Journal of Experimental Botany

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Journal of Experimental Botany, Vol. 52, No. 360, pp. 1581-1585, July 1, 2001
© 2001 Oxford University Press

Short Communications

Mutagenesis and heterologous expression in yeast of a plant ⁶-fatty acid desaturase

Olga Sayanova¹, Frédéric Beaudoin¹, Balázs Libisch^1,2, Aude Castel^1,3, Peter R. Shewry¹ and Johnathan A. Napier^1,4

¹ IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol BS41 9AF, UK
² Department of Plant Anatomy, Eötvös Loránd University, Budapest, Hungary
³ University Paris Sud, CNRS, F-91405 Orsay, France

Received 15 January 2001; Accepted 21 February 2001

Abstract

Membrane-bound microsomal fatty acid desaturases are known to have three conserved histidine boxes, comprising a total of up to eight histidine residues. Recently, a number of deviations from this consensus have been reported, with the substitution of a glutamine for the first histidine residue of the third histidine box being present in the so called ‘front end’ desaturases. These enzymes are also characterized by the presence of a cytochrome b₅ domain at the protein N-terminus. Site-directed mutagenesis has been used to probe the functional importance of a number of amino acid residues which comprise the third histidine box of a ‘front end’ desaturase, the borage ⁶-fatty acid desaturase. This showed that the variant glutamine in the third histidine box is essential for enzyme activity and that histidine is not able to substitute for this residue.

Key words: Microsomal fatty acid desaturase, ‘front end’ desaturation, borage.

Introduction

Plant fatty acid desaturases can be divided into two broad classes. Soluble desaturases present in the plastid act on fatty acyl ACP substrates and convert saturated C16 and C18 substrates into mono-unsaturated derivatives. The prevalent form of this enzyme is the ⁹-stearoyl ACP desaturase which converts stearic acid (C18 : 0) into oleic acid (C18 : 1⁹) and has been characterized at both the molecular and biophysical levels (reviewed in Shanklin and Cahoon, 1998), culminating in the determination of a three-dimensional crystal structure (Lindqvist et al., 1996). These mono-unsaturated C16 and C18 fatty acids then serve as substrates for a second class of desaturases which are membrane-bound, but are less well functionally characterized than the soluble desaturases. Although the use of Arabidopsis mutants defective in fatty acid desaturation has resulted in the identification and cloning of membrane-bound desaturase genes (and the enzymes they encode) (Somerville and Browse, 1996), very little biochemical characterization has been carried out.

The most widely studied membrane-bound plant fatty acid desaturases act on oleic acid (esterified to the glycerol backbone of phosphatidylcholine), sequentially forming bonds at the ¹² and ¹⁵ positions to give linoleic (LA; C18 : 2 ^9.12) and -linolenic acids (ALA; C18 : 3 ^9,12,15), respectively. These desaturases appear to be ubiquitous in higher plants with linoleic and linolenic acid being major components of both membrane lipids and storage triacylglycerols (Miquel and Browse, 1998). However, some plant species also contain a class of desaturase which inserts a double bond between the carboxyl group and the ⁹-position, as distinct from the more prevalent methyl-directed desaturation carried out by the ¹²- and ¹⁵-desaturases. For this reason, this former class of enzymatic reaction has been termed ‘front end’ desaturation (Aitzemuller and Tsevegsuren, 1994; Napier et al., 1997, 1999). The first example of this type of enzyme has been cloned and characterized; a ⁶-fatty acid desaturase from borage (Borago officinalis) (Sayanova et al., 1997); which catalyses the ⁶-desaturation of (glycerolipid) LA and ALA to give -linolenic acid (GLA; C18 : 3 ^6,9,12,) and octadecatetraenoic acids (OTA; C18 : 4 ^6,9,12,15), respectively. This ‘front end’ desaturase differed from previously cloned ¹²-(FAD2) and ¹⁵-(FAD3) microsomal desaturases in that it contained a fused cytochrome b₅ domain at the N-terminus (Sayanova et al., 1997). Microsomal cytochrome b₅ is known to be required as the electron donor for a number of biochemical reactions including plant fatty acyl desaturation (Smith et al., 1990). The absolute requirement of the N-terminal cytochrome b₅ domain in the borage ⁶-fatty acid desaturase was shown by site-directed mutagenesis (Sayanova et al., 1999), which also indicated that free (i.e. not fused) microsomal cytochrome b₅ was unable to substitute for the loss of this domain. Subsequently, N-terminally-fused cytochrome b₅ domains have been observed in other ‘front end’ desaturases such as ⁵- and ⁶-fatty acid desaturases from the nematode Caenorhabditis elegans (Michaelson et al., 1998b; Napier et al., 1998), the fungus Mortierella alpina (Michaelson et al., 1998a; Knutzon et al., 1998) and mammals (Cho et al., 1999). Similar domains are also present in the ⁸-fatty acid desaturase from the alga Euglena gracilis (Wallis and Browse, 1999) and the ⁸-sphingolipid long chain base desaturase present in a number of higher plants (Sperling et al., 1998). A related internal cytochrome b₅ domain is present in the ⁶-fatty acid desaturase of the moss Physcomitrella patens (Girke et al., 1998a).

