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A growing family of cytochrome b5-domain fusion proteins
trends in plant science research news
A growing family of cytochrome b5-domain fusion proteins The field of plant lipid research has expanded rapidly in recent years, and one class of enzymes in particular, the fatty acid desaturases, has attracted considerable interest1. One reason for this is the potential for manipulating desaturases in transgenic crops to create seed oils with altered fatty acid composition. Plants produce a wide variety of modified fatty acids (there are at least 300 different types), the majority of which are considered unusual and are confined to a few plant species2. The formation of (poly)unsaturated fatty acids is catalysed by various enzymes with different regioand stereo-selectivities, introducing double bonds into preformed acyl-chains by oxygendependent desaturation. For many years the biochemical pathways of plant fatty acid desaturation were obscure, because purification of membrane-proteins is extremely difficult: to date, only one plant desaturase protein has been purified from chloroplast envelopes3. The genetic screens undertaken by Somerville and co-workers4, led to the isolation of a number of fad (fatty acid desaturation) mutants, which defined key points in the lipid modification pathway in Arabidopsis. These mutants facilitated the cloning of the respective genes and indicated that a family of membrane-bound desaturases exist, which contain a number of conserved motifs. These motifs, specifically three ‘histidine boxes’, were found to be required for function, and were also found to be present in a number of related enzymes1. Subsequent work has indicated that very small changes in the primary sequences of some of these fatty acid desaturases can dramatically alter the enzyme’s properties. A new class of cytochrome-b5 fusion proteins
Cytochrome b5 functions as an intermediate electron donor in a number of redox reactions in extraplastidial membranes, including NADH-dependent acyl-group desaturation. Importantly, in plants, microsomal cytochrome b5 is thought to be reduced by NADHcytochrome b5 reductase5 and to provide reducing equivalents for phosphatidylcholine desaturation6,7 and ∆12-hydroxylation of oleate to ricinoleate8. One surprising observation of the genome sequencing programmes has been the identification of cytochrome b5-domains in various positions in desaturases and hydroxylases. These proteins represent novel 2
January 1999, Vol. 4, No. 1
members of the cytochrome-b5 superfamily. The b5-domain is also found in a number of other heme-binding proteins, such as yeast cytochrome b2, sulphite oxidase and nitrate reductase9. The first two cytochrome b5-desaturase fusion proteins were discovered in plants and yeast. In a screen for novel desaturases, a PCR-based approach using highly degenerate primers to the conserved histidine boxes of fatty acid desaturases revealed a novel desaturase-like sequence from sunflower (Helianthus annuus)10. This sunflower sequence contained the three histidine boxes that are conserved in all lipid desaturases as well as an N-terminal extension that contained seven of the eight invariant residues characteristic of cytochrome b5. The hemo-protein nature of this N-terminal domain was confirmed by the redox absorbence spectra of the recombinant protein expressed in E. coli. No function was determined for the desaturase
domain, although it showed high similarity to two sequences from Borago officinalis generated by a PCR screen11. In yeast, a similar cytochrome b5-domain was recognized in the ∆9-acyl-CoA desaturase (OLE1), but in this enzyme it was located at the C-terminus10,12. This also explained the surprising observation that deletion of the microsomal cytochrome b5 gene in yeast did not impair fatty acid desaturation13. Furthermore, it was shown that truncation or disruption of the cytochrome b5domain of the OLE1 desaturase, even in cells with wild-type levels of free cytochrome b 5, resulted in unsaturated fatty acid auxotrophy, implying that the C-terminal b5-domain is essential for the OLE1 desaturase reaction. In contrast, the animal ∆9-fatty acid desaturases do not possess this cytochrome b5-extension and require free cytochrome b5 for activity12. Subsequent studies of other ∆9-fatty acid desaturases indicated that the presence of a C-terminal cytochrome b5-domain is
Cytochrome b5 Sulphite oxidase Cytochrome b2 Nitrate reductase ∆9-Fatty acid desaturase OLE1 (S. cerevisiae) ∆5-Fatty acid desaturase (M. alpina) ∆6-Fatty acid desaturase (B. officinalis) ∆8-Sphingolipid desaturase (A. thaliana) α-Acyl-amide hydroxylase FAH1 (S. cerevisiae) ∆6-Fatty acid desaturase (P. patens)
= Cytochrome b5 heme-binding domain
Fig. 1. Examples of the cytochrome b5 superfamily. The position of the diagnostic cytochrome b5 heme-binding domain is indicated, although the exact positions are not to scale. The GenBank accession numbers for the sequences are: Oryza sativa cytochrome b5, X75670; Rattus norvegicus sulphite oxidase, L05084; Saccharomyces cerevisiae cytochrome b2, X03215; Lycopersicon esculentum nitrate reductase, X14060; S. cerevisiae ∆9-fatty acid desaturase (OLE1), J05676; Mortierella alpina ∆5-fatty acid desaturase, AF054824; Borago officinalis ∆6-fatty acid desaturase, U79010; Arabidopsis thaliana ∆8-sphingolipiddesaturase, AJ224161; S. cerevisiae sphingolipid-hydroxylase (FAH1), Z49260; Physco-
1360 - 1385/99/$ – see front matter © 1999 Elsevier Science. All rights reserved.
