Applied Microbiology and Biotechnology

, Volume 66, Issue 3, pp 285–290

Carbon isotope fractionation during cistrans isomerization of unsaturated fatty acids in Pseudomonas putida


    • Department of BioremediationCentre for Environmental Research (UFZ) Leipzig-Halle
  • Grit Neumann
    • Department of BioremediationCentre for Environmental Research (UFZ) Leipzig-Halle
  • Nadja Kabelitz
    • Department of BioremediationCentre for Environmental Research (UFZ) Leipzig-Halle
  • Matthias Kastner
    • Department of BioremediationCentre for Environmental Research (UFZ) Leipzig-Halle
  • Hans Hermann Richnow
    • Department of BioremediationCentre for Environmental Research (UFZ) Leipzig-Halle
Biotechnologically Relevant Enzymes and Proteins

DOI: 10.1007/s00253-004-1734-z

Cite this article as:
Heipieper, H.J., Neumann, G., Kabelitz, N. et al. Appl Microbiol Biotechnol (2004) 66: 285. doi:10.1007/s00253-004-1734-z


The molecular mechanism of the unique cis to trans isomerization of unsaturated fatty acids in the solvent-tolerant bacterium Pseudomonas putida S12 was studied. For this purpose, the carbon isotope fractionation of the cistrans isomerase was estimated. In resting cell experiments, addition of 3-nitrotoluene for activation of the cistrans isomerase resulted in the conversion of the cis-unsaturated fatty acids into the corresponding trans isomers. For the conversion of C16:1 cis to its corresponding trans isomer, a significant fractionation was measured. The intensity of this fractionation strongly depended on the rate of cistrans isomerization and the added concentration of 3-nitrotoluene, respectively. The presence of a significant fractionation provides additional indication for a transition from the sp2 carbon linkage of the cis-double bond to an intermediate sp3 within an enzyme–substrate complex. The sp2 linkage is reconstituted after rotation to the trans configuration has occurred. As cytochrome c plays a major role in the catabolism of Cti polypeptide, these findings favour a mechanism for the enzyme in which electrophilic iron (Fe3+), provided by a heme domain, removes an electron of the cis double bond thereby transferring the sp2 linkage into sp3.


One of the solvent adaptation mechanisms enabling several Pseudomonas strains to grow in the presence of membrane-disrupting compounds is the isomerization of cis to trans unsaturated fatty acids (for review see, Keweloh and Heipieper 1996; Ramos et al. 2002; Heipieper et al. 2003). The extent of the isomerization apparently correlates with the toxicity and the concentration of such organic compounds in the membrane (Heipieper et al. 1992, 1994, 1995). A mutual dependency was also found between the activation of this system and the induction/activation of other stress-response mechanisms (Heipieper et al. 1996).

The cistrans isomerase activity is constitutively present, does not require ATP or other cofactors like NAD(P)H or glutathione, and works in the absence of de novo synthesis of lipids (Heipieper et al. 1992; Morita et al. 1993; Heipieper and de Bont 1994). The enzyme has been purified from the periplasmic fraction of Pseudomonas oleovorans (Pedrotta and Witholt 1999). The cistrans isomerase gene cloned and sequenced from Pseudomonas putida P8 (Holtwick et al. 1997) and P. putida DOT-T1E (Junker and Ramos 1999) made evident that the isomerase has an N-terminal hydrophobic signal sequence, which is cleaved off after targeting the enzyme to the periplasmic space. Holtwick et al. (1999) showed that the enzyme is a cytochrome c-type protein, as they could find a heme-binding site in the predicted Cti polypeptide. For an enzyme preparation from Pseudomonas sp. strain E-3, which is homologous to the cti gene product of P. putida P8, it was suggested that iron (probably Fe3+) plays a crucial role in the catalytic reaction (Okuyama et al. 1998).

Recently, direct evidence was obtained that isomerization does not include a transient saturation of the double bond. In addition, comparison of the amino acid sequences of the seven known Cti proteins identified them as heme-containing proteins of the cytochrome c-type (von Wallbrunn et al. 2003).

Despite the wealth of available information, the biochemical mechanism of the solvent-induced regulation of isomerase activity, in relation to membrane homeostasis (Ramos et al. 1997), is still not completely understood. One technique for an indirect elucidation of the molecular mechanisms of the cistrans isomerization could be the measurement of the stable isotope fractionation of this enzymatic catalysis.

