree with
the UFAs structures reported by Weber et al . (17) . Our
most straightforward interpretation of this difference is that
Weber et al . (17) deduced the double bond position of the
B . subtilis UFAs by comparing the fatty acid profiles
obtained by gas chromatographic analysis with a microbial
identification library generation software (Microbial ID) . Here, we
located the double bond position of the UFAs by GC-MS, which is one
of the most powerful tools used for lipid analysis (5) .
None of the UFAs synthesized by strain JH642 were found in the
isogenic des strain LC5, as shown in Fig . 1B,
confirming that Des is the sole desaturase present in B .
subtilis and that this enzyme has exclusively
5
activity .
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In order to gain some insight into the substrate requirements of the
B . subtilis desaturase in a qualitative sense, a range
of possible fatty acids substrates were supplied to the E . coli
strain AK7/pDM10 (1) . This strain carries a fabB
null mutation that is functionally complemented by the B .
subtilis des gene carried by plasmid pDM10 (1) .
In conjunction with this, analysis of the desaturation products was
undertaken to determine the regioselectiviy of the enzyme . Cultures
of AK7/pDM10 cells grown to exponential phase at 30°C on
Luria-Bertani medium were supplemented with 50 µM of the
straight-chain fatty acids tetradecanoic acid (C14:0),
pentadecanoic acid (C15:0), and octadecanoic acid (C18:0)
and the branched-chain fatty acids 14-methyl pentadecanoic acid
(iso-C16:0), 15-methylhexadecanoic acid (iso-C17:0),
and 14-methylhexadecanoic acid (anteiso-C17:0) . The
positions of the double bond of the UFA products derived from the
exogenous supplemented fatty acids were determined by GC-MS of the
corresponding fatty-acyl DMDS . The results of these experiments
demonstrated the position of the double bond to be the
5
position in all tested substrates (data not shown) . Thus, we conclude
that the B . subtilis desaturase catalyzes the
introduction of a cis-double bond at the
5
position of a wide range of saturated fatty acids, and we have named
this protein
5-Des .
| ACKNOWLEDGMENTS |
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We are grateful to Monica Hourcade for technical support and to
Antonio Uttaro for critically reading the manuscript .
This work was supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and Agencia Nacional de Promoción Científica y Tecnológica (FONCYT) . P . Aguilar was a fellow from CONICET, and D . de Mendoza is a Career Investigator from CONICET and an International Research Scholar from Howard Hughes Medical Institute .
| FOOTNOTES |
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* *Corresponding author . Mailing address: IBR, Universidad
Nacional de Rosario, Suipacha 531, S2002LRK Rosario, Argentina . Phone:
54-341-4350661 . Fax: 54-341-4390465 . E-mail: diegonet@citynet.net.ar.
Present address: Department of Biochemistry and Biophysics,
University of California, San Francisco, CA 94143-0448 .
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