Studies into factors contributing to substrate specificity of membrane-bound 3-ketoacyl-CoA synthases Brenda J. Blacklock and Jan G. Jaworski Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio, USA We are interested in constructing a model for the substrate- binding site of fatty acid elongase-1 3-ketoacyl CoA synthase (FAE1 KCS), the enzyme responsible for production of very long chain fatty acids of plant seed oils. Arabidopsis thaliana and Brassica napus FAE1 KCS enzymes are highly homo- logous but the seed oil content of these plants suggests that their substrate specificities differ with respect to acyl chain length. We used in vivo and in vitro assays of Saccharomyces cerevisiae-expressed FAE1 KCSs to demonstrate that the B. napus FAE1 KCS enzyme favors longer chain acyl sub- strates than the A. thaliana enzyme. Domains/residues responsible for substrate specificity were investigated by determining catalytic activity and substrate specificity of chimeric enzymes of A. thaliana and B. napus FAE1 KCS. The N-terminal region, excluding the transmembrane domain, was shown to be involved in substrate specificity. One chimeric enzyme that included A. thaliana sequence from the N terminus to residue 114 and B. napus sequence from residue 115 to the C terminus had substrate specificity similar to that of A. thaliana FAE1 KCS. However, a K92R substitution in thischimeric enzyme changed the specificityto that of the B. napus enzyme without loss of catalytic activity. Thus, this study was successful in identifying a domain involved in determining substrate specificity in FAE1 KCS andinengineeringanenzymewithnovelactivity. Keywords: Arabidopsis thaliana; Brassica napus; fatty acid elongation; 3-ketoacyl-CoA synthase. The very long chain fatty acids (VLCFA) found in seed oils are derived from the elongation of products of de novo fatty acid biosynthesis [1]. The initial reaction of elonga- tion, i.e. the iterative condensation of acyl units with malonyl-CoA, is catalyzed in the seed by the membrane- bound fatty acid elongase-1 3-ketoacyl-CoA synthase (FAE1 KCS) [2]. Subsequent reduction and dehydration reactions are carried out by distinct and separate enzymes that are just beginning to be characterized [1,3,4]. FAE1 KCS was first identified in Arabidopsis thaliana [5] and homologues have been found in oleaginous species such as Brassica napus, B. juncea, and Simmondsia chinen- sis [6–10]. The functional similarity among these enzymes is demonstrated by the ability of the jojoba FAE1 KCS to complement the canola fatty acid elongation mutation even though jojoba produces wax rather than triacylgly- cerol, as found in other seed oils [6]. Examination of the VLCFA content of the seed oils of A. thaliana and B. napus reveals differences in the levels of eicosenoic (20:1) and erucic (22:1) fatty acids. In A. thaliana seed oil, 20% of the total fatty acids are VLCFA of which 18% of the total fatty acids are in the form of 20:1 and 2% in the form of 22:1 [11]. In B. napus seed oil, 62% of the total fatty acids are monounsaturated VLCFA, 10% as 20:1 and 52% as 22:1 [12]. As FAE1 KCS is responsible for VLCFA production in oilseeds [2,5,6], this diversity in VLCFA content suggests that A. thaliana and B. napus FAE1 KCS enzymes have distinct substrate specificities with the B. napus enzyme favoring longer chain length substrates than the A. thaliana enzyme. The high sequence identity (86%) between these two enzymes further suggests that the determinants responsible for fatty acid substrate specificity in FAE1 KCS are few and potentially identifi- able. The significant amino acid sequence homology of FAE1 KCSs with soluble condensing enzymes, such as chalcone synthase and 3-ketoacyl-acyl carrier protein synthases (KASs) is consistent with a role for FAE1 KCSs as fatty acid condensing enzymes [5,6,13]. Our understand- ing of the structure/function relationships of soluble condensing enzymes has been greatly advanced with the recent crystal structures of KAS I, -II, and -III and chalcone synthase [14–19]. However, only limited information is available about the structure of the membrane-bound KCSs. Secondary structural analysis of the family of FAE1 KCS enzymes reveals two putative transmembrane domains at the N termini of the proteins. Recent work in our laboratory has confirmed that the amino terminus of Arabidopsis FAE1 KCS is involved in anchoring the enzyme to the membrane [20]. The difficulty inherent in crystallizing membrane-bound enzymes required us to take a different approach to probing the structure/function relationships of FAE1 KCS. Here, we report utilization of a domain-swapping approach to investigate the structural domains and residues responsible for substrate specificity in FAE1 KCS. Correspondence to: B. J. Blacklock, Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA. Fax: +1 513 529 5715, Tel.: +1 513 529 1641, E-mail: blacklb@muohio.edu Abbreviations: VLCFA, very long chain fatty acid; FAE1 KCS, fatty acid elongase-1 3-ketoacyl CoA synthase; KAS, 3-ketoacyl-acyl carrier protein synthase; cm-ura, complete minimal dropout media lacking uracil; FAME, fatty acid methyl ester. Note: The SWISS-PROT accession numbers for the FAE1 KCS are: Arabidopsis thaliana, Q38860; Brassica napus, O23738. (Received 31 May 2002, revised 2 August 2002, accepted 12 August 2002) Eur. J. Biochem. 