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INTRODUCTION |
The key of psychrophily in cold-adapted microorganisms, or
psychrophiles, is their ability to produce cold-adapted enzymes (1-5)
and to maintain high membrane fluidity at low temperatures (6-9).
However, the incapacity to grow at moderate temperatures (>20 °C)
remains a paradox and represents a barrier to their use in ecological
and biotechnological processes. Earlier studies, reviewed by Inniss
(10), report that the biochemical basis for the maximum growth
temperature of psychrophiles is likely to be complex, involving a
number of interacting phenomena. Temperature changes cause alterations
in the structural and molecular components of cells, for example
membrane integrity and permeability, functional stability of ribosomes,
and enzyme activity and stability.
Recently, a large body of evidence has been accumulated indicating that
long chain acyl-CoA esters have an important function in the regulation
of a large number of cellular systems and functions, including ion
channels, ion pumps, translocators, enzymes, membrane fusion, and gene
regulation (for review see Ref. 11). Because of the amphipathic nature
of acyl-CoA esters, excessive increases in their concentration can
cause important cellular damage, including membrane disruption and a
nonspecific inhibition of a variety of enzymes (11-14). A number of
recognized metabolic diseases including Reye's syndrome and sudden
infant death syndrome can be attributed to specific enzyme deficiencies
in acyl-CoA catabolic pathways, which result in the accumulation of
toxic acyl-CoA thioesters (15).
The intracellular concentration of free acyl-CoA esters is tightly
controlled by feedback inhibition of the acyl-CoA synthetase and is
buffered and transported by the specific acyl-CoA-binding proteins (11,
16). Under normal physiological conditions the total acyl-CoA ester
content in cells is in the range 5-160 µM, with the free
cytosolic concentration in the low nanomolar range being unlikely to
exceed 200 nM (11). Abnormal increases in the concentration
are expected to be prevented by conversion into acylcarnitines or by
hydrolysis by acyl-CoA hydrolases (17).
At present, for psychrophilic microorganisms, no data are available on
the effects of growth temperature on the cellular metabolism of
acyl-CoA esters.
In this paper we report that moderate temperatures, above 20 °C,
cause an abnormal accumulation of myristoyl-CoA (tetradecanoyl-CoA) in
the psychrophilic yeast Rhodotorula aurantiaca. The
excessive concentration at moderate temperatures is probably one of the principal causes of cell death.
Acyl-CoA thioesterases are enzymes that cleave thioester bonds
of fatty acyl-CoA and liberate free fatty acids and CoASH. Thioesterase
activity is widely distributed in both prokaryotes and eukaryotes (18).
In eukaryotes, acyl-CoA thioesterase activity is detected in various
subcellular organelles (19, 20) including lysosomes (21), peroxisomes,
and mitochondria (22) as well as in the cytosol (23). Although all the
physiological functions of acyl-CoA thioesterases have not yet been
clearly understood, previous data suggest (11, 17, 22) that they are
involved in lipid metabolism and modulation of cellular concentrations of acyl-CoA derivatives.
To assess further the mechanism by which temperature disrupts the
regulation of myristoyl-CoA metabolism, we isolated and characterized a
R. aurantiaca thioesterase with a particular focus on its
thermodynamic properties.
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MATERIALS AND METHODS |
Microorganism and Culture Conditions--
R. aurantiacaA19 was
isolated at the Laboratory of Biochemistry, University of Liege,
Belgium, from ice near the French Antarctic Station at Dumont
d'Urville (66° 40'S, 140° 01'E) and identified by the Mycotheque
of the University of Louvain-la-Neuve, Belgium; the registration number
is 40267.
The growth medium, YPD, contains 2% dextrose, 2% casein peptone, and
1% yeast extract. For thioesterase purification R. aurantiaca A19 was produced in a 400-liter bioreactor (Biolaffite,
France). Culturing was carried out aerobically (0.5 volume of
air per volume of culture per minute) at 12 °C, in the YPD medium.
After 100 h of growth 3.5 kg of cells were recovered by
centrifugation (Sharples centrifuge, Alpha-Laval, Sweden).
