Isolation and characterization of a second subunit of molecular chaperonin from Pyrococcus kodakaraensis KOD1: analysis of an ATPase-deficient mutant enzyme

The cpkA gene encoding a second (alpha) subunit of archaeal chaperonin from Pyrococcus kodakaraensis KOD1 was cloned, sequenced, and expressed in Escherichia coli. Recombinant CpkA was studied for chaperonin functions in comparison with CpkB (beta subunit). The effect on decreasing the insoluble form of proteins was examined by coexpressing CpkA or CpkB with CobQ (cobyric acid synthase from P. kodakaraensis) in E. coli. The results indicate that both CpkA and CpkB effectively decrease the amount of the insoluble form of CobQ. Both CpkA and CpkB possessed the same ATPase activity as other bacterial and eukaryal chaperonins. The ATPase-deficient mutant proteins CpkA-D95K and CpkB-D95K were constructed by changing conserved Asp95 to Lys. Effect of the mutation on the ATPase activity and CobQ solubilization was examined. Neither mutant exhibited ATPase activity in vitro. Nevertheless, they decreased the amount of the insoluble form of CobQ by coexpression as did wild-type CpkA and CpkB. These results implied that both CpkA and CpkB could assist protein folding for nascent protein in E. coli without requiring energy from ATP hydrolysis.     

Two kinds of archaeal chaperonin with different temperature dependency from a hyperthermophile

Thermococcus kodakaraensis KOD1 produces two kinds of chaperonin subunits, CpkA and CpkB. To monitor the expression levels of CpkA and CpkB, anti-CpkA and anti-CpkB antisera were obtained by using synthesized peptides as the haptens. These haptens were prepared based on the carboxyl terminus regions of CpkA and CpkB, which show clear differences in amino acid sequence. Immunoblotting analysis using obtained antisera revealed that the expression levels of CpkA and CpkB changed depending on the cultivation temperature. When cells were grown at 95 degrees C, intracellular amount of CpkA was low, while CpkB was expressed at extremely high level in KOD1. In the case of 70 degrees C cultivation, CpkA existed as the major chaperonin in the cell, whereas CpkB existed as the minor one. Temperature-shift experiments showed that the expression of CpkB was induced by the up-shift and reduced by the down-shift of temperature. In contrast, the expression of CpkA was reduced by the up-shift and induced by the down-shift of temperature. Furthermore, native PAGE and immunoprecipitation experiments revealed that the stoichiometrical ratio of CpkA and CpkB in chaperonin complex changed according to growth temperature.     

In vitro stabilization and in vivo solubilization of foreign proteins by the beta subunit of a chaperonin from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1.

The gene encoding the beta subunit of a molecular chaperonin from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1 (cpkB) was cloned, sequenced, and expressed in Escherichia coli. The cpkB gene is composed of 1,641 nucleotides, encoding a protein (546 amino acids) with a molecular mass of 59,140 Da. The enhancing effect of CpkB on enzyme stability was examined by using Saccharomyces cerevisiae alcohol dehydrogenase (ADH). Purified recombinant CpkB prevents thermal denaturation and enhances thermostability of ADH. CpkB requires ATP for its chaperonin function at a low CpkB concentration; however, CpkB functions without ATP when present in excess. In vivo chaperonin function for the solubilization of insoluble proteins was also studied by coexpressing CpkB and CobQ (cobryic acid synthase), indicating that CpkB is useful for solubilizing the insoluble proteins in vivo. These results suggest that the beta subunit plays a major role in chaperonin activity and is functional without the alpha subunit.     

Isolation and Characterization of a Second Subunit of Molecular Chaperonin from Pyrococcus kodakaraensis KOD1: Analysis of an ATPase-Deficient Mutant Enzyme

The cpkA gene encoding a second (α) subunit of archaeal chaperonin from Pyrococcus kodakaraensis KOD1 was cloned, sequenced, and expressed in Escherichia coli. Recombinant CpkA was studied for chaperonin functions in comparison with CpkB (β subunit). The effect on decreasing the insoluble form of proteins was examined by coexpressing CpkA or CpkB with CobQ (cobyric acid synthase from P. kodakaraensis) in E. coli. The results indicate that both CpkA and CpkB effectively decrease the amount of the insoluble form of CobQ. Both CpkA and CpkB possessed the same ATPase activity as other bacterial and eukaryal chaperonins. The ATPase-deficient mutant proteins CpkA-D95K and CpkB-D95K were constructed by changing conserved Asp⁹⁵ to Lys. Effect of the mutation on the ATPase activity and CobQ solubilization was examined. Neither mutant exhibited ATPase activity in vitro. Nevertheless, they decreased the amount of the insoluble form of CobQ by coexpression as did wild-type CpkA and CpkB. These results implied that both CpkA and CpkB could assist protein folding for nascent protein in E. coli without requiring energy from ATP hydrolysis.     