The intensity of a biological transformation can directly be quantified by the isotope fractionation of the substrates during enzymatic activity, as this leads to an enrichment of the naturally present stable isotopes 13C or 2H, respectively, in the residual fraction. The relation between the concentration and isotopic composition of a compound is described by the kinetic isotope fractionation factor (αC). The isotope effects are capable of providing detailed information about transition-state structure and other aspects of mechanisms not only in organic reactions but in enzymatic reactions as well (O’Leary 1980). The dissociation energy needed to cleave a heavy-isotope-substituted bond is higher and is reflected in a decrease in the reaction rate in the transition state of the reaction (O’Neil 1986). Primary isotope effects are higher when the rate-limiting step in the reaction is directly related to a mass-dependent stability of the bond, which is formed or cleaved in the transition state of the reaction. This is commonly the case for the cleavage of sp3 type chemical linkages.

In this contribution, we report on the analysis of the carbon isotope fractionation of the cistrans isomerization in P. putida. The observed strong isotope fractionation provides evidence that isomerization includes a binding of the substrate to the active center of heme-containing proteins of the cytochrome c-type.

Materials and methods

Strain and chemicals

Pseudomonas putida S12 was isolated as a styrene-degrading organism and has previously been described (Hartmans et al. 1990). All chemicals were reagent grade and obtained from commercial sources.

Culture conditions

Pseudomonas putida S12 was cultivated in a mineral medium as described by Hartmans et al. (1989) with 20 mM Na2-succinate as sole carbon source. Cells were grown in 50-ml shake cultures in a horizontally shaking water bath at 30°C. Growth was monitored by measuring the turbidity (optical density) at 560 nm (OD560).

Preparation of resting cells

Exponentially growing cells (50 ml) were harvested by centrifugation and suspended in the same volume of potassium phosphate buffer (50 mM, pH 7.0). Experiments were started 45 min after suspension of cells, by which time growth had stopped completely.

Incubation with 3-nitrotoluene

For activation of the cistrans isomerase, 3-nitrotoluene in concentrations up to 2.5 mM was added to the resting cells. Cultures were incubated in the presence of 3-nitrotoluene for 1 h in a shaking water bath at 30°C. Cells were then harvested, washed twice with potassium phosphate buffer (50 mM, pH 7.0) and stored at −20°C prior to use.

Lipid extraction, transesterification, and fatty acid analysis

The lipids were extracted with chloroform/methanol/water as described by Bligh and Dyer (1959). Fatty acid methyl esters (FAME) were prepared by incubation for 15 min at 95°C in boron trifluoride/methanol, applying the method of Morrison and Smith (1964). FAME were extracted with hexane.

Analysis of fatty acid composition by GC–MS

FAME in hexane were analyzed using a quadrupole GC–MS System (HP6890, HP5973, Hewlett Packard, Palo Alto, Calif., USA) equipped with a split/splitless injector. A CP-Sil 88 capillary column (Chrompack, Middelburg, The Netherlands; ID: 0.32, 30 m; 0.25 μm film) was used for the separation of the FAME. GC conditions are: injector temperature was held at 240°C. The split flow was 1:10 and carrier gas He. The temperature program was: 80°C, 1 min isotherm, 15°C/min to 140°C; 4°C/min to 240°C. The MS conditions were: ionization mode EI; ionization energy 70 eV. The peak areas of the carboxylic acids in total ion chromatograms (TIC) were used to determine their relative amounts. The fatty acids were identified by GC–MS and co-injection with authentic reference compounds obtained from Suppelco (Bellefonte, USA).

Analysis of the carbon isotope composition of fatty acids by GC–C-IRM–MS

A gas chromatography–combustion-isotope ratio monitoring–mass spectrometer (GC–C-IRM–MS) system was used to determine the isotope composition of fatty acids. The system consists of a gas chromatograph (6890 Series, Agilent Technology, USA) coupled via a Conflow III interface (ThermoFinnigan, Germany) to a MAT 252 mass spectrometer (ThermoFinnigan). The performance and experimental details of this system are described elsewhere (Richnow et al. 2003a,b). A CP-Sil 88 capillary column (Chrompack, Middelburg, The Netherlands; ID: 0.32, 30 m; 0.25-μm film) was used for the separation of FAME. GC conditions consisted of a split injector temperature held at 250°C. The split flow was adjusted between 1:2.5 and 1:10 according to the expected concentrations and He was used as carrier gas. The temperature program was 80°C, 1 min isotherm, 15°C/min up to 140°C; 2°C/min to 280°C. Aliquots (1–2 μl) of n-hexane extracts were used for injection. All samples were measured in at least four replicates.