Comparison of the amino acid sequences of methyl-directed (¹²-, ¹⁵-) membrane-bound desaturases from plants, animals and fungi has revealed the presence of three highly conserved histidine-rich sequences (generically termed ‘histidine boxes’) comprising the general motifs H-X_[3-4]-H, H-X_[2-3]-H-H and H-X_[2]-H-H. Site-directed mutagenesis of the rat microsomal stearoyl acyl CoA ⁹-desaturase and the microsomal ¹²-desaturase of the cyanobacterium Synechocystis have demonstrated that each of the eight histidines is important for catalysis (Shanklin et al., 1994; Avelange-Macherel et al., 1995); replacement of any of these residues with alanine resulted in inactive enzymes. In recent years several exceptions to this consensus pattern have been observed, notably the ‘front end’ desaturases which all contain a variant third histidine box and where the first histidine residue is replaced by glutamine (Sayanova et al., 1997; Girke et al., 1998b). However, the functional significance of this variation has not been experimentally determined.

The presence of a variant third histidine box in ‘front end’ fusion desaturates raises the question of whether this is essential for the function of this group of enzymes. This question has been addressed by constructing mutants in the third histidine box and characterizing them functionally by expression in a yeast system.

Materials and methods

Expression of borage ⁶-desaturase in Saccharomyces cerevisiae
The open reading frame of the borage ⁶-desaturase (Sayanova et al., 1997) was cloned behind the galactose-inducible GAL1 promoter of the yeast expression vector pYES2 (Invitrogen). PCR was used to amplify the entire coding region of the borage enzyme using primers YBF (5'-GCGGATCCATGGCTGCTCAAATCAAG-3' containing a BamHI restriction site (bold) upstream of the underlined initiating methionine codon) and YBR (5'-GCCTCGAGTTAACCATGAGTGTGAAG-3' containing a XhoI site downstream of the underlined stop codon). Amplification was carried out using the following conditions: 2 min denaturation step at 94 °C followed by 35 cycles of 92 °C for 1 min, 55 °C for 1 min, 72 °C for 1.5 min, concluding with a final extension at 72 °C for 10 min. The amplified PCR product was digested with BamHI and XhoI restriction enzymes, gel-purified and cloned into the corresponding restriction sites in pYES2, generating plasmid pYBdes6. This plasmid was transformed into S. cerevisiae by the lithium acetate method and expression of the transgene was induced by the addition of galactose to 1% (w/v) in the presence of 0.003% (w/v) of the corresponding fatty acid and 1% (w/v) of tergitol, as previously described protocols (Napier et al., 1998).

Site-directed mutagenesis
Site-directed mutagenesis was performed using a Quikchange^TM site-directed mutagenesis kit (Stratagene). The coding region of pYBdes6 cDNA was mutated using two synthetic oligonucleotide primers containing the desired mutation. The mutations were: Q³⁷³I, Q³⁷³H, I³⁷⁴L, E³⁷⁵A, L³⁷⁸A, F³⁷⁹W, Q³²⁴H. The oligonucleotides used are shown in Table 1. The letters in bold and underlined indicate altered nucleotides and codons, respectively.

View this table: [in this window] [in a new window]   Table 1. Oligonucleotide primers used to construct site-directed mutants of pYBdes6

 

Fatty acid analyses
Total fatty acids extracted from yeast cultures were analysed by GC of methyl esters (Napier et al., 1998). Fatty acids were identified by comparison with the retention times of FAME standards (Sigma, UK) and by GCMS (Napier et al., 1998; Michaelson et al., 1999a).