Effect of terminal achiral and chiral residues on the conformational behaviour of poly Δ(z)Phe and analysis of various interactions

Conformational properties of the peptides containing (Δ(Z)Phe)6 with achiral (ΔAla, Gly) and chiral (Ala, Leu) residues at both the N- and C-terminal positions have been studied with a view to design a peptide with desired helical screw sense. In all the peptides, the lowest energy conformational state corresponds to Φ = 0° and Ψ = + 90° or - 90° or both +/- 90°. These structures are characterized by rise per residue of 1.94 ?; rotation per residue of 114° and 3.12 residues per turn and are stabilized by: (i) carbonyl-carbonyl interactions with the carbonyl oxygen of ith residue and carbonyl carbon atom of the carbonyl group of ith+1 residue; and (ii) N-H....π interactions between the amino group of Δ(Z)Phe and its own aromatic moiety. The Ala/Leu residues at the N-terminus further stabilized the structure, through C-H....π interactions with the farthest edge of the aromatic ring of ith+3 Δ(Z)Phe residue. For peptides Ac-L-Ala/L-Leu-(Δ(Z)Phe)6-NHMe, the low energy left handed helical structure (approximately 2.5 Kcalmol?1 higher in energy) state corresponds to Φ = -30°, Ψ = 120° for L-residue and Φ = Ψ = 30° for Δ(Z)Phe residues and is in good agreement with the X-ray crystallography results for the peptide Boc-L-Ala-(Δ(Z)Phe)4-NHMe crystals grown from acetonitrile/ethanol mixture. Computational results suggest that the peptides Ac-DAla/D-Leu-(Δ(Z)Phe)6-NHMe adopt a right handed helical structure in polar solvents with Φ = 30°, Ψ = -120° for D-residues and Φ = Ψ = -30° for Δ(Z)Phe residues. Both in the left handed and right handed structures, the carbonyl oxygen of acetyl group is involved in 10-membered hydrogen bonded ring formation with NH of 3rd Δ(z)Phe residue whereas Δ(Z)Phe residues backbone adopts a 3?? helix structure. Computational results also suggest that the conformational state with Φ = 0° and Ψ = 90° can be realized by keeping D-Ala or D-Leu at the C-terminal. There is hardly any effect of achiral residues Gly/ΔAla on the conformational behaviour of poly-Δ(Z)Phe.     

Temperature effects on Agrobacterium phytochrome Agp1

Phytochromes are widely distributed biliprotein photoreceptors with a conserved N-terminal chromophore-binding domain. Most phytochromes bear a light-regulated C-terminal His kinase or His kinase-like region. We investigated the effects of light and temperature on the His kinase activity of the phytochrome Agp1 from Agrobacterium tumefaciens. As in earlier studies, the phosphorylation activity of the holoprotein after far-red irradiation (where the red-light absorbing Pr form dominates) was stronger than that of the holoprotein after red irradiation (where the far red-absorbing Pfr form dominates). Phosphorylation activities of the apoprotein, far red-irradiated holoprotein, and red-irradiated holoprotein decreased when the temperature increased from 25 °C to 35 °C; at 40 °C, almost no kinase activity was detected. The activity of a holoprotein sample incubated at 40 °C was nearly completely restored when the temperature returned to 25 °C. UV/visible spectroscopy indicated that the protein was not denatured up to 45 °C. At 50 °C, however, Pfr denatured faster than the dark-adapted sample containing the Pr form of Agp1. The Pr visible spectrum was unaffected by temperatures of 20-45 °C, whereas irradiated samples exhibited a clear temperature effect in the 30-40 °C range in which prolonged irradiation resulted in the photoconversion of Pfr into a new spectral species termed Prx. Pfr to Prx photoconversion was dependent on the His-kinase module of Agp1; normal photoconversion occurred at 40 °C in the mutant Agp1-M15, which lacks the C-terminal His-kinase module, and in a domain-swap mutant in which the His-kinase module of Agp1 is replaced by the His-kinase/response regulator module of the other A. tumefaciens phytochrome, Agp2. The temperature-dependent kinase activity and spectral properties in the physiological temperature range suggest that Agp1 serves as an integrated light and temperature sensor in A. tumefaciens.     

