Biosynthesis of Isoprenoid Wax Ester in Marinobacter hydrocarbonoclasticus DSM 8798: Identification and Characterization of Isoprenoid Coenzyme A Synthetase and Wax Ester Synthases

Chemicals and materials.CoA trilithium salts were purchased from Roche (Indianapolis, IN). Tris (2-carboxyethyl) phosphine (TCEP) was purchased from EMB Biosciences (La Jolla, CA). Phytanic acid, palmitoyl-CoA (C_16:0), stearoyl-CoA (C_18:0), arachidoyl-CoA (C_20:0), C₁₈ linolenoyl-CoA (C_18:3), myristoyl-CoA (C_14:0), lauroyl (C_12:0) and 5,5′-dithio-bis(2-nitrobenzoic acid) (DNTB), inorganic pyrophosphatase, Triton X-100, triolein, and ATP were purchased from Sigma (St. Louis, MO). Tergitol NP-11 was obtained from Dow Chemical Co. (Midland, MI). Gum arabic, sodium taurocholate, and all solvents (high-pressure liquid chromatography [HPLC] grade) were purchased from Fisher Scientific (Pittsburgh, PA). HPLC-grade water was purchased from Mallinckrodt Chemicals (Phillipsburg, NJ). Restriction endonucleases, polynucleotide kinase, and T4 ligase were purchased from New England Biolabs (Boston, MA).

Strains and growth conditions. Marinobacter hydrocarbonoclasticus strain DSM 8798 was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ). Acinetobacter baylyi ADP1 was kindly provided to us by Nicholas Ornston at Yale University (New Haven, CT). Pseudomonas putida strain U was kindly given to us by José M. Luengo at the University of Léon (Léon, Spain). Cloning and heterologous gene expression was carried out in Escherichia coli strains DH5α and JM109. M. hydrocarbonoclasticus DSM 8798 was grown in Luria-Bertani (LB) medium with sterile, synthetic seawater (Ricca Chemical Company, Arlington, TX) instead of distilled water. E. coli, A. baylyi, and P. putida were grown in LB medium at 30°C unless otherwise specified.

Gene cloning.Genomic DNA was isolated from A. baylyi ADP1, M. hydrocarbonoclasticus DSM 8798, and P. putida U using standard phenol-chloroform DNA extraction techniques described in Sambrook et al. (18). Degenerate rRNA oligonucleotides (TPU1, 5′-AGAGTTTGATCMTGGCTCAG; RTU8, 5′-AAGGAGGTGATCCANCCRCA [6]) were used to amplify the 16S rRNA gene sequences from M. hydrocarbonoclasticus DSM 8798 (referred to as strain 8798). Gene-specific oligonucleotides for cloning of genes from strain 8798 were designed based on gene sequences identified in the rough-draft genome annotation of Marinobacter aquaeolei strain VT8 released by the DOE Joint Genome Institute (http://www.jgi.doe.gov ).

Cloning and DNA manipulations were carried out in E. coli DH5α using the standard molecular biology techniques described by Sambrook et al. (18). Genes encoding WS1, WS2, WS3, Acs1, Acs2, Acs3, and Acs4 were PCR amplified from strain 8798 genomic DNA by using gene-specific oligonucleotides that introduce XbaI and NotI restriction sites. The XbaI/NotI-digested inserts were ligated into plasmid pUCmod for constitutive expression from a modified lac promoter (19). Histidine tags were added to the isolated genes: putative acyl-CoA synthetase genes contain a C-terminal His₆ tag, and WS genes have an N-terminal His₆ tag. Cloned gene sequences were verified by sequencing. The stop codon in the WS4 sequence was verified by sequencing several clones and also the PCR amplification product.