trends in plant science research news common among fungal OLE1 homologues. A microsomal ∆9-desaturase from the red alga, Cyanidiochizon merolae, also contains a Cterminal cytochrome b5-domain14. Analysis of the Saccharomyces cerevisiae genome has revealed the presence of another N-terminal b5-protein with a number of histidine boxes15, and this protein was shown by complementation of fah1/scs7 null mutants16–18 to be involved in the α-hydroxylation of sphingolipid long-chain fatty acids. In contrast to the FAH1/SCS7 genes from S. cerevisiae and Caenorhabditis elegans, which contain an Nterminal cytochrome b5-domain, this domain is lacking in the corresponding FAH1 genes of Schizosaccharomyces pombe and Arabidopsis16. Moreover, the Arabidopsis FAH1 homologue is capable of complementing the S. cerevisiae fah1 mutation16. It is also interesting to note the lack of a b5-domain in the S. cerevisiae SUR2 gene, which is responsible for ∆4-hydroxylation of sphingobases in yeast17. Identification of a plant cytochrome b5-desaturase
The first assignment of a specific biochemical function to a cytochrome b5-desaturase fusion protein of plant origin came as the result of efforts to clone the ∆6-fatty acid desaturase from B. officinalis. This species is similar to evening primrose (Oenothera biennis) in that it produces γ-linolenic acid (GLA; 18:3∆6,9,12), which is an unusual plant polyunsaturated fatty acid that contains a double bond at the C-6 position: GLA is widely marketed as a health supplement and is also registered for pharmaceutical use. The synthesis of GLA from the ubiquitous plant fatty acid, linoleic acid (18:2∆9,12), is catalysed by a ∆6-fatty acid desaturase, which is presumed to be a component of the ER (Ref. 19). However, Arabidopsis (in common with most plants) does not produce GLA and so the desaturases defined by the fad mutants would not include such an enzyme. A PCR-based approach was therefore adopted to isolate the ∆6-fatty acid desaturase from B. officinalis, using degenerate primers to the conserved histidine boxes of microsomal desaturases. A novel desaturaselike sequence was identified, and expression in transgenic tobacco plants resulted in the production of high levels of GLA (Ref. 20). The sequence of this ∆6-fatty acid desaturase contained only small regions of homology to the previously described microsomal desaturases (such as FAD2 and FAD3). Moreover, the ∆6-desaturase was distinguished by the presence of an N-terminal extension which showed homology to the microsomal cytochrome b5. Amino-terminal cytochrome b5-domains have since been identified in a number of other fatty acid desaturases21–23. This may indicate that the N-terminal cytochrome b5-domain is
restricted to the ‘front-end’ class of fatty aciddesaturases (i.e. those that introduce double bonds between the C-terminus and pre-existing bonds). In addition, a novel ∆6-acyl groupdesaturase has been identified in Physcomitrella patens by targeted gene disruption and expression in yeast24. This moss ∆6-desaturase deviates from the others in that it contains an additional N-terminal extension of approximately 100 amino acids, which precedes its cytochrome b5-domain. This is the first example of a desaturase with an internal cytochrome b5-domain that resembles fusion proteins such as nitrate reductase. Novel cytochrome-b5 fusion proteins
The discovery and functional assignment of ∆6- and ∆5-fatty acid desaturases as cytochrome b5-desaturase fusion proteins20–23, is only part of the story. The sunflower b5-fusion protein10 is 59% identical to the borage ∆6desaturase20, and the functionally equivalent borage and C. elegans ∆6-desaturases show only 34% identity20,21. However, sunflower does not accumulate GLA, and so it appeared unlikely that this sunflower protein encoded a ∆6-fatty acid desaturase. A cDNA encoding a similar protein was amplified by PCR from Brassica napus, and experiments indicated that six to eight gene copies were present per haploid genome25. Searches of Arabidopsis EST databases with this sequence