The carbon isotope composition is reported in δ-notation (per mill) relative to Vienna Pee Dee Belemnite standard (V-PDB) according to
$$\delta _{t} ^{{13}} {\text{C}} = {\left( {\frac{{{\left( {^{{13}} {\text{C}}/^{{12}} {\text{C}}} \right)}_{{{\text{Sample}}}} }}{{{\left( {^{{13}} {\text{C}}/^{{12}} {\text{C}}} \right)}_{{{\text{Standard}}}} }} - 1} \right)} \times 1000$$
The isotope fractionation during the isomerization from cis to trans fatty acids was calculated applying the Rayleigh equation (Eq. 2).
$$\frac{{R_{t} }}{{R_{0} }} = {\left( {\frac{{C_{t} }}{{C_{0} }}} \right)}^{{{\left[ {{\left( {1/\alpha } \right)} - 1} \right]}}} $$
Rt and R0 give the isotope composition of the cis fatty acid at times t and zero, Ct and C0 represent the concentration of the cis fatty acid at times t and zero of the experiment. The isotope fractionation factor (α) relates the changes in the isotope composition to changes in the concentration of the residual fraction during the transformation.
The intrinsic isotope fractionation factor αintrinsic was calculated according to Eq. 3. The intrinsic isotope fractionation factor normalizes the isotope fractionation of the isotopomer to the atom at which the biochemical reaction takes place. α is the fractionation factor obtained using Eq. 2. The equation was deduced from the Rayleigh equation (1) to consider the number of carbon atoms (n) that do not undergo isotope fractionation and remain isotopically stable during the reaction.
$$\alpha _{{{\text{intrinsic}}}} = \frac{1}{{{\left[ {{\left( {n/\alpha } \right)} - {\left( {n - 1} \right)}} \right]}}}$$


Cistrans isomerase activity in the cells

It has been previously observed that, in the presence of organic solvents such as 1-nitrotoluene, P. putida S12 cells convert cis-unsaturated fatty acyl chains present in the plasma membrane to the corresponding trans isomers. This mechanism also works in non-growing cells (Heipieper et al. 2001, 2003). Therefore, the experiments were carried out in so-called non-energized resting cells to exclude all other adaptive mechanisms and were very similar to those examined with the isolated enzyme. An addition of 3-nitrotoluene led to an increase of the ratio of C16 trans/cis fatty acid acyl chains from 0.07 in control cells to 2.17 in the presence of 3 mM 3-nitrotoluene (Fig. 1a).
Fig. 1

Effect of 3-nitrotoluene on the conversion of cis-unsaturated to trans-unsaturated fatty acids in resting cells of Pseudomonas putida S12. a Ratio of C16 trans/cis unsaturated fatty acids; b percentages of C16 cis (filled triangles) and trans (filled squares) unsaturated fatty acids, and the isotope ratios percentages of C16 cis (open triangles) and trans (open squares) unsaturated fatty acids given as the δ-13C values; c Rayleigh equation of the conversion of C16 cis-unsaturated to trans-unsaturated fatty acids

At the beginning of the experiment, 93.6% and 6.4% of the unsaturated C16 fatty acids were detected as cis and trans isomers (Fig. 1b). The carbon isotope composition of cis C16 fatty acid was −22.5±0.4‰. The addition of 3-nitrotoluene resulted in a conversion of cis to trans species, which was associated with an enrichment of 13C in the non-transformed fraction. The addition of 3 mM nitrotoluene resulted in a decrease of cis species to 31.5% whereby the isotope composition changed to 21.2±0.1‰. During the reaction, the isotope composition of the cis fatty acids was enriched in 13C by about 1.3 δ ± units. The isotope composition of trans fatty acid at the beginning of the experiment was 21.5±0.1‰, which was slightly heavier than the cis form. Yet, taking into consideration the uncertainty of the isotope measurement, the isotope composition is similar. During the isomerization reaction, the isotope composition of the trans species become isotopically depleted in 13C to −23.3±0.5‰. The isomerization reaction was completed at a concentration of 3 mM nitrotoluene and further addition did not change the cis/trans ratio. The isotope discrimination between precursor and product was about 2.1±0.1 δ units at the end of the experiment, when 68.5% of the unsaturated C16 species was present in the trans form. The ratio of about 32% cis to 69 cis % trans is close to the expected thermodynamic equilibrium for cistrans isomerization of olefinic double bonds (Ferreri et al. 1999; Chatgilialoglu et al. 2000, 2002).

The isotope fractionation was quantified applying the Rayleigh equation (Eq. 2). The change in isotope composition was correlated with the change in concentration. The regression curve showed a reasonable uncertainty (r2=0.9201) and gave an isotope fractionation factor (αC) of 1.0011±0.0001 (Fig. 1c). The isotope fractionation at the reactive center of the C16 fatty acid was calculated assuming that only the carbon atoms at the double bond are involved in the isomerization reaction, which gave an intrinsic fractionation factor of 1.020±0.0024 (Eq. 3).


The cistrans isomerase showed a remarkable isotope fractionation. To our knowledge, this is the first report on isotope fractionation during enzymatic isomerization of fatty acids. The magnitude of the isotopic fractionation factor (αC) 1.0011 is surprisingly high and the intrinsic fractionation factor (αintrinsic 1.020±0.0024) representing the isotope discrimination at the reactive center of the molecule indicates a primary kinetic isotope effect.