Protein expression and purification.Cultures (100 ml) of E. coli JM109 transformed with pUCmod expressing putative His₆-tagged CoA synthetases or WSs were grown in LB media supplemented with 100 μg of ampicillin/ml at 30°C overnight in 500-ml unbaffled flasks. Cells were harvested by centrifugation and resuspended in 10 ml of 50 mM Tris-HCl buffer (pH 8) for CoA synthetase enzymes and in 125 mM sodium phosphate buffer (pH 7.4) for WS enzymes. The cells were lysed by sonication (Branson, Danbury, CT) on ice using a 30% duty cycle consisting of 10 s on and 30 s off for 10 cycles. Cell lysates were spun down at a centrifugal force of 13,763 × g in 50-ml Oakridge tubes in a Beckman J2-HS floor centrifuge equipped with a JA-17 rotor for 30 min at 4°C. The supernatant was applied to immobilized metal affinity chromatography using Talon resin (Clontech, Mountain View, CA) and washed with 10 mM imidazole in 50 mM Tris-HCl buffer (pH 8) or 125 mM sodium phosphate buffer (pH 7.4). The purified proteins were eluted with 300 mM imidazole in either 50 mM Tris-HCl buffer (pH 8) or 125 mM sodium phosphate buffer (pH 7.4). Elutants were desalted with (Amersham, Piscataway, NJ) PD-10 resin columns to remove excess imidazole. The purified proteins were concentrated to 1 ml using Vivaspin (Vivascience, Hannover, Germany) 10,000-Da columns. Protein concentrations were determined by using the bicinchoninic acid protein assay method with bovine serum albumin as a protein standard (Pierce Biotechnology, Inc., Rockford, IL).

CoA synthetase assay.In vitro reactions were performed as described previously (24) with some modifications. CoA synthetase assays were carried out in 250-μl reaction volumes containing 50 mM Tris-HCl buffer (pH 7.5), 0.1% Triton X-100, 10 mM MgCl₂, 5 mM TCEP, 0.1 U of inorganic pyrophosphatase, and as substrates 10 mM phytanic or fatty acids, 10 mM reduced coenzyme A (CoASH), 10 mM ATP, and 0.5 μg of purified Acs (either 1, 2, 3, or 4) protein. The reactions were incubated for 20 min at 37°C and stopped with 25 μl of 5% acetic acid, followed by HPLC analysis of the reaction products.

HPLC and LC/ESI-MS analysis of CoA synthetase reactions.CoA-synthetase assay samples (25 μl) were resolved on an Agilent 1100 HPLC (Agilent Technologies, Palo Alto, CA) equipped with a photodiode array detector set to 259 nm. Samples were separated on a reversed-phase Eclipse XDB-C8 column (Agilent Technologies) at a flow rate of 1 ml min⁻¹. Solvent A consisted of 20 mM ammonium acetate (pH 5.4) in HPLC-grade water, and solvent B contained acetonitrile and methanol (85:15 [vol/vol]). Acyl-CoA and phytanoyl-CoA products were eluted using the following conditions: solvent A-solvent B at 65:35 from 0 to 5 min, followed by a gradient from solvent A-solvent B at 65:35 to 100% solvent B in 30 min. Liquid chromatography-mass spectrometry (LC-MS) analyses of reaction products were done with an LCQ mass spectrophotometer equipped with an electrospray ionization source (ESI) (Thermo Finnigan). Mass fragmentation spectra were monitored in a mass range of m/z 400 to 1,500 with a negative ESI interface.

Preparation of phytanoyl-CoA.Commercially unavailable phytanoyl-CoA for WS assays was synthesized enzymatically with Acs2 under the CoA synthetase assay conditions described above with phytanic acid as the substrate. Enzymatically derived phytanoyl-CoA was purified by preparative HPLC: 100 μl in vitro reaction samples were separated under essentially the same conditions described above for the HPLC analysis of CoA products. Phytanoyl-CoA fractions were collected and dried under nitrogen gas. Phytanoyl-CoA was quantified by comparison to UV/visual (UV/Vis) spectra of lauroyl-CoA, assuming comparable extinction coefficients of the CoA chromophores in phytanoyl- and lauroyl-CoA.

Profiling of WS substrate ranges using a coupled enzyme assay.Stock solutions of various substrates were prepared in 50 mM Tris-HCl buffer (pH 8) containing 1% gum arabic, 12.5 μg bovine serum albumin ml⁻¹, 0.1% taurocholate, and either 100 mM fatty acid, isoprenoid acid, fatty alcohol, or isoprenoid alcohol. Stock solutions were sonicated to disperse the substrates.