Extraction and HPLC1
Analysis of Acyl-CoA Esters--
Culturing was carried out at 0 or
18 °C until cell concentration in cultures reached 107
cells/ml. Cells were harvested by centrifugation, and acyl-CoA esters
were extracted using a method adapted from a previously published
extraction process (24). Cells were washed three times with 100 ml of
50 mM potassium phosphate, pH 7.2, containing 10 mM MgCl2 and 4% (v/v) glacial acetic acid. 3 ml of the same buffer and 40 ml of chloroform:methanol (1:2) were then
added to 3 g of the cells, and the mixture was homogenized using a
blender (Ultra-Turrax T25, Van der Heyden) for 3 min at 8000 rpm. Extraction was completed by adding 12.7 ml of distilled water and
12.7 ml of chloroform. After centrifugation the chloroform phase
containing lipids was carefully removed, and the aqueous phase
containing acyl-CoA esters and proteins was washed three times with 20 ml of chloroform. Proteins were precipitated by addition of 25 ml of
acetonitrile. The mixture was allowed to stand for 20 min at room
temperature before centrifugation. The supernatant was lyophilized before chromatography analysis.
The extracted acyl-CoA esters were analyzed by HPLC on a LiChrospher
100 RP-18 column (Merck). The mobile phases used were solvent A (20%
acetonitrile in 25 mM KH2PO4, pH
5.3) and solvent B (80% acetonitrile in 25 mM
KH2PO4, pH 5.3). The acyl-CoA esters were
eluted with the following gradient of solvent B in solvent A: 0% B for
10 min, 0-60% B for 55 min, 60% B for 10 min, 60-100% B for 10 min, 100% B for 10 min, and 100 to 0% B for 5 min. The flow rate was
1 ml/min. Acyl-CoA esters were detected by UV absorption at 254 nm.
Peaks were identified using standards (Sigma). For quantification of
acyl-CoA esters the recorded chromatograms were integrated, and the
amounts of individual acyl-CoA esters were determined by comparing peak
areas to those obtained by injection of known quantities of commercial
myristoyl-CoA. Data acquisition and integration were performed using
the Softron PC Integration Software.
The peak eluted at the expected myristoyl-CoA elution time (39.7 min)
was analyzed and identified by fast atom bombardment-mass spectrometry
using a method previously described by Norwood et al.
(25).
Cell Disruption and Protein Extraction--
400 g of cells were
suspended in 1 liter of 100 mM phosphate buffer, pH 7.0, containing 3 mM dithiothreitol and 10 mM EDTA and disrupted by pressure at 1500 bar with a Niro homogenizer (Panda).
The cell extract was obtained by centrifugation at 40,000 × g for 60 min. Nucleic acids were removed by addition of
protamine sulfate (0.1% w/v) and centrifugation for 10 min at
10,000 × g.
Thioesterase Precipitation--
Ammonium sulfate salt was added
to the cell extract under constant stirring until 30% saturation was
obtained. The precipitate was removed by centrifugation (30 min,
10,000 × g). The resulting supernatant was saturated
to 80% by addition of (NH4)2SO4.
The resulting precipitate was solubilized in 200 ml of 50 mM phosphate buffer, pH 7.
Hydrophobic Interaction Chromatography with Phenyl-Sepharose
Column--
Samples were applied to a phenyl-Sepharose Fast Flow
XK26/40 column (Amersham Pharmacia Biotech) equilibrated with 50 mM phosphate buffer, pH 7.0, containing 0.1 M
NaCl. A flow rate of 3 ml/min was used. The unadsorbed material was
washed successively with 1 liter of the equilibration buffer and with
600 ml of distilled water. Desorption of the bound proteins was
performed with 200 ml of 20 mM Tris-HCl buffer at pH
8.5.