269, 4789–4798 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03176.x The redesign of a number of plant lipid metabolic enzymes by swapping domains between related yet func- tionally divergent enzymes has proven useful in obtaining catalysts with novel substrate specificity. An understanding of the regions of enzymes that contribute to substrate specificity and catalytic activity has also been gleaned from these studies [21–25]. By replacing only five amino acid residues, Cahoon and coworkers engineered a soluble fatty acid desaturase with D 6 -16:0 substrate specificity to one that functions principally as a D 9 -18:0 desaturase [21]. Similarly, specificity for a 16:0 substrate was imparted to a D 9 -18:0 desaturase by replacement of a single residue [22]. An analogous approach was taken to deciphering the architecture of the substrate-binding site of membrane- bound desaturases and related enzymes [24,26]. Libisch et al. constructed chimeras of Borago officinalis D 6 -fatty acid and D 8 -sphingolipid desaturases and analyzed effects on substrate specificity [24]. These studies were unsuccessful in identifying a discrete domain that can differentiate between a phospholipid-conjugated substrate and a cera- mide-conjugated substrate. However, the authors were successful in modifying the substrate specificity of the D 6 -fatty acid desaturase to a preference for shorter chain fatty acids [24]. A similar study demonstrated a switch in the oxidative reactions catalyzed by membrane-bound oleate desaturases and hydroxylases upon site-directed mutagenesis [26]. When the amino acid sequences of these highly homologous enzymes were compared, seven residues were identified that were conserved in the desaturases but divergent in the hydroxylases. The mutation of these residues in the desaturases to the corresponding hydroxylase residues resulted in the conversion of desaturase activity to hydroxy- lase activity. The reciprocal experiment allowed the conver- sion of a hydroxylase to a desaturase [26]. The objective of our work was to examine the substrate specificity of A. thaliana and B. napus FAE1 KCSs and to study the determinants of fatty acyl chain length specificity in 3-ketoacyl CoA condensing enzymes. We were able to map residues and regions of primary structure involved in substrate specificity in KCS enzymes. These studies repre- sent the first steps toward a characterization of the substrate- binding site of the membrane-bound KCS enzymes. EXPERIMENTAL PROCEDURES Construction of yeast expression vectors and cell lines A. thaliana (var. WS) FAE1 and B. napus (var. Golden) FAE1.1 genes were amplified from plasmids containing the cDNAs with Vent DNA polymerase (New England Biolabs) using primers (Table 1) containing restriction sites convenient for subcloning into the pYES2 expression vector (Invitrogen). PCR products and DNA fragments were purified by agarose gel electrophoresis followed by Gene Clean (Bio101, Vista, CA). A. thaliana, B. napus and At114K92R genes were subcloned into a pYES2 vector engineered to encode a (His) 6 GlySer fusion protein through BamHI/EcoRI restriction enzyme sites. Purified, digested insert and pYES2 vector were ligated with T4 DNA ligase (Life Technologies Gibco BRL) and transformed into competent XL-1 Blue Escherichia coli (Stratagene) by standard techniques [27]. Insert-containing plasmids were sequenced by in-house automated DNA sequencing to ensure they were mutation-free. Plasmids were transformed into competent Saccharomyces cerevisiae (strain InvSc1, Invitrogen) by the lithium acetate method of Geitz et al. [28]. Domain swapping and site-directed mutagenesis by overlap extension Chimeric genes were constructed either by restriction enzyme digestion when convenient sites for domain swap- ping were present, or by thermostable DNA polymerase- mediated overlap extension and PCR essentially as described [29,30]. Briefly, gene fragments were amplified from plasmids containing A. thaliana or B. napus FAE1 using 5¢ or 3¢ specific primers with restriction sites and overlapping primers either straddling the desired splice site of the chimeric gene or containing the desired site-directed mutation (Table 1). Purified fragments were mixed and the full-length chimeric gene was constructed by extension of the overlapping fragments and PCR amplification with extreme 5¢ and 3¢ primers in the same reaction. Amplifica- tion products were subcloned into pYES2 and sequenced as described above to confirm the correct sequence. Expression of FAE1 KCSs in yeast and preparation of microsomes Transformed yeast was grown overnight with shaking in rich media at 30 °C and was used to inoculate complete minimal dropout media lacking uracil (cm-ura) [31] supple- mented with 2% galactose to give an initial D 600 of 0.01– 0.04. The cm-ura + gal cultures were grown to 1.5–2 D 600 units and harvested. Microsomes were prepared as des- cribed previously [32] using ice-cold isolation buffer (80 m M Hepes/KOH pH 7.2, 5 m M EGTA, 5 m M EDTA, 10 m M KCl, 320 m M sucrose, 2 m M dithiothreitol). Protein con- centrations were determined after the method of Bradford [33] using BSA as a standard and adjusted to 2.5 lgÆlL )1 by the addition of glycerol to 15% and isolation buffer. Microsomes were frozen on dry ice, stored at )80 °C until use and, once thawed, were not refrozen. GC/MS analysis of yeast lipids Yeast transformed with pYES2 or pYES2 with FAE1 insert were grown in cm-ura plus 2% raffinose and were induced by 2% galactose as described above. Cells were harvested by centrifugation and washed with dH 2 O; methyl esters of cellular lipids were prepared by incubation of the cell pellet in 2% H 2 SO 4 in methanol at 80 °C for 1–2 h. Fatty acid methyl esters (FAMEs) were extracted into hexanes, concentrated by evaporation under an N 2 stream and dissolved in a small volume of hexanes. FAMEs were separated by GC (Thermoquest Trace GC) on an RTX- 5MS 0.25 lm column (Restek Corp. Bellefonte, PA) and identifiedbyMSonaninlineFinniganPolarismass spectrometer. Assay of elongation activity of FAE1 KCSs Fatty acid elongase activity was measured essentially as described previously [34]. The elongation reaction consisted 4790 B. J. Blacklock and J. G. Jaworski (Eur. J. Biochem. 