Substrate profiles of the three WS were tested using coupled enzyme in vitro reactions in which the CoA synthetases Asc1 and Asc2 are added to synthesize the CoA-activated fatty acid phytanic acid substrates from corresponding acid precursors for the WS reactions. Assays were carried out in 500-μl reactions containing in 50 mM Tris-HCl buffer (pH 8.0), 12.5 μl of each acid, and alcohol substrate stock solution (final concentrations of each substrate of 250 μM), 10 mM MgCl₂, 10 mM CoASH, 10 mM ATP, 5 mM TCEP, 0.1 U of inorganic pyrophosphatase, 0.25 μg of Acs1 and Acs2 CoA synthetase, and 0.5 μg of WS to be tested. Assays were incubated at 37°C overnight before thin-layer chromatography (TLC) analysis of the reaction products.

TLC.In vitro WS assay samples were extracted with 500 μl of chloroform-methanol (1:1 [vol/vol]), and extracts were analyzed by TLC with Whatman normal phase silica gel 60 plates and developed using hexane-diethyl ether-acetic acid (90:10:1 [vol/vol/vol]). TLC plates were stained with either iodine vapor or anisaldehyde solution as described earlier (10). Palmitoyl palmitate and triolein were used as WE and TAG reference compounds, respectively.

DGAT assay.The DGAT activity of WS enzymes was measured using the coupled WS enzyme assay conditions described above with oleic acid as acyl donor and 1,2 dipalmitoyl-sn glycerol as the acyl acceptor (final concentration of each substrate of 250 μM) and 0.25 μg of Acs2 isoprenoid/acyl-CoA synthetase to generate CoA activated oleic acid.

GC-MS analysis of WE.GC electron impact MS analyses were performed with a Hewlett-Packard 6890 series gas chromatograph connected to an HP 5973 mass spectrometer. GC conditions consisted of a column (30 m by 0.25 mm [inner diameter] by 1.5 μm coated with 5% phenylmethyl silicone) with the injector temperature set to 250°C. The oven was set to a temperature gradient of 30°C min⁻¹ from 60 to 130°C, followed by slowing of the gradient from 130 to 300°C at 4°C min⁻¹ using helium as a carrier gas. The MS conditions used an electron energy of 70 eV and a source temperature set to 170°C. Mass spectra were scanned in a range of m/z 40 to 600 at 1-s intervals.

Kinetic WS assay.WS activity was determined by monitoring CoA release using Ellman's reagent [5,5′-dithio-bis(2-nitrobenzoic acid); DTNB] at 412 nm (ε = 13,600 M⁻¹ cm⁻¹) (5). Kinetic in vitro assays were performed in triplicate in 125 mM sodium phosphate buffer (pH 7.4) containing 0.1% Tergitol NP-11 detergent, 10 mM MgCl₂, 1 mM DNTB, 250 μM palmitoyl-CoA, 1 to 250 μM hexadecanol, and 0.5 μg of WS enzyme. Assay reactions were preincubated at 37°C for 5 min before the reactions were started by the addition of enzyme. Heat-denatured enzyme (99°C for 15 min) was used as a negative control.

Measurement of acyl-CoA and fatty/isoprenoid alcohol specificity.Acyl-CoA and fatty/isoprenoid alcohol specificity of WS2 was determined in the same manner as described for the kinetic WS assay with assumed saturating conditions containing both the fatty/isoprenoid alcohol and acyl-CoA substrates at a concentration of 1 mM. Acyl-CoA specificity was measured with hexadecanol as acyl acceptor, whereas palmitoyl-CoA was used as acyl donor to determine alcohol specificity. Isoprenoid WS activity of WS2 was determined with 250 μM HPLC-purified phytanoyl-CoA and 250 μM phytol as substrates.