Ion-exchange Chromatography with Mono Q Column--
Active
fractions from the phenyl-Sepharose column were pooled, dialyzed
overnight with distilled water, and lyophilized. The dried sample was
solubilized in 20 mM Tris-HCl buffer, pH 7.7, and applied
to a Mono Q HR5/5 column (Amersham Pharmacia Biotech) previously
equilibrated with the same buffer. Proteins were eluted with a gradient
of NaCl in 20 mM Tris-HCl buffer, pH 7.7, as follows: 0 M NaCl for 20 min, 0-0.15 M NaCl for 20 min,
0.15 M NaCl for 30 min, 0.15-1 M NaCl for 5 min, and 1 M NaCl for 10 min. The flow rate was 0.8 ml/min,
and fractions of 2 ml were collected.
Hydrophobic Interaction Chromatography with Phenyl-Superose
Column--
Active fractions eluted from the Mono Q column were
pooled, concentrated by ultrafiltration on a Amicon YM 10 membrane, and mixed with a 4 M
(NH4)2SO4 solution to give a final
(NH4)2SO4 concentration of 1 M. The sample was then applied to a phenyl-Superose column
HR5/5 previously equilibrated with 50 mM phosphate buffer, pH 7, containing 1 M
(NH4)2SO4. Proteins were eluted
with a gradient of (NH4)2SO4 in 50 mM phosphate buffer, pH 7, as follows: 1 M (NH4)2SO4 for 20 min, 1 to 0.6 M (NH4)2SO4 for 20 min,
0.6 M (NH4)2SO4 for 30 min, 0.6-0 M (NH4)2SO4
for 5 min, and 0 M
(NH4)2SO4 for 10 min. The flow rate
was 0.5 ml/min, and fractions of 2 ml were collected.
Protein Assay--
Protein concentrations were determined by the
Bradford method (26) using Coomassie Blue G.
Electrophoresis--
Analysis of column fractions during
purification was performed by gel electrophoresis using the LKB
Multifor II electrophoresis system (Amersham Pharmacia Biotech).
Polyacrylamide gels (ExcelGel SDS 8-18, Amersham Pharmacia Biotech)
were used for SDS-PAGE following the standard procedure of Laemmli
(27). 15-40 µg of protein per sample were deposited on the gel.
Proteins were revealed by silver nitrate staining.
Thioesterase Assays--
Thioesterase activity was assayed
spectrophotometrically at 25 °C by monitoring the increase in
absorbance at 212 nm resulting from the generation of free CoASH in the
presence of 5,5'-dithiobis(2-nitrobenzoic acid) (Sigma). Activities
were determined with an
412 nm of 13.6 mM
1 cm
1
for the 2-nitrobenzoate anion. A typical reaction mixture contained 100 µM myristoyl-CoA substrate in 20 mM Tris-HCl
buffer, pH 7.4. A unit of enzyme activity is defined as the amount of
enzyme required to hydrolyze 1 µmol of myristoyl-CoA per min.
Molecular Mass Determination--
The molecular mass of the
native protein was determined by gel filtration on a Sephacryl S-200
column (Amersham Pharmacia Biotech). An aliquot of the purified enzyme
from the phenyl-Superose chromatography step was applied to the column.
Running buffer was 20 mM Tris-HCl, pH 7.7, and 150 mM NaCl. The flow rate was fixed at 0.3 ml/min, and
fractions of 3 ml were collected. The molecular mass of the
thioesterase was also estimated by SDS-PAGE under the conditions
described above.
Substrate Specificity--
Kinetic parameters for hydrolysis of
acyl-CoAs were determined at concentrations of 10, 25, 50, 75, 100, and
150 µM in 20 mM Tris-HCl buffer, pH 7.2, as
described for the thioesterase assay.
Thioesterase Thermodependence--
The thermodependence of the
kinetic parameters was determined in the temperature range from 5 to
35 °C. The substrate used was myristoyl-CoA at concentrations of 10, 25, 50, 75, 100, and 150 µM in 20 mM Tris-HCl
buffer, pH 7.2. The study was carried out using a computer controlled
LKB Ultrospec III spectrophotometer equipped with a thermostated cell
changer base plate (Amersham Pharmacia Biotech). To calculate
Km and Vmax values
experimental data points were computer-fitted (Enzyme Kinetic
Application Software 2.01, Amersham Pharmacia Biotech) to the Hanes
transformation of the Michaelis-Menten equation.