Identification of the genes affecting the regulation of riboflavin synthesis in the flavinogenic yeast Pichia guilliermondii using insertion mutagenesis

Pichia guilliermondii is a representative of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B(2)) in response to iron limitation. Using insertion mutagenesis, we isolated P. guilliermondii mutants overproducing riboflavin. Analysis of nucleotide sequence of recombination sites revealed that insertion cassettes integrated into the genome disrupting P. guilliermondii genes similar to the VMA1 gene of Ashbya gossypii and Saccharomyces cerevisiae and FES1 and FRA1 genes of S. cerevisiae. The constructed P. guilliermondiiΔvma1-17 mutant possessed five- to sevenfold elevated riboflavin production and twofold decreased iron cell content as compared with the parental strain. Pichia guilliermondiiΔfra1-45 mutant accumulated 1.8-2.2-fold more iron in the cells and produced five- to sevenfold more riboflavin as compared with the parental strain. Both Δvma1-17 and Δfes1-77 knockout strains could not grow at 37 °C in contrast to the wild-type strain and the Δfra1-45 mutant. Increased riboflavin production by the wild-type strain was observed at 37 °C. Although the Δfes1-77 mutant did not overproduce riboflavin, it showed partial complementation when crossed with previously isolated P. guilliermondii riboflavin-overproducing mutant rib80-22. Complementation analysis revealed that Δvma1-17 and Δfra1-45 mutants are distinct from previously reported riboflavin-producing mutants hit1-1, rib80-22 and rib81-31 of this yeast.     

Correcting for heat capacity and 5'-TA type terminal nearest neighbors improves prediction of DNA melting temperatures using nearest-neighbor thermodynamic models

Nearest-neighbor thermodynamic (NNT) models currently provide some of the most accurate predictions of melting thermodynamics, including melting temperature (T(m)) values, for short DNA duplexes. Inherent to all existing NNT models is the assumption that ΔH° and ΔS° for the helix-to-coil transition are temperature invariant. Here we investigate the impact that this zero-ΔC(p) assumption has on the accuracy of T(m) predictions for 128 DNA duplexes. Previous and new melting thermodynamic data are analyzed to establish an estimate of ΔC(p)(bp), the heat capacity change per base pair, of 42 ± 16 cal mol(-1) K(-1) bp(-1), as well as an optimal thermodynamic reference temperature (T(ref)) of 53 ± 5 °C. These results were used to modify the unified NNT model to properly account for the temperature dependence of ΔH° and ΔS° and thereby extend the range over which T(m) is accurately predicted. This new approach is shown to be especially useful for duplexes that melt at a T(m) greater than 70 °C. Thermodynamic data collected by differential scanning calorimetry (DSC) for 16 duplexes designed to melt over a broad temperature range were used to verify the values of ΔC(p)(bp) and T(ref) and to show that ΔC(p)(bp) is essentially constant above 37 °C. Additional DSC analysis of 12 duplex sequences containing all 10 nearest neighbors allowed for errors associated with different terminal nearest neighbors to be examined and showed that duplexes containing one or more terminal 5'-TA groups are significantly more stable than predicted by the unified NNT model. A correction to improve prediction of the hybridization thermodynamics of duplexes with terminal 5'-TA groups is provided.     

Compatible solutes contribute to heat resistance and ribosome stability in Escherichia coli AW1.7