Identification and cloning of putative isoprenoid WE biosynthetic genes. M. hydrocarbonoclasticus DSM 8798 (strain 8798) was shown previously to synthesize isoprenoid WE from phytol (17). Based on the recent characterization of a fatty acid WS (WS/DGAT) from Acinetobacter baylyi ADP1 that condenses a CoA activated fatty acid and a fatty alcohol to make fatty acid WE storage compounds, we reasoned that isoprenoid WE synthesis may follow a similar pathway with an isoprenoid specific acyl-CoA synthetase and WS as key enzymes (Fig. 1). Because no sequence information is available for strain 8798, we used a recent draft genome sequence (released by the DOE Joint Genome Institute [http://www.jgi.doe.gov ] in October 2005) of the alkane hydrocarbon metabolizing M. aquaeolei VT8 strain (8, 13) (referred to as strain VT8 below) for the identification and cloning of isoprenoid WE biosynthetic genes in strain 8798. Sequence analysis of the 16S rRNA gene of strain 8798 showed it to be 99.4% identical to that of strain VT8, suggesting a high degree of genomic conservation between the two strains.

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FIG. 1.

Proposed pathway for isoprenoid WE biosynthesis. Phytol is transported into M. hydrocarbonoclasticus by an unknown system and is reduced to phytenal by an alcohol dehydrogenase. Phytenal is further oxidized into phytenic acid by an aldehyde dehydrogenase. Concurrently, phytanic acid (saturated by an unknown reductase (indicated as “reductase?”, a saturated form of phytenic acid) is activated to phytanoyl-CoA by an isoprenoid/acyl-CoA synthetase. Phytanoyl-CoA and phytol are substrates for a WS to form the isoprenoid WE product. The isoprenoid/acyl-CoA synthetase (CoA synthetase) and isoprenoid WS are the focus of this research (boxed region).

A BLAST homology search of the draft VT8 genome sequence (released in October 2005) using the known WS/DGAT amino acid sequence from A. baylyi ADP1 identified four putative WS homologues (WS1, -2, -3, and -4) (Table 1 and Fig. 2). Two of the homologues, WS1 and WS2, were located in two putative alkane degradation gene clusters that share some sequence similarity with a known alkane degradation cluster described from P. oleovorans (26). However, while the present study was under review, the sequence of Marinobacter aquaeolei VT8 genome was reassembled, and a final draft of the genome sequence was released 28 December 2006. In the previous annotation, WS1 was associated with gene cluster 1, which is no longer the case in the new genome assembly. Now, WS1 is located approximately 250 kb upstream of this cluster (see Fig. S1 in the supplemental material, which maintains the gene organization in the first genome assembly but now also shows the new gene localizations). WS1 and alkane gene cluster 1 are each flanked by inverted transposase sequences (of which there are three 100% conserved copies in the genome), resulting in a contiguous assembly in the first draft. Similarly, we hypothesized that WS2 was also associated with an alkane degradation operon (see Fig. S1, gene cluster 2, in the supplemental material) since both were located near a physical sequence gap. This gap has now been closed, and WS2 is not associated with any alkane degradation gene cluster. If the current genome sequence is correct, none of the WS homologues are clustered any longer with any obvious alkane-utilizing metabolic operons.

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FIG. 2.

Multiple protein sequence alignment of Acinetobacter WS/DGAT (ADP1) with four putative WS cloned from M. hydrocarbonoclasticus strain 8798 (WS1 to -4). Identical and similar amino acids are marked black and gray, respectively. The WS4 gene contains a stop codon signified with an “X” at position 350 (denoted with an asterisk). The region outlined by a black box corresponds to the putative conserved acyltransferase catalytic domain, HHXXXDG (11).