revealed the presence of a similar N-terminal b5-domaincontaining desaturase-like protein26. Neither of these plant species accumulates GLA, which again calls into question the function of these unusual desaturase-like proteins. A functional analysis of the homologous cDNAs from B. napus and Arabidopsis was performed in yeast. Analysis of the transformed and wild-type cells fed with various fatty acids as possible substrates for desaturation, revealed identical fatty acid patterns, indicating that the plant enzymes do not encode fatty acid desaturases. However, significant changes in the composition of sphingolipid long-chain bases were observed. The transformed cells converted a large proportion of phytosphinganine, which predominates in wild-type yeast cells, to 8- and 9-cis/transphytosphingenine; the latter regioisomer (∆9phytosphingenine) is not normally present in plant long chain bases. The introduction of a double bond suggests that the expressed cytochrome b5-fusion protein is a stereo-unselective sphingolipid desaturase26, which preferentially forms a trans-double bond. The introduction of cis/trans ∆8- and ∆9- double bonds in phytosphingenine can be explained by assuming that the sphingolipid desaturase ‘measures’ a ∆6-distance from the last oxygen-carrying carbon atom of the alkyl chain, before (C-3 of sphinganine in yeast and plants) and after C4-hydroxylation (C-4 of phytosphinganine in
d9Cm d9Sc d2Sc d5Ma
d6Pp d8At d8Bn d6Ce
d8Ha d6Bo
d5Ce
Fig. 2. Phylogenetic tree analysis of cytochrome b5-fusion proteins involved in proximal fatty acid modification. Alignments were generated by the CLUSTAL-X program, and the phylogenetic tree was made with ‘TreeView’. Sequences analysed are: d9Cm 5 Cyanidioschyzon merolae (AB006677); d9Sc 5 Saccharomyces cerevisiae ∆9-fatty acid desaturase (OLE1); d5Ma 5 Mortierella alpina ∆5fatty acid desaturase; d6Pp 5 Physcomitrella patens ∆6-fatty acid desaturase; d6Ce 5 Caenorhabditis elegans ∆6-fatty acid desaturase (AF031477); d5Ce 5 C. elegans ∆5-fatty acid desaturase (Z81122); d6Bo 5 Borago officinalis ∆6fatty acid desaturase; d8Ha 5 Helianthus annuus ∆8-sphingolipid-desaturase (X87143); d8Bn 5 Brassica napus ∆8sphingolipid-desaturase (AJ224160); d8At 5 Arabidopsis thaliana ∆8-sphingolipid-desaturase (AJ224161); d2Sc 5 S. cerevisiae sphingolipid-hydroxylase (FAH1). Accession numbers for sequences not described in Fig. 1 are also included.
yeast) of the long chain bases. This is in contrast to the ∆6-fatty acid desaturases, which depend on the presence of an additional 9-cisdouble bond in an allylic position, whereas the sphingolipid desaturase accepts a saturated substrate. The high similarity between the two types of desaturases implies that regioselectivity, rather than the absence of double bonds in the substrate, was the criterion responsible for evolution of the sphingolipid desaturases. Evolution of fatty acid and sphingolipid desaturases
The discovery of cytochrome b5-fused desaturases and hydroxylases (see Fig. 1) has interesting implications for both the evolution of multifunctional, multidomain enzymes and the specificity (i.e. stereochemistry, regioselectivity and substrate specificity) of desaturases. Southern blot analysis of the tobacco genome indicates that cytochrome b5 is present as a small gene family27. However, plants January 1999, Vol. 4, No. 1
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trends in plant science research news such as B. officinalis express at least four distinct cytochrome b5-domain proteins: the ‘free’ cytochrome b5 protein, an internal domain of nitrate reductase and two different N-terminal sequences in the ∆6-acyl group- and the ∆8-sphingolipid desaturases. All of these cytochrome b5-domain sequences have significantly diverged, although without modification of the His-Pro-Gly-Gly motif involved in heme-binding9,15. It may be that the fusion of the mobile electron carrier (cytochrome b5) to various positions on an acceptor protein provides a kinetic advantage. However, cytochrome b5 and its reductase are