269) Ó FEBS 2002 of 20 m M Hepes/KOH pH 7.2, 20 m M MgCl 2 , 500 l M NADPH, 10 l M CoASH, 100 l M malonylCoA, 15 l M [1- 14 C]18:1CoA, and 6 lg protein of prepared microsomes in a final volume of 25 lL. [1- 14 C]18:1CoA was synthesized from [1- 14 C]18:1 free fatty acid (50–55 lCiÆlmol )1 ;ICN, Costa Mesa CA) as described by Taylor et al.[35].Methyl esters of the radiolabeled acyl CoA elongation products were prepared as described above, separated by reversed phase silica gel TLC (Alltech, Deerfield, IL) with acetonit- rile/methanol/H 2 O (65 : 35 : 0.5, v/v) developing solvent [36] and analyzed by phosphorimaging; each band was then quantitated by ImageQuant software (Molecular Dynam- ics, Inc.). Solubilization and isolation of (His) 6 -tagged fusion proteins (His) 6 fusion proteins were solubilized and isolated as described [20]. Microsomes (2 lgproteinÆlL )1 ) expressing (His) 6 -tagged fusion proteins were solubilized by incubation on ice for 2 h in solubilization buffer (320 m M NaCl, 0.5% Triton X-100) and insoluble material was pelleted by ultracentrifugation at 4 °C for 1 h at 235 000 g. Superna- tants were combined with binding buffer (25 m M sodium phosphate pH 8.0, 0.5% Triton X-100, 150 m M NaCl, 10% glycerol) at a ratio of 0.43 : 1 (supernatant : binding buffer) and applied to a 300-lL column of Ni +2 -pentadentate chelator (Ni-PDC, Affiland, Ans-Liege, Belgium). The column was washed with 1 mL binding buffer, 1 mL wash buffer (25 m M sodium phosphate pH 8.0, 0.5% Triton X-100, 500 m M NaCl, 10% glycerol, 20 m M imidazole) and 1 mL binding buffer. Proteins were eluted from the column with 300 lL elution buffer (25 m M sodium phosphate pH 8.0, 0.5% Triton X-100, 150 m M NaCl, 10% glycerol, 300 m M imidazole), dithiothreitol was added to 2 m M , and samples were frozen on dry ice and stored at )80 °C. Condensation assay of (His) 6 -FAE1 KCS Condensation activity of (His) 6 -FAE1 KCSs was assayed as previously described [37]. Briefly, 2.5 lL purified sample was incubated with condensation reaction mix (10 m M sodium phosphate, pH 7.2, 0.05% Triton X-100, 15 l M acyl CoA, 20 l M [3- 14 C]malonyl CoA) where the acyl CoA Table 1. Primers used in subcloning and overlap PCR. Primer Name Sequence A. thal.5¢ BamHI 5¢-GGGGATCCATGACGTCCGTTAACGTTA3¢ A. thal.3¢ EcoRI 5¢-CCCGAATTCTTAGGACCGACCGTTTTGGACATGAGTCTT-3¢ B. nap.5¢ BamHI 5¢-GGGGATCCATGACGTCCATTAACGTAAAGCTCC-3¢ B. nap.3¢ EcoRI 5¢-CCGAATTCTTAGGACCGACCGTTTTGG-3¢ At173sense 5¢-GCGCTCGAAAATCTATTCAAGAACACC-3¢ At173anti 5¢-GTTCTTGAATAGATTTTCGAGCGCACCGATGATAAC-3¢ Bn173sense 5¢-GCCCTAGAAAATCTATTCAAGAACACC-3¢ Bn173anti 5¢-GTTCTTGAATAGATTTTCTAGCGCACCATT-3¢ At399sense 5¢-GCCGGAGGCAGAGCCGTGATCGAT-3¢ At399anti 5¢-ATCGATCACGGCTCTGCCTCCGGC-3¢ Bn399sense 5¢-GCCGGAGGCAGAGCCGTGATCGAT-3¢ Bn399anti 5¢-ATCGATCACGGCTCTGCCTCCGGC-3¢ At74sense 5¢-CCCAAACCGGTTTACCTCGTTGA-3¢ At74anti 5¢-TCAACGAGGTAAACCGGATTGGG-3¢ At114sense 5¢-CGGAACGGCACGTGTGATGATTCGTCCT-3¢ At114anti 5¢-GGACGGATCATCACACGCGACGTTCCG-3¢ At114D81Eanti 5¢-GTAACACG GTACTCAACGAGATAAAC-3¢ At114D81Esense 5¢-GTTTATCTCGTTGAGTACTCGTGTTAC3¢ At114P89Tanti 5¢-AACTTTGAGATGCGTTGGCGGAAGGTA-3¢ At114P89Tsense 5¢-TACCTTCCGCCAACGCATCTCAAAGTT-3ı ´ At114L91Canti 5¢-GACACTAACTTTACAATGCGTGGCGG3¢ At114L91Csense 5¢-CCGCCACCGCATTGTAAAGTTAG GTC-3¢ At114K92Ranti 5¢-AGAGACACTAACTCTGAGATGCGGTGG-3¢ At114K92Rsense 5¢-CCACCGCATCTCAGAGTTAGTGTCTCT-3¢ At114V93Santi 5¢-TTTAGAGACACTTGATTTGAGATGCGG3¢ At114V93sense 5¢-CCGCATCTCAAATCAAGT GTCTCTAAA-3¢ At114V95Ianti 5¢-CATGACTTTAGAGATACTAACTTTGAG-3¢ At114V95Isense 5¢-CTCAAAGTTAGTATCTCTAAAGTCATG-3¢ At114I105Vanti 5¢-ATCAGCTTTTCTTACTTGGAGAAAAT-3¢ At114I105Vsense 5¢-ATTTTCTACCAAGTAAGAAAAGCTGAT-3¢ At114T110Panti 5¢-GTTCCGTGAAGAAGGATCAGCTTTTCT-3ı ´ At114T110Psense 5¢-AGAAAAGCTGACCTTCTTCACGGAAC-3¢ At114S112-anti 5¢-CGTGCCGTTCCGAGAAGTATCAGC-3¢ At114S112-sense 5¢-GCTGATACTTCTCGGAACGGCACG-3¢ AtK92R sense 5¢-CCACCGCATCTCAGAGTTAGTGTCTCT-3¢ AtK92R anti 5¢-AGAGACACTAACTCTGAGATGCGGTGG-3¢ Ó FEBS 2002 Substrate specificity of FAE1 KCSs (Eur. J. Biochem. 269) 4791 was either 18:1CoA or 20:1CoA for 10 min at 30 °C. Radiolabeled malonyl CoA was prepared as described previously [38]. 3-ketoacyl CoA reaction products were reduced to the diols with NaBH 4 , extracted into petroleum ether, concentrated by evaporation under an N 2 stream, and quantified by liquid scintillation analysis. RESULTS This report describes studies of the fatty acid chain length substrate specificity of FAE1 KCS. We tested the hypothe- sis that A. thaliana and B. napus FAE1 KCS enzymes have divergent substrate specificity and that determinants of that specificity can be identified by domain swapping experi- ments. We have developed a facile biochemical method to characterize fatty acid elongase activities using an S. cere- visiae expression system. Endogenous yeast fatty acids serve as elongation substrates for in vivo analysis [2] and exogenously supplied radiolabeled 18:1CoA is readily taken up by microsomal fractions for in vitro assays [39]. As a first step, we established that the A. thaliana and B. napus FAE1 KCSs were active in the S. cerevisiae expression system and indeed had