We searched the draft genome sequence for putative acyl-CoA synthetases and identified four open reading frames (ORFs) annotated as medium-chain (Acs1) and long-chain (Asc2, -3, and -4) acyl-CoA synthetases (Table 1). Acs2, -3, and -4 are not clustered with any obvious gene functions. Acs1 is part of one of the putative alkane degradation gene cluster 1 (see Fig. S1 in the supplemental material), previously annotated to also include WS1. Gene cluster 2 contained in the first draft genome sequence a partial acyl-CoA synthetase ORF flanking the physical gap. As stated above, this gap has been closed in the new genome assembly, and this ORF is now annotated as a medium-chain CoA synthetase. Oligonucleotides were designed from the VT8 sequences of the putative WS and Acs genes found in the genome annotation and used for PCR amplification and cloning of the homologues from strain 8798. PCR products were obtained for Acs1 to -4 and WS1 to -4 ORFs and cloned into pUCmod for sequencing. The putative acyl-CoA synthase of gene cluster 2, for which only a partial sequence was available until very recently (see above), could not be amplified using a C-terminal oligonucleotide derived from the Acs1 sequence. All cloned WS and Acs genes from strain 8798 share a >97% peptide sequence identity with those identified in the genome sequence of strain VT8 (Table 1). The peptide sequence identities of the cloned WS homologues to the experimentally characterized WS/DGAT from A. baylyi range from 27 to 45%, with WS4 being the least similar and WS1 having the highest identity (Table 1). However, the cloned WS4 from strain 8798 is a pseudogene with a stop codon that truncates its ORF, whereas the corresponding ORF of WS4 from strain VT8 appears to be intact based on the released draft genome sequence. Acs1 to Acs4 show greater than 50% peptide sequence identity to experimentally characterized medium- and long-chain acyl-CoA synthetases from different Pseudomonas strains (Table 1).

Identification of Marinobacter isoprenoid CoA synthetase.To determine the substrate specificities and to test whether any of the cloned putative acyl-CoA synthetases can catalyze CoA-activation of isoprenoid acids, purified recombinant enzymes (Acs1, -2, -3, and -4) were assayed with various saturated fatty acid substrates containing different acyl chain lengths (C₁₀, C₁₂, C₁₄, C₁₆, C₁₈, and C₂₀) and with the isoprenoid phytanic acid. HPLC analysis of the reaction products confirmed Acs1 to be a medium-chain acyl-CoA synthetase that accepts fatty acids with chain lengths ranging from C₁₀ to C₁₆, while Acs2, -3, and -4 were found to be long-chain acyl-CoA synthetases that act on fatty acids with chain lengths ranging from C₁₂ to C₂₀ (data not shown). The long-chain acyl-CoA synthetases (Acs2, -3, and -4) showed the most activity when palmitic acid (C₁₆) was the substrate. Only Acs2 converted phytanic acid into phytanoyl-CoA, a finding confirmed by LC-MS (Fig. 3). This enzyme, now referred to as isoprenoid/acyl-CoA synthetase, shows 63% peptide sequence identity with a previously described acyl-CoA synthetase (FadD) found in P. putida that accepts aromatic alkanoic acids (13). The broad substrate range of FadD prompted us to investigate whether this enzyme would also accept phytanic acid. We cloned FadD and assayed the purified recombinant protein with phytanic acid. However, unlike the Marinobacter enzyme, FadD does not synthesize phytanoyl-CoA (data not shown).

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FIG. 3.

HPLC-MS analysis of Asc2 in vitro reaction with phytanic acid and CoA as substrates. HPLC chromatogram of in vitro reaction showing the phytanoyl-CoA product with a retention time of 22.5 min and the corresponding mass spectrum of the product peak (inset). The observed parent ion (1,062 m/z) is consistent with that of the calculated mass of phytanoyl-CoA. Ions of 664, 708, and 752 m/z correspond to Triton X-100 detergent present in the reaction mixture.

Substrate profiles of Marinobacter WSs.The activities of the three cloned putative WS with various CoA-activated fatty acids, phytanic acid, and primary alcohols, including the isoprenoid alcohols farnesol and phytol, were investigated by using a coupled enzyme assay. A total of 54 in vitro reactions containing fatty acids and alcohols with various degrees of saturation and carbon chain lengths were arrayed for each WS enzyme. Purified WS proteins and Marinobacter CoA synthetases were incubated with CoA and different combinations of acid and alcohol substrates, and product formation was analyzed by TLC. CoA activation of medium-chain fatty acids (C₁₀ to C₁₄) in these assays was conducted with Acs1, while long-chain fatty acids (C₁₆ to C₂₀) and phytanic acid were esterified with CoA by Acs2. The identification of Acs2 as an isoprenoid/acyl-CoA synthetase made it possible to synthesize commercially unavailable phytanoyl-CoA from available phytanic acid as a substrate for testing with WS1, -2, and -3.