usually found in excess in microsomal membranes28, although this may imply that they are not particularly efficient. Another intriguing aspect, is why acyl-desaturases with ∆12- and ∆15-regioselectivities appear to lack a fused cytochrome b5 domain, even though these desaturases are much more prevalent in the plant kingdom. It may be that the presence of the b5-domain is restricted to enzymes that modify the proximal portion of lipid components facing the membrane surface (∆2-∆9 in acyl-CoA; O-acyl groups of glycerolipids; N-acyl groups and long-chain bases of ceramides). These features also call into question whether cytochrome b5 was independently fused to desaturases that had already acquired their different specificities, or whether an ancestral fusion protein for proximal lipid modification duplicated and subsequently evolved into different desaturase/hydroxylase enzymes. Phylogenetic analysis indicates that these fusion events may have happened independently at least twice, with one branch comprising the ∆5-, ∆6- and ∆8-glycerolipid/sphingolipiddesaturases (Fig. 2). With regard to the possible ancestral gene for this branch, it is interesting to note that ∆8-unsaturated long-chain bases are much more wide-spread in present day plants than ∆5- or ∆6-unsaturated fatty acids; this may imply that the sphingolipiddesaturase evolved first. It will be interesting to assess whether there is any change in the fitness of a plant in which the capacity to perform ∆8-sphingolipid-desaturation has been disrupted. Johnathan A. Napier* and Olga Sayanova IACR-Long Ashton Research Station, Dept of Agricultural Sciences, University of Bristol, Long Ashton, Bristol, UK BS41 9AF Petra Sperling and Ernst Heinz Institut für Allgemeine Botanik, University of Hamburg, Ohnhorststr. 18, D-22609 Hamburg, Germany
*Author for correspondence (e-mail [email protected]) 4
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References 1 Shanklin, J. and Cahoon, E.B. (1998) Desaturation and related modifications of fatty acids, Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 611–641 2 van de Loo, F.J., Fox, B.G. and Somerville, C.R. (1993) Unusual fatty acids, in Lipid Metabolism in Plants (Moore, T.S., ed.), pp. 91–126, CRC Press 3 Schmidt, H. et al. (1994) Purification and PCR-based cDNA cloning of a plastidial ∆6-desaturase, Plant Mol. Biol. 26, 631–642 4 Somerville, C.R. and Browse, J.A. (1996) Dissecting desaturation; plants prove advantageous, Trends Cell Biol. 6, 148–153 5 Donaldson, R.P. and Luster, D.G. (1991) Multiple forms of plant cytochromes P450, Plant Physiol. 96, 669–674 6 Smith, M.A. et al. (1990) Electon-transport components of the 1-acyl-2-oleoyl-sn-glycero-3phosphocholine ∆12-desaturase (∆12-desaturase) in microsomal preparations from developing safflower (Carthamus tinctorius L.) cotyledons, Biochem. J. 272, 23–29 7 Kearns, E.V., Hugly, S. and Somerville, C.R. (1991) The role of cytochrome b5 in ∆12 desaturation of oleic acid by microsomes of safflower (Carthamus tinctorius L.), Arch. Biochem. Biophys. 284, 431–436 8 Smith, M.A. et al. (1992) Evidence for cytochrome b5 as electron donor in ricinoleic acid biosynthesis in microsomal preparations from developing castor bean (Ricinus communis L.), Biochem. J. 287, 141–144 9 Lederer, F. (1994) The cytochrome b5-fold: an adaptable molecule, Biochimie 76, 674–692 10 Sperling, P., Schmidt, H. and Heinz, E. (1995) A cytochrome-b5-containing fusion protein similar to acyl lipid desaturases, Eur. J. Biochem. 232, 798–805 11 Schmidt, H., Sperling, P. and Heinz, E. (1995) PCR-based cloning of membrane-bound desaturases, in Plant Lipid Metabolism (Kader, J.C. and Mazliak, P., eds), pp. 21–23, Kluwer Academic Publishers, Dordrecht 12 Mitchell, A.G. and Martin, C.E. (1995) A novel cytochrome b5-like domain is linked to the carboxyl terminus of the Saccharomyces cerevisiae ∆9- fatty acid desaturase, J. Biol. Chem. 270, 29766–29772 13 Truan, G. et al. (1994) Cloning and characterization of a yeast cytochrome b5-encoding gene which suppresses ketoconazole hypersensivity in a NADPH-P-450 reductase–deficient strain, Gene 149, 123–127 14 Ito, R. et al. (1998) ∆9- fatty acid desaturase gene containing a carboxyl-terminal cytochrome b5 domain from the red alga Cyanidioschyzon merolae, Curr. Genet. 33, 165–170