Results and discussion

Functional expression of the borage ⁶-desaturase in yeast
To examine the enzyme activity of the borage ⁶-desaturase in S. cerevisiae further, the coding region of the cDNA (Sayanova et al., 1997) was cloned into the yeast expression vector pYES2 (Invitrogen) to create the plasmid pYBdes6. Since S. cerevisiae does not contain the polyunsaturated fatty acids substrates normally utilized by the ⁶-desaturase enzyme, the growth medium was supplemented with either LA or ALA. Analysis of the total lipids from yeast cells transformed with pYBdes6 and supplemented with LA showed high levels of ⁶-desaturation, with GLA comprising 26.4% of total fatty acids. Very similar levels of ⁶-desaturation were obtained when ALA was used as a substrate with 26.5% OTA (Table 2 ). Thus, the borage ⁶-fatty acid desaturase is capable of recognizing both n-6 (i.e. LA) and n-3 (i.e. ALA) substrates with very similar levels for the percentage conversion rates for both fatty acids (44% and 41%, respectively). The ⁶-desaturated fatty acids GLA and OTA were not detected in cells containing the empty vector or in cells expressing the borage ⁶-desaturase, but grown without exogenous substrate. Yeast cells expressing the borage enzyme also contained ⁶-desaturated C16 fatty acids, with C16 : 2 ^6,9 accounting for 4.5% of total fatty acids in cells grown without exogenous substrates but less than 1% in cells provided with either LA or ALA. This may indicate that C16 and C18 substrates may compete for the ⁶-desaturase. No ⁶-desaturation was observed when a range of exogenous C20 unsaturated substrates were provided (data not shown).

View this table: [in this window] [in a new window]   Table 2. Fatty acid compositions (expressed as moles % of total fatty acid methyl esters) of yeast cells containing the pYES2 and pYBdes6 plasmids and grown on media with and without exogenously supplied fatty acid substrates Values represent means obtained from triplicated experimental runs (n=5), with all SE values<3% of total.

 
Interestingly, a study of the related ⁵-fatty acid desaturase of Caenorhabditis elegans (which preferentially desaturates C20 trienoic substrates) revealed activity towards endogenous C18 : 1 ⁹ (but not endogenous C16 : 1 ⁹) fatty acids (Watts and Browse, 1999). It has previously been suggested that the observed range of ‘front end’ desaturases has arisen through gene-duplication events followed by functional divergence (Napier et al., 1999). Evidence for this is indicated by the C. elegans ⁵- and ⁶-fatty acid desaturases, which exist as a tandem gene pair with conserved intron-exon junctions (Michaelson et al., 1998b). Gene duplication is a common phenomenon in both plant and animal genomes and can be considered a prerequisite for enzyme diversification, at least in the case of essential genes required for primary metabolism (Pichersky and Gang, 2000). Thus, the natural variation in substrate specificity displayed by members of the ‘front end’ desaturase class may provide insights into the both evolutionary history and proliferation of this gene family (Napier et al., 1999).

Site-directed mutagenesis of the variant third histidine box
The sequences of the third histidine boxes of a range of ⁵-, ⁶-(‘front end’) fatty acid desaturases and ⁸-sphingolipid desaturases are compared in Fig. 1A. The consensus sequence differs from that of the membrane-bound methyl-directed ¹²- and ¹⁵-desaturases (Fig. 1B) in the substitution of glutamine (residue Q³⁷³ in the borage ⁶-desaturase enzyme) for histidine at the first position of this motif. In order to determine whether this mutation is important for catalytic activity, two mutant forms of the borage ⁶-desaturase were generated by site-directed mutagenesis. These were the replacement of Q³⁷³ by histidine (i.e. conversion to the consensus motif for methyl-directed, non-cytochrome b₅-fusion desaturases; Q³⁷³H), or by the uncharged amino acid isoleucine (Q³⁷³I). Neither of these two mutant forms of the borage enzyme resulted in the accumulation of any ⁶-desaturated fatty acids when expressed in yeast, either in the presence or absence of exogenous substrate (Table 3; for clarity, data are given as percentage conversion of substrate, as well as relative percentage when compared to wild-type activity). However, Northern blot analyses indicated that steady-state mRNA levels for the two mutants were equivalent to that of wild-type borage ⁶-desaturase expressed in yeast (data not shown). Thus, it is clear that the third histidine boxes of ‘front end’ or methyl directed desaturases require different residues (glutamine versus histidine) in the primary position.

View larger version (74K): [in this window] [in a new window]   Fig. 1. Amino acid sequence comparison of the domain containing the third histidine box (H3) present in microsomal fatty acid desaturases. All sequences are present on GenBank. (A) Comparison of ‘front end’ desaturases from plants and animals. Sequences are derived from the following organisms: Boof, Borago officinalis, Ceel, Caenorhrabditis elegans, Moal, Mortierella alpina, Phpa, Physcomitrella patens, and Eugr, Euglena gracilis. The deduced consensus for the third histidine box is Q-X-X-H-H. In the case of the borage 6-sequence, this Q is residue 373. (B) Comparison of methyl-directed desaturases (FAD2/12-desaturase and FAD3/15-desaturase) from higher plants. Sequences are derived from the following organisms: Arth, Arabidopsis thaliana, Nita, Nicotiana tabacum, Trae, Tricticum aestivum, Glma, Glycine max, and Boof, Borago officinalis. The deduced consensus for the third histidine box is H-V-X-H-H.