Table 2 summarizes WE product formation detected on TLC plates for the tested putative WSs from strain 8798. The TLC substrate profiles show that WS1 and WS2 catalyzed ester bond formation between various activated fatty acids and fatty alcohols or isoprenoid alcohols. Figure 4 shows representative TLC results for reactions with palmitic acid and hexadecanol and with phytanic acid and phytol. WS2 appears to have a broader substrate range and a higher preference for longer-chain fatty alcohols than WS1. WE products derived from short chain acyl-CoA (C₁₀ and C₁₂) substrates were only detected using the overnight assay conditions. No WE formation was detected using WS3 with any of the substrate combinations tested.

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FIG. 4.

TLC plate with iodine-stained WE products. TLC analysis of coupled in vitro reactions containing isoprenoid CoA synthetase (Asc2) and WS (WS1, -2, or -3) and either palmitic acid-hexadecanol or phytanic acid-phytol as substrates. A wax standard consisting of palmitoyl palmitate (marked “wax”) shows the position of the expected acyl and isoprenoid WE products (marked with an arrow). Products were observed only with WS1 and WS2; no products were observed with WS3.

WS1 and WS2 also esterified activated fatty acids with isoprenoid alcohols (phytol, farnesol), thereby producing hybrid acyl-isoprenoid WEs (see Fig. S2 in the supplemental material). However, fatty alcohols were not condensed to phytanoyl-CoA by either enzyme. Synthesis of hybrid isoprenoid WE was therefore only possible between an activated fatty acid and an isoprenoid alcohol. Notably, both enzymes produced isoprenoid WEs from phytanoyl-CoA and the isoprenoid alcohols phytol and farnesol, although WS2 was considerably more active with these substrates.

The structure of the synthesized isoprenoid WE was confirmed by GC-MS. Figure 5 shows the results of the GC-MS analysis of chloroform extracts of a reaction with WS2 and phytanic acid and phytol as substrates. A product peak with a retention time of 41 min and a mass of 590 m/z was detected that was not present in a control using heat-denatured WS2. Its mass and fragmentation pattern match those of the isoprenoid WE previously isolated from strain 8798 by Rontani et al. (17). Together, these results suggest that WS1 and WS2, along with Acs1, are involved in the synthesis of isoprenoid WE storage compounds in M. hydrocarbonoclasticus DSM 8798. Because the two Marinobacter WSs displayed novel WS activity, the previously described Acinetobacter WS/DGAT was also tested but did not show isoprenoid WE formation using phytanic acid and phytol as substrates.

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FIG. 5.

GC-MS analysis of isoprenoid WE product synthesized in a coupled enzyme reaction with isoprenoid CoA-synthetase Asc2 and WS2 containing phytanic acid and phytol as substrates. (A) Total ion chromatogram showing isoprenoid WE product peak at a retention time of 41.9 min (arrow). (B) Electron impact mass spectrum of isoprenoid WE product peak. The masses of the parent ion at 590 m/z and fragment ions (365, 311, and 278 m/z) match those reported for the phytanoyl-phytol ester (17).

WS/DGAT activity of Marinobacter WSs.It has been reported that Acinetobacter WS/DGAT has DGAT activity (11). To test whether any of the three Marinobacter WSs can catalyze this reaction, WS1, WS2, and WS3 were tested with oleoyl-CoA as the acyl donor and dipalmitoyl-glycerol as the acyl acceptor, and the products were resolved on TLC plates. TAG products were only detected for WS1, whereas WS2 did not show DGAT activity (see Fig. S3 in the supplemental material).