15 Napier, J.A et al. (1997) A new class of cytochrome b5 fusion proteins, Biochem. J. 328, 717–720 16 Mitchell, A.G. and Martin, C.E. (1997) Fah1p, a Saccharomyces cerevisiae cytochrome b5 fusion protein, and its Arabidopsis thaliana homolog that lacks the cytochrome b5 domain both function in the α-hydroxylation of sphingolipid-associated very long chain fatty acids, J. Biol. Chem. 272, 28281–28288 17 Haak, D. et al. (1997) Hydroxylation of Saccharomyces cerevisiae ceramides requires Sur2p and Scs7p, J. Biol. Chem. 272, 29704–29710 18 Dunn, T.M. et al. (1998) Synthesis of monohydroxylated inositolphosphorylceramide (IPC-C) in Saccharomyces cerevisiae requires Scs7p, a protein with both a cytochrome b5-like domain and a hydroxylase/desaturase domain, Yeast 14, 311–321 19 Stymne, S. and Stobart, A.K. (1986) Biosynthesis of γ-linolenic acid in cotyledons and microsomal preparations of the developing seeds of common borage (Borago officinalis), Biochem. J. 240, 385–393 20 Sayanova, O. et al. (1997) Expression of a borage desaturase cDNA containing an N-terminal cytochrome b5 domain results in the accumulation of high levels of ∆6desaturated fatty acids in transgenic tobacco, Proc. Natl. Acad. Sci. U. S. A. 94, 4211–4216 21 Napier, J.A. et al. (1998) Identification of a Caenorhabditis elegans ∆6-fatty-acid-desaturase by heterologous expression in Saccharomyces cerevisiae, Biochem. J. 330, 611–614 22 Michaelson L.V. et al. (1998) Isolation of a ∆5-desaturase gene from Caenorhabditis elegans, FEBS Lett. 439, 215–218 23 Michaelson L.V. et al. (1998) Isolation of a ∆5fatty acid desaturase gene from Mortierella alpina, J. Biol. Chem. 273, 19055–19059 24 Girke, T. et al. (1998) Identification of a novel ∆6-acyl-group desaturase by targeted gene disruption in Physcomitrella patens, Plant J. 15, 39–48 25 Scheffler, J.A. et al. (1997) Desaturase multigene families of Brassica napus arose through genome duplication, Theor. Appl. Genet. 94, 583–591 26 Sperling, P., Zähringer, U. and Heinz, E. (1998) A sphingolipid desaturase from higher plants: identification of a new cytochrome b5 fusion protein, J. Biol. Chem. 273, 28590–28596 27 Smith, M.A. et al. (1994) Tobacco cytochrome b5: cDNA isolation, expression analysis and in vitro protein targeting, Plant Mol. Biol. 25, 527–537 28 Sperling, P. et al. (1990) High oleic sunflower: studies on composition and desaturation of acyl groups in different lipids and organs, Z. Naturforsch., C 45, 166–172
A growing family of cytochrome b5-domain fusion proteins The field of plant lipid research has expanded rapidly in recent years, and one class of enzymes in particular, the fatty acid desaturases, has attracted considerable interest1. One reason for this is the potential for manipulating desaturases in transgenic crops to create seed oils with altered fatty acid composition. Plants produce a wide variety of modified fatty acids (there are at least 300 different types), the majority of which are considered unusual and are confined to a few plant species2. The formation of (poly)unsaturated fatty acids is catalysed by various enzymes with different regioand stereo-selectivities, introducing double bonds into preformed acyl-chains by oxygendependent desaturation. For many years the biochemical pathways of plant fatty acid desaturation were obscure, because purification of membrane-proteins is extremely difficult: to date, only one plant desaturase protein has been purified from chloroplast envelopes3. The genetic screens undertaken by Somerville and co-workers4, led to the isolation of a number of fad (fatty acid desaturation) mutants, which defined key points in the lipid modification pathway in Arabidopsis. These mutants facilitated the cloning of the respective genes and indicated that a family of membrane-bound desaturases exist, which contain a number of conserved motifs. These motifs, specifically three ‘histidine boxes’, were found to be required for function, and were also found to be present in a number of related enzymes1. Subsequent work has indicated that very small changes in the primary sequences of some of these fatty acid desaturases can dramatically alter the enzyme’s properties. A new class of cytochrome-b5 fusion proteins