The optimum temperature for activity was determined over the range
5-60 °C with the thermostated LKB Ultrospec III spectrophotometer (Amersham Pharmacia Biotech).
For thermostability, aliquots of purified thioesterase (28 µg per ml
of 20 mM Tris buffer, pH 7.0) were incubated at 40, 50, and
60 °C, and samples were periodically withdrawn and chilled on ice
before being assayed at 25 °C by the standard method.
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RESULTS |
Influence of Culture Temperature on Growth and Acyl-CoA Ester
Metabolism--
The Antarctic yeast R. aurantiaca A19 is
unable to grow above 20 °C and is therefore referred as a
psychrophilic strain (28). Cultures are highly temperature-sensitive,
and cell density drastically decreases when temperature increases (29).
Moreover, temperatures higher than those normally experienced by the
strain (
2 to 4 °C) have pronounced effects on physiological
processes and cell morphology. Examination by phase contrast microscopy
shows major cell morphological changes when the cultures are grown near
20 °C (Fig. 1). Budding is inhibited,
and cells are larger than at low temperatures. Membrane integrity could
also be affected since cellular content release was observed
(arrows in Fig. 1B).

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Fig. 1.
Morphological effect of growth temperature in
R. aurantiaca A19. Cells were grown at 0 (A) and 18 °C (B), stained by ethylene blue
for 5 min, and visualized by contrast phase microscopy. The
arrows in B indicate released cellular contents
stained by ethylene blue. Bar represents 10 µm.
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A similar behavior is reported for several temperature-sensitive
mutants of Saccharomyces cerevisiae. The mutants are
affected in long chain acyl-CoA metabolism and showed a
temperature-dependent auxotrophy for long chain saturated fatty
acids (30-35). A thermal shift from 24 to above 30 °C causes growth
arrest associated with an increase in cell size and membrane lysis
(33). With R. aurantiaca A19, exogenous fatty acids (C12,
C14, C16, and C18) do not improve cell growth at nonpermissive
temperatures (Fig. 2).

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Fig. 2.
Effect of fatty acid addition on cell growth
at nonpermissive temperatures. Cultures are incubated at 18 °C
on YPD medium with or without 500 µM of each fatty acid.
, control cultivated at 4 °C on YPD medium without fatty acid;
, YPD without fatty acid; , YPD+C12; , YPD+C14; , YPD+C16;
, YPD+C18.
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To determine whether acyl-CoA metabolism is affected by growth
temperature, intracellular acyl-CoA esters were extracted and analyzed
by HPLC (Fig. 3). The extract of cells
grown at 18 °C showed an important peak at a retention time of 39.7 min. The peak was identified as myristoyl-CoA (C14-CoA) by comparison
with an authentic standard. Fast atom bombardment-mass spectrometry confirmed the identification. The mass spectrum and suggested fragmentation pattern are shown in Fig.
4. The peak at m/z 978 corresponds to the protonated molecular ion (MH+).
Cleavages producing ions at m/z 471, 508, 428, and 330 are significance. The ion at m/z 471 is of particular
significant because it preserves the identity of the acyl group (25).
Cleavage at the adenine-ribose bond produces a positive ion at
m/z 136.

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Fig. 3.
Reverse-phase HPLC of R. aurantiaca A19 extract. Cell extracts of cells
cultivated at 0 (heavy line) and 18 °C (fine
line) were chromatographed on a LiChrospher 100 RP-18 column. The
mobile phases are as follows: buffer A (20% acetonitrile in 25 mM KH2PO4, pH 5.3) and buffer B
(80% acetonitrile in 25 mM KH2PO4,
pH 5.3). The acyl-CoA esters were detected by UV absorption at 254 nm.
The arrow indicates the putative myristoyl-CoA.
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Fig. 4.
Full mass spectrum and suggested
fragmentation pattern for myristoyl-CoA eluted at 39.7 min on
reverse-phase chromatography (Fig. 3) and analyzed by
positive ion fast atom bombardment.