Kinetic measurement of WS activities.A spectrophotometric assay was developed to determine the kinetic properties of WS1 and WS2. The concentration of sulfhydryl groups of CoA released during the condensation reaction between fatty/isoprenoid CoA activated acids and alcohols was determined by using Ellman's reagent (DTNB) (5). The specific activities of WS1, WS2, and Acinetobacter WS/DGAT were measured with palmitoyl-CoA and hexadecanol, palmitoyl-CoA and phytol, and phytanoyl-CoA and phytol. Phytanoyl-CoA for these assays was enzymatically synthesized from phytanic acid and CoA using the above-characterized isoprenoid/acyl-CoA synthetase Acs2. Approximately 5 mM phytanoyl-CoA was purified by preparative HPLC, which was used to determine the specific activity of the most active Marinobacter enzyme WS2 (Table 3). Because the Acinetobacter WS/DGAT did not show activity with phytanic acid and phytol in the coupled enzyme assay, its specific activity with phytanoyl-CoA and phytol was not measured.

As shown in Table 3, WS2 was more active than either of the other two enzymes tested. The specific activity of WS2 measured with palmitoyl-CoA and hexadecanol as substrates was 61 mmol mg⁻¹ min⁻¹ versus 1.3 mmol mg⁻¹ min⁻¹ for WS1 and 0.38 mmol mg⁻¹ min⁻¹ for Acinetobacter WS/DGAT. WS2 was ∼20-fold more active than WS1 or Acinetobacter WS/DGAT in creating the hybrid WE using palmitoyl-CoA and the isoprenoid alcohol phytol as substrates. WS2 activity with phytanoyl-CoA and phytol was determined to be 0.397 mmol mg⁻¹ min⁻¹.

The kinetic constants of WS2 with palmitoyl-CoA and hexadecanol as substrates were determined under saturating palmitoyl-CoA conditions (at 250 μM) and various concentrations of hexadecanol. WS2 activity followed typical Michaelis-Menten kinetics with a K_m of 44 μM, a V _max of 10 mmol mg⁻¹ min⁻¹, and a k _cat of 4,794 s⁻¹ (Fig. 6A and B).

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FIG. 6.

Kinetic measurement of WS2 activity using a spectrophotometric assay. (A and B) Plot of reaction velocity versus hexadecanol concentration (0, 10, 25, 50, 150, and 250 μM) with palmitoyl-CoA (250 μM) (A) and corresponding double reciprocal plot of WS2 activity (B). (C and D) Comparison of WS2 specific activities for various chain lengths of CoA-activated fatty acids (C₁₂ to C₂₀) and hexadecanol (C) and various fatty/isoprenoid alcohols (C₁₀ to C₁₈; F, farnesol, P, phytol) and palmitoyl-CoA (D). Values are averages of three experiments; error bars correspond to one standard deviation.

Acyl-CoA and fatty/isoprenoid alcohol specificity of WS2.Acyl-CoA and fatty/isoprenoid alcohol specificity of WS2 was determined by using the developed spectrophotometric assay. Acyl-CoA specificity of this enzyme was investigated using acyl-CoAs with various acyl chain lengths (C₁₂ to C₂₀) and hexadecanol as substrates (Fig. 6C). Long-chain fatty acyl-CoA derivatives arachidoyl-CoA (C₂₀) and stearoyl-CoA (C_18:0) were readily accepted as substrates by WS2, as was the polyunsaturated acyl-CoA linolenoyl-CoA (C_18:3). Also, WS2 showed a clear preference for palmitoyl-CoA (C₁₆), whereas the shorter acyl-chain substrate myristoyl-CoA (C₁₄) was poorly converted and lauroyl-CoA (C₁₂) was not accepted at all by the enzyme.

The relative substrate activity of WS2 was also tested against various fatty/isoprenoid alcohols and palmitoyl-CoA as substrates (Fig. 6D). Compared to WS2's preference for acyl-CoA substrates with medium and long chains, the enzyme displayed a broad activity with alcohols of various chain lengths. Decanol and dodecanol were more readily taken up for WE synthesis than the equivalent-chain-length acyl-CoA carbon chain.