Cytochrome b5 functions as an intermediate electron donor in a number of redox reactions in extraplastidial membranes, including NADH-dependent acyl-group desaturation. Importantly, in plants, microsomal cytochrome b5 is thought to be reduced by NADHcytochrome b5 reductase5 and to provide reducing equivalents for phosphatidylcholine desaturation6,7 and ∆12-hydroxylation of oleate to ricinoleate8. One surprising observation of the genome sequencing programmes has been the identification of cytochrome b5-domains in various positions in desaturases and hydroxylases. These proteins represent novel 2
January 1999, Vol. 4, No. 1
members of the cytochrome-b5 superfamily. The b5-domain is also found in a number of other heme-binding proteins, such as yeast cytochrome b2, sulphite oxidase and nitrate reductase9. The first two cytochrome b5-desaturase fusion proteins were discovered in plants and yeast. In a screen for novel desaturases, a PCR-based approach using highly degenerate primers to the conserved histidine boxes of fatty acid desaturases revealed a novel desaturase-like sequence from sunflower (Helianthus annuus)10. This sunflower sequence contained the three histidine boxes that are conserved in all lipid desaturases as well as an N-terminal extension that contained seven of the eight invariant residues characteristic of cytochrome b5. The hemo-protein nature of this N-terminal domain was confirmed by the redox absorbence spectra of the recombinant protein expressed in E. coli. No function was determined for the desaturase
domain, although it showed high similarity to two sequences from Borago officinalis generated by a PCR screen11. In yeast, a similar cytochrome b5-domain was recognized in the ∆9-acyl-CoA desaturase (OLE1), but in this enzyme it was located at the C-terminus10,12. This also explained the surprising observation that deletion of the microsomal cytochrome b5 gene in yeast did not impair fatty acid desaturation13. Furthermore, it was shown that truncation or disruption of the cytochrome b5domain of the OLE1 desaturase, even in cells with wild-type levels of free cytochrome b 5, resulted in unsaturated fatty acid auxotrophy, implying that the C-terminal b5-domain is essential for the OLE1 desaturase reaction. In contrast, the animal ∆9-fatty acid desaturases do not possess this cytochrome b5-extension and require free cytochrome b5 for activity12. Subsequent studies of other ∆9-fatty acid desaturases indicated that the presence of a C-terminal cytochrome b5-domain is
Cytochrome b5 Sulphite oxidase Cytochrome b2 Nitrate reductase ∆9-Fatty acid desaturase OLE1 (S. cerevisiae) ∆5-Fatty acid desaturase (M. alpina) ∆6-Fatty acid desaturase (B. officinalis) ∆8-Sphingolipid desaturase (A. thaliana) α-Acyl-amide hydroxylase FAH1 (S. cerevisiae) ∆6-Fatty acid desaturase (P. patens)
= Cytochrome b5 heme-binding domain
Fig. 1. Examples of the cytochrome b5 superfamily. The position of the diagnostic cytochrome b5 heme-binding domain is indicated, although the exact positions are not to scale. The GenBank accession numbers for the sequences are: Oryza sativa cytochrome b5, X75670; Rattus norvegicus sulphite oxidase, L05084; Saccharomyces cerevisiae cytochrome b2, X03215; Lycopersicon esculentum nitrate reductase, X14060; S. cerevisiae ∆9-fatty acid desaturase (OLE1), J05676; Mortierella alpina ∆5-fatty acid desaturase, AF054824; Borago officinalis ∆6-fatty acid desaturase, U79010; Arabidopsis thaliana ∆8-sphingolipiddesaturase, AJ224161; S. cerevisiae sphingolipid-hydroxylase (FAH1), Z49260; Physco-
1360 - 1385/99/$ – see front matter © 1999 Elsevier Science. All rights reserved.