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The intracellular concentration of myristoyl-CoA in cells cultivated at
18 °C is 500 µM and is 28-fold higher than in cells grown at 0 °C. Retention times for octanoyl-CoA, decanoyl-CoA, palmitoyl-CoA, and stearoyl-CoA are 16.2, 25.1, 45.2, and 52.1 min,
respectively, and no significant difference is observed in their
intracellular concentrations at 0 and 18 °C.
Several mechanisms have been suggested for the regulation of cellular
acyl-CoA ester concentration. Berge and Aarsland (17) proposed that
acyl-CoA pools are regulated by acyl-CoA thioesterase, thus we purified
and characterized the R. aurantiaca A19 thioesterase.
Purification and Characterization of Long Chain Acyl-CoA
Thioesterase--
Despite the negative effect of the increasing
temperature on cell growth, thioesterase production is not directly
influenced by temperature, and it decreases proportionally with cell
density (data not shown). At all experimental temperatures production was 1 unit/109 cells.
Following cultivation of R. aurantiaca A19 at 12 °C, the
intracellular long chain acyl-CoA thioesterase was released by mechanic cell disruption and purified by ammonium sulfate precipitation and
three successive fast protein liquid chromatography steps. Recoveries
calculated after each purification step are given in Table
I. A final yield of 32% was obtained,
and the purified enzyme had a specific activity of 108 units/mg
of protein. Specific activity is 351 times higher after purification
than in the crude extract.
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Table I
Purification of thioesterase from the psychrophilic yeast R. aurantiaca
A19
Activity was determined with myristoyl-CoA as the substrate.
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SDS-PAGE analysis shows that the enzyme is pure at homogeneity, with an
apparent molecular mass of about 80 kDa (Fig.
5). Gel filtration on Sephacryl S-200
shows an apparent molecular mass of 85 ± 6 kDa indicating that
the purified thioesterase is monomeric. The pI of the enzyme is 4.4, and the enzyme is stable and active over a broad pH range (5-10) with
an optimal activity at pH 8 (data not shown). Enzyme activity is
completely inhibited by diisopropyl fluorophosphates, indicating that
the thioesterase has a serine residue in its active site. The
thioesterase is a highly glycosylated protein, and the glycosidic
portion, removed by endoglycosidase H, represents about 19% of its
molecular weight.

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Fig. 5.
SDS-PAGE analysis. Fractions pooled
after each purification step of acyl-CoA thioesterase are analyzed by
SDS-PAGE and silver-stained for protein revelation. Lane 1, pooled fractions after ammonium sulfate precipitation; lane
2, pooled fractions after phenyl-Sepharose chromatography;
lane 3, pooled fractions after anion exchange (Mono Q)
chromatography; lane 4, pooled fractions after
phenyl-Superose chromatography; lane 5, molecular mass
standards in kDa (LMW calibration kit, Amersham Pharmacia
Biotech).
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The intact enzyme has an N terminus blocked to protein sequencing.
However, analysis of two internal peptide sequences obtained by
endoproteinase Lys-C digestion (LREIALMRY and QAAYDTTAEFA) exhibited,
respectively, 77 and 72% identity with two amino acid sequences
located between residues 179-187 and 252-262 of the S. cerevisiae Mvp1 protein (36). This protein interacts, by unknown
mechanism, with Vps1p; a protein essential for vacuolar protein sorting
and cell growth at high temperature (36, 37).
Substrate specificity was determined using several acyl-CoA ester
derivatives. Kinetic parameters for hydrolysis are given in Table
II. Thioesterase is active on thioesters
with carbon chain lengths ranging from 8 to 18. No activity is detected
with C2-CoA or C4-CoA. The preferred substrates are C14-CoA, C16-CoA, and C18-CoA, and their Km values range from 18 to 24 µM. The best substrate is myristoyl-CoA which is
hydrolyzed with the higher catalytic efficiency
(kcat/Km) of about 10 s
1·µM
1.