 

View this table: [in this window] [in a new window]   Table 3. Percentage conversion of exogenously provided C18 : 2 9,12and endogenous C16 : 19, respectively, by the wild type and mutant borage 6-desaturase enzymes expressed in yeast The relative percentage conversion rate (compared to that obtained with the wild type) is also given. Percentage conversions are derived from experimental samples statistically analysed as for Table 2.

 
The presence of a HQ substitution in the third histidine box of cytochrome b₅-fusion class of membrane desaturases is intriguing. Histidine residues are often involved in binding metal ions, such as iron or zinc. The three histidine boxes present in the soluble fatty acid desaturases are expected to be ligands for two iron molecules (Lindqvist et al., 1996) and a similar role has been suggested for the membrane-bound desaturases (Shanklin et al., 1994; Shanklin and Cahoon, 1998). Replacement of one of the histidine residues might, therefore, be expected to weaken the iron binding and lead to reduced catalytic activity, as reported for other metal-containing enzymes. For example, carbonic anhydrase II (CAII) requires the divalent metal zinc for enzymatic activity (Hunt et al., 1999), and replacement of the active site histidine by glutamine destabilized the zinc-bound hydroxide resulting in decreased catalytic activity, though the mutation had no apparent effect on the structure of the CAII protein. Although the carboxamide side chain of glutamine could also serve to co-ordinate zinc, this interaction might be expected to be weaker than co-ordination via histidine (Hunt et al., 1999). The substitution of glutamine for histidine in the ‘front end’ desaturases implies that there are subtle differences in the active site chemistry of the two major types of membrane-bound desaturases with any weakening of iron binding in the ‘front end’ enzymes having little or no effect on their catalytic activity.

Mutagenesis of other residues within the third histidine box
Comparison of amino acids in close proximity to the variant third histidine box of ‘front end’ desaturases reveals a generally high level of sequence identity but some substitutions can be observed (Fig. 1A). Residue I³⁷⁴ is conserved in all ⁶-fatty acid desaturases and in the ⁸-fatty acid desaturase from Euglena (Wallis and Browse, 1999) but is replaced by alanine in the M. alpina ⁵-fatty acid desaturase (Michaelson et al., 1998a) and by leucine in the ⁸-sphingolipid desaturase (Sperling et al., 1998). Residue E³⁷⁵ is also highly conserved, being present in all the ‘front end’ desaturases apart from the M. alpina ⁵-fatty acid desaturases where it is replaced by valine. Similarly, residue L³⁷⁸ is conserved in all of the enzymes while F³⁷⁹ is substituted by another aromatic residue (tryptophan) in the ⁸-fatty acid desaturase from Euglena.

To investigate the significance of these conserved amino acids, four mutations were generated in the third histidine box (I³⁷⁴L, E³⁷⁵A, L³⁷⁸A and F³⁷⁹W) by site-directed mutagenesis of the borage ⁶-desaturase cDNA and they were expressed in yeast. None of these mutations resulted in major changes to the specificity of borage ⁶-fatty acid desaturase (Table 3), but all showed reduced total levels of ⁶-desaturation (of either LA or 16 : 1⁹). In particular, the I³⁷⁴L and L³⁷⁸A mutants desaturated GLA with only 71% and 61% of the activity of the wild-type enzyme, respectively. Therefore a double mutant was generated in which these substitutions were combined (I³⁷⁴L, L³⁷⁸A). Functional characterization of this double mutant showed total enzyme activity and activity towards 16 : 1⁹ substrates equivalent to those of the single mutations, with no synergistic interaction (Table 3).

Conclusions

In this study it is demonstrated that replacement of the glutamine residue by histidine or isoleucine in the third histidine box of the cytochrome b₅-fusion ‘front end’ ⁶-fatty acid desaturase abolishes enzyme activity. It is also shown that mutations within and around the third histidine box reduce the activity, but do not alter the specificity of this enzyme. The experimental data therefore indicate that the consensus motif for the third histidine box of ‘front end’ desaturases should be amended to Q-X_[2-3]-H-H, since substitution of this glutamine residue by histidine is not tolerated.

Acknowledgments

IACR receives grant-aided support from the Biotechnology and Biological Sciences Research Council (BBSRC) of the United Kingdom.

Notes

⁴ To whom correspondence should be addressed. Fax: +44 1275 549225. E-mail: jon.napier{at}bbsrc.ac.uk

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