distinct substrate speci- ficities. Elongation of yeast endogenous fatty acids by FAE1 KCSs was examined by expressing plant FAE1 KCSs in yeast and then analyzing the fatty acid content of cellular lipids. Table 2 shows the C20 to C26 fatty acid content of cellular lipids when yeast transformed with pYES2 with A. thaliana or B. napus FAE1 inserts or the empty vector was grown under galactose induction conditions. The VLCFA content of yeast carrying the empty vector was principally limited to 26:0 with little or no C20, C22 or C24 fatty acids present. In contrast, expression of either A. thaliana or B. napus FAE1 KCS resulted in the production of significant levels of both saturated and unsaturated C20 to C26 VLCFA. A. thaliana FAE1 KCS expression caused an increase in VLCFA methyl esters to 32% of the total FAMEs: 19.3% C20, 5.8% C22, 2.6% C24 and 4.3% C26. B. napus FAE1 KCS expression, on the other hand, produced 11% of the total FAMEs as VLCFA: 3.5% C20, 2.1% C22, 0.8% C24 and 4.1% C26. These data demonstrate that plant FAE1 KCS enzymes were expressed and have activity in this yeast system. Further, these results show that in yeast, A. thaliana FAE1 KCS produces more C20, C22 and C24 fatty acids than B. napus FAE1 KCS and that the majority of the VLCFA products are C20. B. napus FAE1 KCS appeared to have more activity toward C20 than A. thaliana FAE1 KCS as indicated by near equal levels of C20 and C22 products when B. napus FAE1 KCS was expressed. While these results are, in general, consistent with the apparent substrate specificity of the A. thaliana and B. napus FAE1 KCS enzymes in seeds, the B. napus enzyme appeared to be less active than the A. thaliana FAE1 KCS in yeast. The substantial levels of saturated and unsaturated fatty acid methyl esters demonstrated that FAE1 KCSs have activity toward both monounsaturated and saturated fatty acids. The FAE1 KCS-derived saturated fatty acid prod- ucts appear to feed into the yeast saturated VLCFA pathway. VLCFA up to C26 as 24:0 and 26:0 were produced at elevated levels when either A. thaliana or B. napus FAE1 KCS were expressed compared to the empty vector control. While in vivo assays indicated that the plant FAE1 KCSs were able to couple to the yeast fatty acid elongation complex, an assay of the actual catalytic activity of the FAE1 KCSs was required to characterize the enzymes accurately. Microsomes were prepared from galactose- induced yeast transformed with empty vector (pYES2) or pYES2 with A. thaliana or B. napus FAE1 insert. Elonga- tion of [1- 14 C]18:1CoA to [3- 14 C]20:1CoA and [5- 14 C]22:1CoA by the microsomes was measured by incubation with malonyl CoA and required cofactors, followed by transacylation of the CoA conjugates to methyl esters [34,39]. Elongation activity of both A. thaliana and B. napus FAE1 KCS reached a plateau by 20 min but the catalytic activity of B. napus FAE1 KCS was lower than that of A. thaliana FAE1 KCS (Fig. 1). When the activities of the two enzymes were compared in numerous experi- ments with individual transformants, this difference between A. thaliana and B. napus FAE1 KCSs was consis- tently observed. As the same expression vector and yeast strain were used, this appears to reflect actual differences in catalytic activity rather than differences in expression level although we do not know the amount of FAE1 KCS protein present in the microsomes. It appears that the A. thaliana FAE1 allele codes for a more active enzyme than the B. napus FAE1 allele used here. Subsequently, we were able to isolate (His) 6 -tagged fusion proteins and directly assay condensation activity of A. thaliana and B. napus (His) 6 -FAE1 KCSs [A. thaliana, 538.5 ± 68.1 pmolÆlg )1 protein (± SD) 20:1 productand 126.8 ± 14.7 22:1 product; B. napus 135.0 ± 17.5 20:1 product and 64 ± 4.4 22:1 product]. These observations are consistent with results obtained in in vivo experiments. Comparison of total elongation activities demonstrated that the A. thaliana enzyme was more active than the Table 2. Effect of the expression of A. thaliana and B. napus FAE1 KCS on yeast cellular lipids. S. cerevisiae transformed with expression vectors encoding A. thaliana or B. napus FAE1 KCS or the empty vector (pYES2) were pre-grown on raffinose (2%) and induced with galactose (2%) for 2 days. Total fatty acid composition was determined by GC/MS of FAMEs prepared by transacylation. Results are reported as the percentage total fatty acid methyl esters and are the mean ± SD of five independent transformants. FAME (%) 20:1 20:0 22:1 22:0 24:1 24:0 26:0 pYES2 0.17 ± 0.03 0.23 ± 0.05 0.07 ± 0.01 0.00 ± 0.01 0.00 ± 0.00 0.06 ± 0.01 1.57 ± 0.33 A. thaliana 17.29 ± 2.52 2.01 ± 0.46 4.32 ± 0.67 1.46 ± 0.29 0.60 ± 0.16 1.99 ± 0.39 4.29 ± 1.30 B. napus 2.47 ± 0.6 1.02 ± 0.24 1.61 ± 1.06 0.50 ± 0.22 0.19 ± 0.08 0.59 ± 0.19 4.13 ± 1.26 4792 B. J. Blacklock and J. G. Jaworski (Eur. J. Biochem. 