The Km and
kcat/Km values calculated for
the hydrolysis of the unsaturated palmitoleoyl-CoA (C16:1) are 2-fold higher and 3.5-fold lower, respectively, than those calculated for the
corresponding saturated thioester (C16:0).
Thermodependence of Thioesterase Activity--
Thermal stability
of the thioesterase activity was examined by heating the enzyme
solution to 40, 50, and 60 °C for different times (Fig.
6A). The remaining activities
after 60 min of incubation are 82, 52, and 3%, respectively. The
temperature profile of thioesterase activity was determined at
temperatures ranging from 5 to 60 °C (Fig. 6B) and shows
an optimal activity at 45 °C.

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Fig. 6.
Thermal stability and optimal temperature of
long chain acyl-CoA thioesterase. A, thermal
inactivation of the acyl-CoA thioesterase from R. aurantiaca
A19 at 40 °C ( ), 50 °C ( ), and 60 °C ( ). Residual
activity was determined using myristoyl-CoA as the substrate.
B, effect of temperature on the activity of acyl-CoA
thioesterase from R. aurantiaca A19. Activity was determined
at different temperatures as described under "Material and
Methods." Myristoyl-CoA was used as the substrate.
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Thermodependence of the kinetic parameters Km and
catalytic efficiency
(kcat/Km) for myristoyl-CoA
hydrolysis shows that the best physiological efficiency of the
thioesterase is reached near 0 °C (Fig.
7). The Km value is
0.4 µM at 5 °C and increases exponentially with
temperature; at 20 °C it is 59-fold higher. Catalytic efficiency is
also largely affected by a temperature increase; its value is 28-fold
higher at 5 than at 20 °C (Fig. 7).

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Fig. 7.
Thermodependence of
Km and catalytic efficiency. The kinetic
parameters (Km ( ) and
kcat/Km ( )) were
determined at the temperature range from 5 to 35 °C. The substrate
was myristoyl-CoA at the concentrations of 10, 25, 50, 75, 100, and 150 µM in 20 mM Tris-HCl buffer, pH 7.2. Initial
velocity values were fitted to the Hanes transformation of the
Michaelis-Menten equation.
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DISCUSSION |
The inability to grow above 20 °C is common in psychrophilic
microorganisms (10, 28, 38) and seems to be thermodynamically a
paradox, especially if one considers the favorable effect of moderate
temperatures on enzyme reactions and biological processes. Temperatures
higher than 5 °C have a negative effect on the growth of the
antarctic yeast R. aurantiaca A19, and cell lysis occurs when cultures are grown close to the upper temperature limit for growth.
Because of their amphipathic and toxic property, an excess of acyl-CoA
esters is likely to disrupt membrane bilayers and to impair several
cellular enzymes and functions (11, 16). HPLC analysis of the
intracellular acyl-CoA esters from R. aurantiaca A19 reveals
an abnormal accumulation of myristoyl-CoA (C14-CoA) in cells cultivated
close to the nonpermissive temperature (20 °C). Its concentration is
500 µM (28-fold higher than at 0 °C), whereas it has
been reported that the total cellular acyl-CoA ester content is
unlikely to exceed 160 µM, even under the most extreme
conditions (11).
Myristoyl-CoA is an essential compound for cell growth because it
contributes to the activation, by N-myristoylation, of
several proteins regulating cell growth and signal transduction (31, 33).
In S. cerevisiae there are at least two metabolic pathways
that produce myristoyl-CoA, de novo synthesis or activation
of free myristate by acyl-CoA synthetases (30-33). The de
novo pathway uses malonyl-CoA produced by acetyl-CoA carboxylase
(39) to generate long chain saturated acyl-CoAs through the cytosolic fatty-acid synthetase complex (40). Palmitoyl-CoA and stearoyl-CoA are
the main products of fatty-acid synthetase, whereas myristoyl-CoA represents only 3-5% of the total acyl-CoAs synthesized (41, 42).
In R. aurantiaca A19 myristoyl-CoA accumulation at
nonpermissive temperatures could not be due to an increase in the
fatty-acid synthetase activity or acyl-CoA synthetase as it represents
a minor product of these enzymes in comparison with palmitoyl-CoA and
stearoyl-CoA (41-43).