This study investigated the mechanisms of heat resistance in Escherichia coli AW1.7 by quantification of cytoplasmic solutes, determination of ribosome denaturation, and by determination of protein denaturation. To assess the contribution of heat shock proteins and compatible solutes, experiments were conducted after exposure to sublethal heat shock, and with cultures grown at NaCl concentrations ranging from 0 to 6%. Heat resistance of E. coli AW1.7 was compared to the heat sensitive E. coli GGG10 and a plasmid-cured, heat sensitive derivative of E. coli AW1.7 named E. coli AW1.7ΔpHR1. Sublethal heat shock improved survival at 60°C of E. coli GGG10 and AW1.7ΔpHR1 but not of E. coli AW1.7. Addition of NaCl increased the heat resistance of all three strains, but only E. coli AW1.7 exhibited high heat resistance when grown in NaCl concentrations ranging from 2 to 6%. E. coli AW1.7 and GGG10 accumulated 16.1±0.8 and 8.8±0.8mmolL(-1) amino acids when grown at 0% NaCl, and 1.47±0.07 and 0.78±0.06mmolL(-1) carbohydrates when grown at 6% NaCl, respectively. Ribosome denaturation was determined by differential scanning calorimetry. After growth in the presence of 0% NaCl, the 30S subunit denatured at 63.7±0.8°C and 60.7±0.3°C in E. coli AW1.7 and GGG10, respectively. Fourier-transformed-infrared-spectroscopy did not indicate differences in protein denaturation between the strains during heating. In conclusion, heat resistance in E. coli AW1.7 correlates to ribosome stability at 60°C and is dependent on accumulation of cytoplasmic solutes.     

Characterization of Campylobacter jejuni RacRS reveals roles in the heat shock response, motility, and maintenance of cell length homogeneity

Campylobacter jejuni commensally colonizes the cecum of birds. The RacR (reduced ability to colonize) response regulator was previously shown to be important in avian colonization. To explore the means by which RacR and its cognate sensor kinase RacS may modulate C. jejuni physiology and colonization, ΔracR and ΔracS mutations were constructed in the invasive, virulent strain 81-176, and extensive phenotypic analyses were undertaken. Both the ΔracR and ΔracS mutants exhibited a ~100-fold defect in chick colonization despite no (ΔracS) or minimal (ΔracR) growth defects at 42 °C, the avian body temperature. Each mutant was defective for colony formation at 44°C and in the presence of 0.8% NaCl, both of which are stresses associated with the heat shock response. Promoter-reporter and real-time quantitative PCR (RT-qPCR) analyses revealed that RacR activates racRS and represses dnaJ. Although disregulation of several other heat shock genes was not observed at 38°C, the ΔracR and ΔracS mutants exhibited diminished upregulation of these genes upon a rapid temperature upshift. Furthermore, the ΔracR and ΔracS mutants displayed increased length heterogeneity during exponential growth, with a high proportion of filamented bacteria. Filamented bacteria had reduced swimming speed and were defective for invasion of Caco-2 epithelial cells. Soft-agar studies also revealed that the loss of racR or racS resulted in whole-population motility defects in viscous medium. These findings reveal new roles for RacRS in C. jejuni physiology, each of which is likely important during colonization of the avian host.     

Effects of flow and temperature on growth and photophysiology of scleractinian corals in Moorea, French Polynesia

To test for threshold effects in the response of coral physiology to increasing seawater flow, field and laboratory experiments were conducted in Moorea. First, the growth of juvenile massive Porites spp. and branching P. irregularis was compared among habitats differing in water motion. Growth of massive Porites spp. responded to flow in a pattern consistent with a threshold effect, whereas growth of P. irregularis increased linearly with flow. Second, a recirculating flume was used to test the effect of flow on photophysiology (ΔF/F(m)', effective photochemical efficiency) for massive Porites spp.; ΔF/F(m)' displayed a threshold response at 23 cm s(-1) and 28 °C, but not at 31 °C. Finally, intra-colony variation in the response of ΔF/F(m)' to flow and temperature was explored to evaluate the functional significance of colony shape in small corals. ΔF/F(m)' on the top and upstream surfaces of massive Porites spp. responded with a threshold effect of flow at 28 °C (but not 31 °C), but ΔF/F(m)' on downstream surfaces was unresponsive to flow. ΔF/F(m)' for P. irregularis was less responsive to flow than for massive Porites spp., suggesting that the photophysiological response of corals to varying flow speeds may differ between species and morphologies. Together, these results emphasize that flow can have diverse effects on the physiology of corals, with the outcome depending on flow speed, temperature, location on the colony, and perhaps morphology.     