269) Ó FEBS 2002 B. napus enzyme (Table 3). Analysis of the individual acyl products indicated differences in the substrate specificity of the two FAE1 KCSs. Relative substrate specificity of FAE1 KCSs can be compared by expressing the ratio of 22:1 produced over 20:1 produced. An enzyme which favors the production of 22:1 would therefore have a higher 22:1/ 20:1 product ratio than one which favors the production of 20:1 fatty acids. Table 3 shows the product ratios of A. thaliana and B. napus FAE1 KCSs after 10 min The larger 22:1/20:1 product ratio of B. napus FAE1 KCS demonstrated that B. napus FAE1 KCS was indeed pro- portionally more active toward 20:1 as a substrate than the A. thaliana FAE1 KCS. A. thaliana FAE1 KCS had a substrate specificity directed primarily toward an 18:1 substrate. Both in vivo and in vitro data indicate that A. thaliana and B. napus FAE1 KCS enzymes have distinct substrate specificities and that differences in seed oil composition reflect these substrate specificities. The primary sequence alignment (Fig. 2) demonstrates that there is a high degree of homology between A. thaliana and B. napus FAE1 KCS (86% identity). This similarity suggested to us that factors involved in substrate specificity were potentially identifiable. As little is known about the structure of FAE1 KCSs, we approached the problem with no attempt to predict where residues or regions important in substrate specificity would be found. Swap- ping of domains between A. thaliana and B. napus FAE1 KCSs by ligation of restriction digest fragments or by overlap PCR produced cognate full-length chimeric FAE1 genes. Fig. 3 is a schematic representation of a number of the chimeric enzymes engineered in this study. Chimeric enzymes were characterized based on activity and the 22:1/20:1 product ratio. Our goal was to identify the smallest domain required to retain a particular substrate specificity and to engineer an enzyme with novel activity. Twenty-nine chimeras were prepared for this study. The first group of constructs produced pairs of chimeras with switchover points between A. thaliana FAE1 KCS sequence and B. napus FAE1 KCS sequence at residues 399, 254 and 173. These constructs represent swaps in the C-terminal domain, at the midpoint and in the N-terminal domain (Fig. 3). Chimeras were named based on the origin of the N terminus and the residue at which the switchover occurred. For example, for At254, residues 1–254 are derived from A. thaliana FAE1 KCS and residues 255–506 are derived from B. napus FAE1 KCS. When in vitro elongation activity was measured, the initial FAE1 KCS chimeras segregated into two classes: those with measurable elongase activity and those with little or no activity (Fig. 4A). All of the chimeric enzymes with activity had A. thaliana sequence at the N terminus while enzymes with B. napus coding sequence in the N terminus had little or no activity. All of the active chimeras had A. thaliana-like substrate specificity as indicated by low 22:1/20:1 product ratios (Fig. 4B). The FAE1 KCS chimera with the smallest region containing A. thaliana FAE1 KCS sequence in this group was At173 indicating that at least one determinant of Table 3. Elongation activity and product ratio (22:1/20:1) for A. thaliana and B. napus FAE1 KCS. Reactions were carried out for 10 min and results presented are the mean ± SD of five individual assays using four separate microsomal preparations. Elongation activity (pmolÆlg )1 protein) 20:1 + 22:1 20:1 22:1 22:1/20:1 A. thaliana 12.04 ± 1.80 10.72 ± 0.60 1.32 ± 0.36 0.12 ± 0.01 B. napus 7.39 ± 0.60 5.52 ± 1.2 1.87 ± 0.90 0.34 ± 0.1 16 14 12 10 8 6 4 2 0 elongation activity (pmol/ µ g protein) 50403020100 time (min) Fig. 1. Elongase activity of microsomes prepared from S. cerevisiae expressing A. thal iana or B. napus FAE1 KCS. Microsomes prepared from induced S. cerevisiae transformed with empty vector (pYES2, n) or vectors encoding A. thaliana (h)orB. napus (s)FAE1 KCSswere assayed for the conversion of [1- 14 C]18:1CoA to [3- 14 C]20:1CoA and [5- 14 C]22:1CoA as described in Experimental procedures. Time points were taken between 5 and 45 min and are plotted as total elongation products (20:1 + 22:1, pmolÆlg )1 protein) vs. time. The presented results are the mean of five assays using four separate microsomal preparations and error bars indicate standard deviation. Fig. 2. Alignment of A. thaliana (top) and B. napus (bottom) FAE1 KCS sequences. Identical residues are included in the shaded box. Ó FEBS 2002 Substrate specificity of FAE1 KCSs (Eur. J. Biochem. 269) 4793 fatty acyl chain length specificity in FAE1 KCSs resides in the N terminal one-third of the protein. We further dissected the N-terminal region of the FAE1 KCSs by preparing chimeras with switchover points at residues 114 and 74 (At114 and At74). Although the catalytic activity was lower in these chimeras, At114 had A. thaliana FAE1 KCS-like substrate specificity while At74 more closely resembled B. napus FAE1 KCS in substrate specificity (Table 4). At74 had primarily B. napus sequence (85%) with A. thaliana sequence inclu- ded only in the extreme N-terminal putative transmem- brane domains. Thus, a shift in substrate specificity from A. thaliana FAE1 KCS-like to B. napus FAE1 KCS-like occurred between chimera At114 and At74 indicating that determinants of substrate specificity reside between resi- dues 74 and 114. Furthermore, as At74 is encoded entirely by B. napus sequence except for the transmembrane domain, the transmembrane domain appears to have little or no role in the determination of substrate specificity in FAE1 KCS. The stretch of primary sequence between residues 74 and 114 contains nine nonidentical residues (Fig. 2). Site-direc- ted mutagenesis of chimera At114 was utilized to dissect this region further in an attempt to reveal specific residues involved in imparting specificity toward the 20:1 substrate in FAE1 KCSs. Some of these chimeras were inactive (At114D81E, At114P89T, At114L91C, At114V93S), while others had activity similar to that of the parent enzymes (Fig. 5A). When the 22:1/20:1 product ratio of the active chimeras were compared, all chimeras in this group had A. thaliana-like product ratios except At114K92R, which had the substrate