trends in plant science research news common among fungal OLE1 homologues. A microsomal ∆9-desaturase from the red alga, Cyanidiochizon merolae, also contains a Cterminal cytochrome b5-domain14. Analysis of the Saccharomyces cerevisiae genome has revealed the presence of another N-terminal b5-protein with a number of histidine boxes15, and this protein was shown by complementation of fah1/scs7 null mutants16–18 to be involved in the α-hydroxylation of sphingolipid long-chain fatty acids. In contrast to the FAH1/SCS7 genes from S. cerevisiae and Caenorhabditis elegans, which contain an Nterminal cytochrome b5-domain, this domain is lacking in the corresponding FAH1 genes of Schizosaccharomyces pombe and Arabidopsis16. Moreover, the Arabidopsis FAH1 homologue is capable of complementing the S. cerevisiae fah1 mutation16. It is also interesting to note the lack of a b5-domain in the S. cerevisiae SUR2 gene, which is responsible for ∆4-hydroxylation of sphingobases in yeast17. Identification of a plant cytochrome b5-desaturase
The first assignment of a specific biochemical function to a cytochrome b5-desaturase fusion protein of plant origin came as the result of efforts to clone the ∆6-fatty acid desaturase from B. officinalis. This species is similar to evening primrose (Oenothera biennis) in that it produces γ-linolenic acid (GLA; 18:3∆6,9,12), which is an unusual plant polyunsaturated fatty acid that contains a double bond at the C-6 position: GLA is widely marketed as a health supplement and is also registered for pharmaceutical use. The synthesis of GLA from the ubiquitous plant fatty acid, linoleic acid (18:2∆9,12), is catalysed by a ∆6-fatty acid desaturase, which is presumed to be a component of the ER (Ref. 19). However, Arabidopsis (in common with most plants) does not produce GLA and so the desaturases defined by the fad mutants would not include such an enzyme. A PCR-based approach was therefore adopted to isolate the ∆6-fatty acid desaturase from B. officinalis, using degenerate primers to the conserved histidine boxes of microsomal desaturases. A novel desaturaselike sequence was identified, and expression in transgenic tobacco plants resulted in the production of high levels of GLA (Ref. 20). The sequence of this ∆6-fatty acid desaturase contained only small regions of homology to the previously described microsomal desaturases (such as FAD2 and FAD3). Moreover, the ∆6-desaturase was distinguished by the presence of an N-terminal extension which showed homology to the microsomal cytochrome b5. Amino-terminal cytochrome b5-domains have since been identified in a number of other fatty acid desaturases21–23. This may indicate that the N-terminal cytochrome b5-domain is
restricted to the ‘front-end’ class of fatty aciddesaturases (i.e. those that introduce double bonds between the C-terminus and pre-existing bonds). In addition, a novel ∆6-acyl groupdesaturase has been identified in Physcomitrella patens by targeted gene disruption and expression in yeast24. This moss ∆6-desaturase deviates from the others in that it contains an additional N-terminal extension of approximately 100 amino acids, which precedes its cytochrome b5-domain. This is the first example of a desaturase with an internal cytochrome b5-domain that resembles fusion proteins such as nitrate reductase. Novel cytochrome-b5 fusion proteins
The discovery and functional assignment of ∆6- and ∆5-fatty acid desaturases as cytochrome b5-desaturase fusion proteins20–23, is only part of the story. The sunflower b5-fusion protein10 is 59% identical to the borage ∆6desaturase20, and the functionally equivalent borage and C. elegans ∆6-desaturases show only 34% identity20,21. However, sunflower does not accumulate GLA, and so it appeared unlikely that this sunflower protein encoded a ∆6-fatty acid desaturase. A cDNA encoding a similar protein was amplified by PCR from Brassica napus, and experiments indicated that six to eight gene copies were present per haploid genome25. Searches of Arabidopsis EST databases with this sequence revealed the presence of a similar N-terminal b5-domaincontaining desaturase-like protein26. Neither of these plant species accumulates GLA, which again calls into question the function of these unusual desaturase-like proteins. A functional analysis of the homologous cDNAs from B. napus and Arabidopsis was performed in yeast. Analysis of the transformed and wild-type cells fed with various fatty acids as possible substrates for desaturation, revealed identical fatty acid patterns, indicating that the plant enzymes do not encode fatty acid desaturases. However, significant changes in the composition of sphingolipid long-chain bases were observed. The transformed cells converted a large proportion of phytosphinganine, which predominates in wild-type yeast cells, to 8- and 9-cis/transphytosphingenine; the latter regioisomer (∆9phytosphingenine) is not normally present in plant long chain bases. The introduction of a double bond suggests that the expressed cytochrome b5-fusion protein is a stereo-unselective sphingolipid desaturase26, which preferentially forms a trans-double bond. The introduction of cis/trans ∆8- and ∆9- double bonds in phytosphingenine can be explained by assuming that the sphingolipid desaturase ‘measures’ a ∆6-distance from the last oxygen-carrying carbon atom of the alkyl chain, before (C-3 of sphinganine in yeast and plants) and after C4-hydroxylation (C-4 of phytosphinganine in
d9Cm d9Sc d2Sc d5Ma
d6Pp d8At d8Bn d6Ce
d8Ha d6Bo
d5Ce
Fig. 2. Phylogenetic tree analysis of cytochrome b5-fusion proteins involved in proximal fatty acid modification. Alignments were generated by the CLUSTAL-X program, and the phylogenetic tree was made with ‘TreeView’. Sequences analysed are: d9Cm 5 Cyanidioschyzon merolae (AB006677); d9Sc 5 Saccharomyces cerevisiae ∆9-fatty acid desaturase (OLE1); d5Ma 5 Mortierella alpina ∆5fatty acid desaturase; d6Pp 5 Physcomitrella patens ∆6-fatty acid desaturase; d6Ce 5 Caenorhabditis elegans ∆6-fatty acid desaturase (AF031477); d5Ce 5 C. elegans ∆5-fatty acid desaturase (Z81122); d6Bo 5 Borago officinalis ∆6fatty acid desaturase; d8Ha 5 Helianthus annuus ∆8-sphingolipid-desaturase (X87143); d8Bn 5 Brassica napus ∆8sphingolipid-desaturase (AJ224160); d8At 5 Arabidopsis thaliana ∆8-sphingolipid-desaturase (AJ224161); d2Sc 5 S. cerevisiae sphingolipid-hydroxylase (FAH1). Accession numbers for sequences not described in Fig. 1 are also included.