Intracellular concentrations of acyl-CoA esters are regulated by their
rate of synthesis, utilization, and degradation, and excessive
increases in their concentration are prevented by hydrolysis by
thioesterases (11, 16, 17, 22). At present, myristoyl-CoA metabolism is
not completely understood; however, the increase in concentration at
18 °C indicates a deficiency in its utilization and/or hydrolysis at
high temperatures.
Because of their putative contribution to the control of myristoyl-CoA
levels, an 80-kDa long chain acyl-CoA thioesterase was isolated and
characterized. The intracellular accumulation of myristoyl-CoA could
not be related to a thermal inhibition of the genetic expression of the
purified thioesterase because its production is about 1 unit/109 cells and varies proportionally with cell density
at various culture temperatures.
On the other hand, great structural flexibility is a rather
common characteristic of the psychrophilic enzymes (1-5). Hence, it is
tempting to speculate that the lower thermal stability of R. aurantiaca thioesterase could be a cause of the intracellular accumulation of myristoyl-CoA at nonpermissive temperatures. However, the thermostability of R. aurantiaca thioesterase contrasts
with this suggestion. Enzyme-substrate interactions may also exhibit marked temperature dependence. The affinity and the catalytic efficiency generally give much information on this, especially in the
case of regulatory intracellular enzymes that catalyze reactions at
substrate concentration close to the Km value (2,
44, 45). The temperature at which the affinity and the catalytic
efficiency toward the myristoyl-CoA are highest closely coincides with
the habitat temperature of the strain. At nonpermissive temperatures
both parameters dramatically decrease, and the loss of catalytic
efficiency correlates with the intracellular increase in myristoyl-CoA
concentration. This behavior is consistent with the physiological
temperature of the source organism; however, among the cold-adapted
enzymes studied so far, this is the first intracellular enzyme found to
display such a large variation in the Km and
catalytic efficiency with temperature. If one admits that the
kcat/Km ratio is the
operational parameter for regulatory intracellular enzymes, this
finding is very important and reveals one of the probable mechanisms
for the intracellular accumulation of myristoyl-CoA and cell death at
moderate temperatures.
On the other hand, thioesterase is described as a "scavenger" in
acyl-CoA metabolism, and its regulation role should only be utilized to
avoid excessive concentrations (11, 16). Consequently, the pathological
accumulation of myristoyl-CoA at high temperatures would not only be
caused by the decrease in its hydrolysis by thioesterase but also by
other, as yet unknown, factors. One such factor could be a deficiency
in N-myristoyltransferase activity, which is an essential
enzyme for cell growth and represents an important myristoyl-CoA
utilizer. Recent work indicates that in mesophilic yeasts several
N-myristoyltransferase mutations can cause a
temperature-dependent affinity decrease for myristoyl-CoA and growth arrest above 30 °C (30, 31, 33). Moreover, a high
myristoyl-CoA concentration without increase of the concentration of
other long chain acyl-CoA esters, particularly palmitoyl-CoA, well
known as the major product of the fatty acid synthase (41-43), suggests that the principal pathway of C14-CoA use in the psychrophilic R. aurantiaca is presumably temperature-sensitive and could
be different from the metabolic pathways using other long chain
acyl-CoA esters.
In conclusion, our results show for the first time that moderate
temperatures cause a deficiency in the myristoyl-CoA metabolism in the
psychrophile R. aurantiaca. Growth arrest at high
temperatures is associated with a toxic intracellular accumulation of
myristoyl-CoA. The purified long chain acyl-CoA thioesterase shows a
temperature-dependent decrease in its catalytic efficiency
and in its affinity for myristoyl-CoA. This result reveals one of the
probable mechanisms by which high temperatures induce growth arrest.
Finally, our work should stimulate further studies of other enzymes
that are implied in myristoyl-CoA metabolism and contribute to the
understanding of the biochemical basis of the inability of
psychrophilic microorganisms to grow at moderate temperatures.