specificity of B. napus FAE1 KCS (Fig. 5B). This suggests that residue 92 has some role in determining substrate specificity of FAE1 KCSs. Catalytic activity of At114K92R, however, was similar to that of A. thaliana FAE1 KCS (Fig. 5A) while At114 had lower catalytic activity than A. thaliana FAE1 KCS. This dem- onstrated a novel activity in At114K92R and suggested that residue 92 is involved in catalytic activity as well as substrate specificity. We further examined the role that residue 92 plays in substrate specificity by replacing K92 with R in wild-type A. thaliana FAE1 KCS. Both catalytic activity and 25 20 15 10 5 0 elongation activity (pmol/ µ g protein) pYES2 A. thaliana B. napus At399 Bn399 At254 Bn254 At173 Bn173 A 0.6 0.5 0.4 0.3 0.2 0.1 0.0 product ratio 22:1/20:1 A. thaliana B. napus At399 At254 At173 B Fig. 4. (A) Elongation activity (20:1 + 22:1, pmolÆlg )1 protein) for A. thaliana, B. napus, At399, Bn399, At254, Bn254, At173, and Bn173 FAE1 KCS and (B) product ratio (22:1/20:1) for A. thaliana, B. napus, At399, At254, and At173 FAE1 KCS. (A) Reactions were carried out for 10 min and results presented are the mean of one to three indi- vidual assays using three microsomal preparations (± SD). (B) Reactions were carried out for 10 min and results presented are the mean of one to three individual assays using three microsomal preparations (± SD). Fig. 3. Schematic representation of chimeric FAE1 KCS polypeptides. A. thaliana FAE1 KCS sequence is represented as an open bar and B. napus FAE1 KCS sequence is represented as a shaded bar. The chimeric alleles were named for the FAE1 KCS sequence in the N-terminal domain followed by the residue number at which the sequence shifts to the other FAE1 KCS sequence. Point mutations are named by the convention of the wild-type residue followed by the residue number and the amino acid that has been substituted at that residue. Table 4. Elongation activity and product ratio (22:1/20:1) for A. t hali- ana, B. napus,At114andAt74FAE1KCS.Reactions were carried out for 10 min and results presented are the mean ± SD of two or three individual assays for each of four microsomal preparations. Elongation activity (pmolÆlg )1 protein) 20:1 + 22:1 22:1/20:1 A. thaliana 16.7 ± 2.4 0.13 ± 0.01 B. napus 11.9 ± 3.6 0.33 ± 0.10 At74 7.2 ± 2.3 0.36 ± 0.06 At114 10.0 ± 2.7 0.15 ± 0.03 4794 B. J. Blacklock and J. G. Jaworski (Eur. J. Biochem. 269) Ó FEBS 2002 substrate specificity of AtK92R were essentially identical to the wild-type A. thaliana FAE1 KCS (Fig. 5). A procedure for the isolation of (His) 6 -tagged FAE1 KCSs was developed in our laboratory, subsequent to the analysis of the entire set of chimeras prepared in this study, which allowed us to assay directly the condensation activity of the isolated (His) 6 -FAE1 KCSs [20]. We used this assay to verify that the product ratios observed in the elongation assay of microsomal preparations were an actual measure of the activity of the FAE1 KCS and were not an artifact of differential interactions with yeast enzymes required to produce the elongation product. When the condensation activity of isolated (His) 6 -tagged A. thaliana, B. napus, and At114K92R FAE1 KCSs were measured with 18:1CoA and 20:1CoA substrates, 22:1/20:1 product ratios were: (His) 6 -A. thaliana FAE1 KCS, 0.25 ± 0.07; (His) 6 -B. napus FAE1 KCS, 0.78 ± 0.13; (His) 6 - At114K92R, 0.71 ± 0.10 (product ratio ± SD, n ¼ 9). The product ratio of condensation activity of (His) 6 - B. napus FAE1 KCS and (His) 6 -At114K92R was therefore similar and distinct from that of (His) 6 -A. thaliana FAE1 KCS as was demonstrated by the elongation assay of FAE1 KCSs in the yeast microsomal system. DISCUSSION In this study, we attempted to gain insights into the structural basis for substrate specificity in FAE1 KCS enzymes. As no crystal structure of any membrane- bound FAE1 KCS is available, we used a biochemical approach with molecular genetic tools. We reasoned that the difference in oil content of A. thaliana and B. napus seeds is a consequence of divergent substrate specificity of the key enzyme responsible for VLCFA production in seeds, FAE1 KCS. The high degree of homology between these enzymes allowed us to use a domain-swapping approach to identify regions/residues involved in substrate specificity. Our first step was to establish the substrate specificity of A. thaliana and B. napus FAE1 KCSs with both in vivo and in vitro assays of elongation activity. Analysis of the effect of expression of A. thaliana and B. napus FAE1 KCS on yeast cellular lipids indicated that these enzymes have activity toward both monounsaturated and saturated fatty acids. This is consistent with the observation by Millar and Kunst that over-expression of FAE1 KCS in Arabidopsis resulted in an increase in elongation products of both 18:0 and 18:1 fatty acids [2]. In addition, James et al. showed that disruption of expression of FAE1 KCS in Arabidopsis resulted in a decrease in the levels of both saturated and monounsaturated VLCFA in seed [5]. In the in vivo experiment presented here, C18 and C20 monounsaturated fatty acids were elongated by the FAE1 KCSs to a greater degree than were saturated fatty acids. This may reflect the available substrates in this yeast system and/or the ability of the enzymes further down the metabolic pathway to use the products of the FAE1 KCS condensation activity. Indeed, in the S. cerevisiae strain used in these studies, monoun- saturated C16 and C18 fatty acids were at least twice as abundant as the saturated C16 and C18 fatty acids. The in vivo