For enzymatic reactions that proceed in a single step, such as the cistrans isomerase, the magnitude of a heavy-atom isotope effect on that reaction may reflect the structure of the transition state for the reaction. Qualitatively, the magnitude of the isotope effect is related to the change in bonding to the isotopic atom ongoing from ground state to transition state (O’Leary 1980).

The major open question in all investigations dealing with Cti concerns the molecular mechanism of the isomerization reaction. Though the mechanism is not completely understood, cleavage of the double bond is presumably facilitated by the formation of a substrate–catalyst complex, enabling rotation of the carbon–carbon single bond. In all the Cti sequences of the seven bacteria compared (belonging to the genera Pseudomonas and Vibrio), a heme-binding site of the cytochrome c type is present, which strongly supports a mechanism of cistrans isomerization by forming an enzyme-substrate complex as outlined above. These results are in good accordance with site-directed mutagenesis experiments carried out to destroy the heme-binding motif in Cti of P. putida P8 (Holtwick et al. 1999). In previous investigations, we have shown that during isomerization in resting cells no hydrogen exchange takes place at the double bond (von Wallbrunn et al. 2003). This may imply that a hydrogenation of the double bond to form a sp3 bond prior to rotation can be ruled out as an intermediate step. A radical mechanism induced by thiyl-like radicals would not lead to a hydrogenation of the double bond in a transition state to change the sp3 bond to sp2 in order to facilitate the rotation (Chatgilialoglu et al. 2002). This radical mechanism may resemble a nucleophilic reaction mechanism.

In contrast, we propose a direct attack on the double bond in unsaturated fatty acids by the electrophilic heme (Fe3+) iron that results in a disruption of an electron from the double bond to form a transient radical complex covalently bound to iron. This mechanism transforms the sp2 linkage of the double bond to the sp3 type in the transition state enabling rotation. The concomitantly arising positive charge of one of the involved C atoms possibly may be stabilized by a negatively charged amino acid of the enzyme. Although the precise chemical mechanism is not known, the solvatization of the sp3 type iron–carbon bond may be a rate-limiting reaction step causing the primary carbon isotope effect.

Heme-containing cytochrome P450 enzymes catalyze the hydroxylation of unsaturated fatty acids; they are also instrumental in desaturating fatty acids, reactions which are, however, NADPH dependent (Murataliev et al. 1997; Guan et al. 1998). Cti activity is independent of additional factors such as ATP, NADPH, or O2 (Diefenbach and Keweloh 1994; Okuyama et al. 1998); it differs in this respect from all other known heme-containing enzymes acting on fatty acids as substrates. There is, however, no need of a cofactor because no net electron power is consumed. The energy difference between cis and trans configurations is about 3.1 kJ mol−1 and shows a trans/cis ratio of about 83.7/16.3% at the thermodynamic equilibrium measured at 22°C (Chatgilialoglu et al. 2002). Therefore, the cistrans isomerization in this order is an exergonic reaction that does not consume energy (Chatgilialoglu et al. 2002). The trans/cis ratio in our experiment was about 68/32%, suggesting that the isomerization process was exergonic but close to being completed.

It will be difficult to propose a more detailed mechanism in the absence of structural data. Nonetheless, the observed isotope fractionation implies that the breaking (or the formation) of a covalent bond is implicated in the mechanism (O’Leary 1980; Yoshizawa et al. 2000a,b).

Figure 2 presents a scheme of the proposed mechanism of Cti. The initial attack of the cis-configured double bond occurs by the Fe2+ of the cytochrome c that is known to be present in Cti and necessary for its catabolic activity (Holtwick et al. 1999; Heipieper et al. 2003; von Wallbrunn et al. 2003). A covalent binding occurs between the intermediately oxidized iron (Fe3+) and one of the two carbon atoms of the double bond. Thus, an enzyme–substrate complex is formed whereupon the electrophilic iron (probably Fe3+), provided by the heme domain present in the enzyme, removes an electron from the cis double bond transferring the sp2 linking into a sp3 one. The double bond is then reconstituted after rotation to the trans configuration has occurred. The formation or, more likely, the breaking of the intermediate σ bond may explain the carbon isotope fractionation. Future research should therefore focus on crystallographic studies with the overexpressed, purified and crystalloid Cti.
Fig. 2

Scheme of a possible enzymatic mechanism of the cistrans isomerase of unsaturated fatty acids


This work was partially supported by contract no. QLRT-2001-00435 of the European Commission within its Fifth Framework Programme. We thank M. Gehre and U. Günther for technical support in the isotope laboratory of the UFZ.

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© Springer-Verlag 2004