Pseudouridine at position 55 in tRNA controls the contents of other modified nucleotides for low-temperature adaptation in the extreme-thermophilic eubacterium Thermus thermophilus

Pseudouridine at position 55 (Ψ55) in eubacterial tRNA is produced by TruB. To clarify the role of the Ψ55 modification, we constructed a truB gene disruptant (ΔtruB) strain of Thermus thermophilus which is an extreme-thermophilic eubacterium. Unexpectedly, the ΔtruB strain exhibited severe growth retardation at 50 °C. We assumed that these phenomena might be caused by lack of RNA chaperone activity of TruB, which was previously hypothetically proposed by others. To confirm this idea, we replaced the truB gene in the genome with mutant genes, which express TruB proteins with very weak or no enzymatic activity. However the growth retardation at 50 °C was not rescued by these mutant proteins. Nucleoside analysis revealed that Gm18, m(5)s(2)U54 and m(1)A58 in tRNA from the ΔtruB strain were abnormally increased. An in vitro assay using purified tRNA modification enzymes demonstrated that the Ψ55 modification has a negative effect on Gm18 formation by TrmH. These experimental results show that the Ψ55 modification is required for low-temperature adaptation to control other modified. (35)S-Met incorporation analysis showed that the protein synthesis activity of the ΔtruB strain was inferior to that of the wild-type strain and that the cold-shock proteins were absence in the ΔtruB cells at 50°C.     

Thermodynamics of insertion and self-association of a transmembrane helix: a lipophobic interaction by phosphatidylethanolamine

Thermodynamic parameters for the insertion and self-association of transmembrane helices are important for understanding the folding of helical membrane proteins. The lipid composition of bilayers would significantly affect these fundamental processes, although how is not well understood. Experimental systems using model transmembrane helices and lipid bilayers are useful for measuring and interpreting thermodynamic parameters (ΔG, ΔH, ΔS, and ΔC(p)) for the processes. In this study, the effect of the charge, phase, acyl chain unsaturation, and lateral pressure profile of bilayers on the membrane partitioning of the transmembrane helix (AALALAA)(3) was examined. Furthermore, the effect of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE) on the thermodynamics for insertion and self-association of the helix in host membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) was investigated in detail. Interbilayer transfer of the helix monomer from POPC to POPC/POPE (1/1) bilayers was unfavorable (ΔG = +4.5 ± 2.9 kJ mol(-1) at 35 °C) due to an increase in enthalpy (ΔH = +31.1 ± 2.1 kJ mol(-1)). On the other hand, antiparallel dimerization of the helices in POPC/POPE (1/1) bilayers was enhanced compared with that in POPC bilayers (ΔΔG = -4.9 ± 0.2 kJ mol(-1) at 35 °C) due to a decrease in enthalpy (ΔΔH = -33.2 ± 1.5 kJ mol(-1)). A greater thickness of POPC/POPE bilayers only partially explained the observed effects. The residual effects could be related to changes in other physical properties such as higher lateral pressure in the hydrocarbon core in the PE-containing membrane. The origin of the enthalpy-driven "lipophobic" force that modulates the insertion and association of transmembrane helices will be discussed.     

Production and characterization of two N-terminal truncated esterases from Thermus thermophilus HB27 in a mesophilic yeast: effect of N-terminus in thermal activity and stability

Two N-terminally truncated variants of the esterase E34Tt from Thermus thermophilus HB27 (YP_004875.1) were expressed in Kluyveromyces lactis. Production and biochemical properties of both recombinant proteins were investigated. The esterase activity was greatly increased compared to the wild-type strain. In particular, the extracellular production of the ΔN16 variant (KLEST-3S) was 50-fold higher than that obtained with T. thermophilus HB27. Response surface methodology was applied to describe the pH and temperature dependence of both activity and stability. When compared with the wild type esterase, the optimal temperature of reaction decreased 35 and 15 °C for ΔN16 and ΔN26, respectively. KLEST-3S showed a maximum of activity at pH 7.5 and 47.5 °C, and maximal stability at pH 8.1 and 65 °C. KLEST-5A (ΔN26) did not show an absolute maximum of activity. However, best results were obtained at 40 °C and pH 8.5. KLEST-5A showed also a lower stability. In the presence of a surfactant, both proteins showed lower stability at 85 °C (t(?)< 5 min) than the wild-type enzyme (t(?)=135 min). However, in the absence of detergent, the stability of KLEST-3S was higher (t(?)=230 min, at 85 °C) than that of the mutant KLEST-5A (12 min) or the wild type enzyme (19 min). Minor differences were observed in the substrate specificity. Our results suggest that the N-terminal segment is critical for maintaining the hyperthermophilic function and stability.     