activities presented here demonstrated that both FAE1 KCS enzymes were active in the yeast expres- sion system and were able to couple to the yeast fatty acid elongation complex. These results show the usefulness of this in vivo system as a quick screen for activities of FAE1 KCSs. However, the results are reflective of the entire yeast metabolic pathways from the synthesis of 3-ketoacyl CoAs by a condensing enzyme to the incorporation of fatty acids into lipids such as triacylglycerol or membrane phospholipids. This in vivo system is therefore inadequate for assigning substrate specificity and catalytic activity to an exogenous KCS as activities and specificities of subsequent yeast enzymes, potential compartmentalization and avail- ability of substrates could interfere with interpretation of the results. This may be especially important in examining the activity of other plant KCS enzymes such as those encoded by KCS1 and CER6 which use longer VLCFA as substrates [39,40]. A detailed examination of activity and substrate specificity of a KCS is possible only with an in vitro approach. We found that in both in vivo and in vitro assays, A. thaliana FAE1 KCS had more activity toward a C18 substrate than toward a C20 substrate. The B. napus enzyme, on the other hand, had similar activity toward C18 and C20 fatty acyl groups. The data presented here demonstrate that A. thaliana and B. napus FAE1 KCS enzymes have distinct substrate specificities 20 15 10 5 0 elongation products (pmol/ µ g protein) A. thaliana B. napus At114D81E At114P89T At114L91C At114K92R At114V93S At114V95I At114I105V At114T110P At114S112- AtK92R 0.5 0.4 0.3 0.2 0.1 0.0 product ratio 22:1/20:1 A. thaliana B. napus At114K92R At114V95I At114I105V At114T110P At114S112- AtK92R B A Fig. 5. (A) Elongation activity (20:1 + 22:1, pmolÆlg )1 protein) for A. thaliana, B. na pus, At114D81E, At114P89T, At114L91C, At114K92R, At114V93S, At114V95I, At114I105 V, At114T110P, At114S112-, AtK92R FAE1 KCS and (B) product ratio (22:1/20:1) for A. thaliana, B. napus, At114K92R, At114V95I, At114I105 V, At114T110P, At114S112-, AtK92R FAE1 KCS. (A) Reactions were carried out for 10 min and results presented are the mean of one to three individual assays each of at least seven separate microsomal preparations (± SD). (B) Reactions were carried out for 10 min and results presented are the mean of one to three individual assays each of at least seven separate microsomal preparations (± SD). Ó FEBS 2002 Substrate specificity of FAE1 KCSs (Eur. J. Biochem. 269) 4795 and are consistent with our hypothesis that differences in seed oil composition reflect these substrate specificities. Through the preparation and assay of chimeric enzymes, we were able to define the region between residue 74 and 173 as an important domain in conferring substrate specificity to FAE1 KCSs. This domain excludes the putative transmem- brane domain predicted to be to the N-terminal side of residue 72. It is not surprising that the transmembrane domain of FAE1 KCSs is not involved in substrate specificity and therefore substrate binding because the fatty acyl CoA substrates are expected to be delivered from the cytosol and available at the membrane interface [1]. This is in contrast to membrane-bound fatty acid- and sphingo- lipid-desaturases, which appear to utilize phospholipid- or sphingolipid-conjugated fatty acids. Chimerigenesis studies of the borage D 6 -fatty acid and D 8 -sphingolipid desaturases indicate that a transmembrane portion of these enzymes is involved in substrate binding which is consistent with the membrane localization of the substrates of these desaturases [24]. If the region from the end of the transmembrane domain to residue 173 is indeed involved in imparting substrate specificity to KCS condensing enzymes, we would expect this region to be of particular heterogeneity among the family of plant ketoacyl-CoA condensing enzymes. The completion of the sequence of the Arabidopsis genome [41] presents an opportunity to compare the sequences of all predicted KCS-like genes in the genome. A recent database search revealed at least 15 distinct KCS-like genes in the Arabidopsis genome. The gene product of four of these have been assigned physiological functions in mutant analysis and expression disruption studies; FAE1, CER6, KCS1, and FDH [2,5,39,40,42,43]. Alignment of these sequences demonstrated regions of high homology distributed throughout the enzymes. The region corresponding to residues 74–173 of A. thaliana FAE1 KCS, however, is relatively heterogeneous. Recent work in our laboratory has identified the active site cysteine of A. thaliana FAE1 KCS as residue 223 and has suggested that His391 is also involved in the active site [44]. These two catalytically important residues are found in regions of high homology and are distant from the region that has been identified to be important in substrate specificity in this study. Given the similarity of the two interchanged residues, it is surprising that the change of K92 to R in At114 resulted in a shift of substrate specificity from A. thaliana-like to one more specific for a 20:1 substrate. When the substitution of amino acids in families of proteins such as globins and cytochrome c was calculated, arginine and lysine were often substituted for each other and were considered ÔsafeÕ substitutions [45]. The absence of an effect of the K92R mutation in wild-type A. thaliana FAE1 KCS points to a role for residue 92 in determining substrate specificity that is specific to At114 FAE1 KCS. Arg92 in At114K92R appears to