yeast) of the long chain bases. This is in contrast to the ∆6-fatty acid desaturases, which depend on the presence of an additional 9-cisdouble bond in an allylic position, whereas the sphingolipid desaturase accepts a saturated substrate. The high similarity between the two types of desaturases implies that regioselectivity, rather than the absence of double bonds in the substrate, was the criterion responsible for evolution of the sphingolipid desaturases. Evolution of fatty acid and sphingolipid desaturases
The discovery of cytochrome b5-fused desaturases and hydroxylases (see Fig. 1) has interesting implications for both the evolution of multifunctional, multidomain enzymes and the specificity (i.e. stereochemistry, regioselectivity and substrate specificity) of desaturases. Southern blot analysis of the tobacco genome indicates that cytochrome b5 is present as a small gene family27. However, plants January 1999, Vol. 4, No. 1
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trends in plant science research news such as B. officinalis express at least four distinct cytochrome b5-domain proteins: the ‘free’ cytochrome b5 protein, an internal domain of nitrate reductase and two different N-terminal sequences in the ∆6-acyl group- and the ∆8-sphingolipid desaturases. All of these cytochrome b5-domain sequences have significantly diverged, although without modification of the His-Pro-Gly-Gly motif involved in heme-binding9,15. It may be that the fusion of the mobile electron carrier (cytochrome b5) to various positions on an acceptor protein provides a kinetic advantage. However, cytochrome b5 and its reductase are usually found in excess in microsomal membranes28, although this may imply that they are not particularly efficient. Another intriguing aspect, is why acyl-desaturases with ∆12- and ∆15-regioselectivities appear to lack a fused cytochrome b5 domain, even though these desaturases are much more prevalent in the plant kingdom. It may be that the presence of the b5-domain is restricted to enzymes that modify the proximal portion of lipid components facing the membrane surface (∆2-∆9 in acyl-CoA; O-acyl groups of glycerolipids; N-acyl groups and long-chain bases of ceramides). These features also call into question whether cytochrome b5 was independently fused to desaturases that had already acquired their different specificities, or whether an ancestral fusion protein for proximal lipid modification duplicated and subsequently evolved into different desaturase/hydroxylase enzymes. Phylogenetic analysis indicates that these fusion events may have happened independently at least twice, with one branch comprising the ∆5-, ∆6- and ∆8-glycerolipid/sphingolipiddesaturases (Fig. 2). With regard to the possible ancestral gene for this branch, it is interesting to note that ∆8-unsaturated long-chain bases are much more wide-spread in present day plants than ∆5- or ∆6-unsaturated fatty acids; this may imply that the sphingolipiddesaturase evolved first. It will be interesting to assess whether there is any change in the fitness of a plant in which the capacity to perform ∆8-sphingolipid-desaturation has been disrupted. Johnathan A. Napier* and Olga Sayanova IACR-Long Ashton Research Station, Dept of Agricultural Sciences, University of Bristol, Long Ashton, Bristol, UK BS41 9AF Petra Sperling and Ernst Heinz Institut für Allgemeine Botanik, University of Hamburg, Ohnhorststr. 18, D-22609 Hamburg, Germany
*Author for correspondence (e-mail [email protected]) 4
January 1999, Vol. 4, No. 1
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