StHsp14.0, a small heat shock protein of Sulfolobus tokodaii strain 7, protects denatured proteins from aggregation in the partially dissociated conformation

The small heat shock protein (sHsp), categorized into a class of molecular chaperones, binds and stabilizes denatured proteins for the purpose of preventing aggregation. The sHsps undergo transition between different oligomeric states to control their nature. We have been studying the function of sHsp of Sulfolobus tokodaii, StHsp14.0. StHsp14.0 exists as 24meric oligomer, and exhibits oligomer dissociation and molecular chaperone activity over 80°C. We constructed and characterized StHsp14.0 mutants with replacement of the C-terminal IKI to WKW, IKF, FKI and FKF. All mutant complexes dissociated into dimers at 50°C. Among them, StHsp14.0FKF is almost completely dissociated, probably to dimers. All mutants protected citrate synthase (CS) from thermal aggregation at 50°C. But, the activity of StHsp14.0FKF was the lowest. Then, we examined the complexes of StHsp14.0 mutants with denatured CS by SAXS. StHsp14.0WKW protects denatured CS by forming the globular complexes of 24 subunits and a substrate. StHsp14.0FKF also formed similar complex but the number of subunits in the complex is a little smaller. These results suggest that the dimer itself exhibits low chaperone activity, and a partially dissociated oligomer of StHsp14.0 protects a denatured protein from interacting with other molecules by surrounding it.     

Thermodynamic and structural properties of the acid molten globule state of horse cytochrome C

To understand the stabilization, folding, and functional mechanisms of proteins, it is very important to understand the structural and thermodynamic properties of the molten globule state. In this study, the global structure of the acid molten globule state, which we call MG1, of horse cytochrome c at low pH and high salt concentrations was evaluated by solution X-ray scattering (SXS), dynamic light scattering, and circular dichroism measurements. MG1 was globular and slightly (3%) larger than the native state, N. Calorimetric methods, such as differential scanning calorimetry and isothermal acid-titration calorimetry, were used to evaluate the thermodynamic parameters in the transitions of N to MG1 and MG1 to denatured state D of horse cytochrome c. The heat capacity change, ΔC(p), in the N-to-MG1 transition was determined to be 2.56 kJ K(-1) mol(-1), indicating the increase in the level of hydration in the MG1 state. Moreover, the intermediate state on the thermal N-to-D transition of horse cytochrome c at pH 4 under low-salt conditions showed the same structural and thermodynamic properties of the MG1 state in both SXS and calorimetric measurements. The Gibbs free energy changes (ΔG) for the N-to-MG1 and N-to-D transitions at 15 °C were 10.9 and 42.2 kJ mol(-1), respectively.     

Caenorhabditis elegans Delta12-desaturase FAT-2 is a bifunctional desaturase able to desaturate a diverse range of fatty acid substrates at the Delta12 and Delta15 positions

Caenorhabditis elegans FAT-2 has been characterized as fatty acid Δ12-desaturase able to desaturate C16 and C18 fatty acids. However, in this report we show that when expressed in yeast cells this enzyme can also catalyze Δ15 desaturation. This results in the production of both linoleic acid (ω6 C18:2Δ9,12) and linolenic acid (ω3 C18:3Δ9,12,15) from oleic acid (C18:1Δ9) substrate, and hexadecadienoic acid (ω4 C16:2Δ9,12) and hexadecatrienoic acid (ω1 C16:3Δ9,12,15) from palmitoleic acid (C16:1Δ9) substrate. In addition, this enzyme can also produce C14:2Δ9,12, C15:2Δ9,12, C17:2Δ9,12, and C18:4Δ6,9,12,15 when C14:1Δ9, C15:1Δ9, C17:1Δ9, and C18:3Δ6,9,12 substrates are available in yeast cells. Mass spectrometry analysis of 2,4-dimethyloxazoline modification of fatty acid methyl esters confirms the positions of all newly formed double bonds. These results indicate that when expressed in yeast the C. elegans Δ12-desaturase CeFAT-2 shows a characteristic of a bifunctional Δ12/Δ15-desaturase and has a great deal of elasticity with respect to fatty acid chain length in being able to accept fatty acids ranging from C14 to C18. Interestingly, despite possessing a bifunctional Δ12/Δ15 desaturation activity, phylogenetic analysis suggests that C. elegans Δ12-desaturase CeFAT-2 might have arisen independently from other reported dual Δ12/Δ15-desaturases from fungi and protozoa.