interact with B. napus residues that are not present in A. thaliana FAE1 KCS. These results also indicate that residue 92 is not the sole residue involved in conferring substrate specificity in FAE1 KCS. The chimera At114K92R will therefore be invaluable in further studies of the domains/residues that are involved in substrate speci- ficity in FAE1 KCS as this enzyme may now be utilized in domain-swapping experiments that focus on the C termini of the proteins. We noted the levels of enzyme activity of A. thaliana and B. napus FAE1 KCSs with interest. B. napus seed oil has a larger proportion of VLCFA compared to total fatty acids than A. thaliana seed oil. The incongruity of the level of seed oil VLCFA and the activity of the B. napus FAE1 KCS assayed here may have several explanations. A less active FAE1 KCS in B. napus seeds could be compensated by the efficiency of the remainder of the biosynthetic pathway. At least five very similar genes encoding FAE1 KCS enzymes have been found in B. napus cultivars [7,8,46,47]. The fatty acid content of the seed oils of the cultivars from which these genes were cloned may reflect differences in elongation activity. The FAE1 KCS from B. napus (var. Golden) used in this study may have a relatively low level of activity compared to other B. napus FAE1 KCS homologues. In addition, the amphidiploidy of Brassica species such as B. napus [12] may result in a higher seed oil VLCFA level than that predicted from the in vitro activities of individual FAE1 KCSs. In the plant, one highly active FAE1 KCS may dominate or the additive effect of expression of two different FAE1 KCSs may result in high VLCFA production. In this work we also noted the level of activity found in many of the chimeric enzymes. Many of the changes we made resulted in chimeric enzymes with substantially lower activity than the wild-type parents. This was especially apparent when the N terminus of the chimeras was derived from B. napus FAE1 KCS. In addition, chimeras At74 and At114 had relatively poor activity compared to the wild-type enzymes. When similar experi- ments were conducted with plant acyl-acyl carrier protein thioesterases and acyl-acyl carrier protein desaturases, a decrease in the activity of the engineered enzymes often resulted, regardless of a high level of identity between the two enzymes examined [21–23]. On the other hand, several of the changes made in our study resulted in enzymes with excellent retention of activity. For example, At114K92R had even more activity than the At114 parent chimera. The crystal structures of related condensing enzymes, E. coli KAS I, -II, and -III, alfalfa chalcone synthase and S. cerevisiae 3-ketoacyl-CoA thiolase, have been solved [15– 19,48]. Although the overall sequence homology is very low, these enzymes exhibit a common fold with a five-layered core structure; a-b-a-b-a, where a comprises two a-helices and each b is a five-stranded, mixed b-sheet [16,18,19]. Alignment of the secondary structural features of the crystal structures with PHD predicted secondary structure of FAE1 KCS (data not shown) suggesting that there are common structural elements among these condensing enzymes. The substrate-binding site of KAS II may repre- sent the best available model for that of FAE1 KCS, as KAS II catalyzes the condensation of a 16:1 moiety with malonyl-ACP. The crystal structure of KAS II alone and with the mycotoxin inhibitor, cerulenin, revealed that the substrate binding pocket of KAS II is lined with hydro- phobic residues predominantly from the N-terminal domain of the enzyme that are essential for the binding of long chain substrates such as 16:1 [16,49]. The research presented here demonstrates that residues important to substrate binding for FAE1 KCS also include N-terminal residues and may suggest similar substrate-binding pockets for the two enymes. 4796 B. J. Blacklock and J. G. Jaworski (Eur. J. Biochem. 269) Ó FEBS 2002 In the redesign of a soluble fatty acid desaturase from one with D 6 -16:0 activity to one with D 9 -18:0 activity, Cahoon et al. replaced only five amino acids in the D 6 -16:0 specific enzyme with corresponding residues from the D 9 -18:0 specific desaturase [21]. The X-ray crystal structure of the D 9 -18:0 desaturase revealed that many of the residues which were identified in the mutagenesis study to be responsible for chain length substrate specificity, line the substrate- binding pocket [21,50]. The substitution of only two residues is sufficient for the conversion of an 18:0-specific desaturase to one that strongly prefers a 16:0 substrate albeit with less than half of the total catalytic activity of the parent enzyme [21]. Taken together with our studies, these results suggest that the substrate-binding site of the soluble desaturases is more rigid in the nature of acceptable substrates than that of FAE1 KCS. The KCS enzymes studied here appear to have a flexible substrate-binding pocket as demonstrated by the in vivo activities in yeast. The conversion of an 18:1-specific condensing enzyme to one that uses only 20:1 substrates may not therefore be feasible with the replacement of a small number of residues. In summary, this work was successful in identifying, for the first time, regions and residues important in fatty acyl chain length specificity in a membrane-bound condensing enzyme. 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Blacklock and. obtaining catalysts with novel substrate specificity. An understanding of the regions of enzymes that contribute to substrate specificity and catalytic activity has