A Small Membrane-peripheral Region Close to the Active Center Determines Regioselectivity of Membrane-bound Fatty Acid Desaturases from Aspergillus nidulans (2007)

Fatty acid desaturases catalyze the introduction of double bonds at specific positions of an acyl chain and are categorized according to their substrate specificity and regioselectivity. The current understanding of membrane-bound desaturases is based on mutant studies, biochemical topology analysis, and the comparison of related enzymes with divergent functionality. Because structural information is lacking, the principles of membrane-bound desaturase specificity are still not understood despite of substantial research efforts. Here we compare two membrane-bound fatty acid desaturases from Aspergillus nidulans: a strictly monofunctional oleoyl-Δ12 desaturase and a processive bifunctional oleoyl-Δ12/linoleoyl-ω3 desaturase. The high similarities in the primary sequences of the enzymes provide an ideal starting point for the systematic analysis of factors determining substrate specificity and bifunctionality. Based on the most current topology models, both desaturases were divided into nine domains, and the domains of the monofunctional Δ12 desaturase were systematically exchanged for their respective corresponding matches of the bifunctional sister enzyme. Catalytic capacities of hybrid enzymes were tested by heterologous expression in yeast, followed by biochemical characterization of the resulting fatty acid patterns. The individual exchange of two domains of a length of 18 or 49 amino acids each resulted in bifunctional Δ12/ω3 activity of the previously monofunctional parental enzyme. Sufficient determinants of fatty acid desaturase substrate specificity and bifunctionality could, thus, be narrowed down to a membrane-peripheral region close to the catalytic site defined by conserved histidine-rich motifs in the topology model. Fatty acid desaturases catalyze the introduction of double bonds at specific positions of an acyl chain and are categorized according to their substrate specificity and regioselectivity. The current understanding of membrane-bound desaturases is based on mutant studies, biochemical topology analysis, and the comparison of related enzymes with divergent functionality. Because structural information is lacking, the principles of membrane-bound desaturase specificity are still not understood despite of substantial research efforts. Here we compare two membrane-bound fatty acid desaturases from Aspergillus nidulans: a strictly monofunctional oleoyl-Δ12 desaturase and a processive bifunctional oleoyl-Δ12/linoleoyl-ω3 desaturase. The high similarities in the primary sequences of the enzymes provide an ideal starting point for the systematic analysis of factors determining substrate specificity and bifunctionality. Based on the most current topology models, both desaturases were divided into nine domains, and the domains of the monofunctional Δ12 desaturase were systematically exchanged for their respective corresponding matches of the bifunctional sister enzyme. Catalytic capacities of hybrid enzymes were tested by heterologous expression in yeast, followed by biochemical characterization of the resulting fatty acid patterns. The individual exchange of two domains of a length of 18 or 49 amino acids each resulted in bifunctional Δ12/ω3 activity of the previously monofunctional parental enzyme. Sufficient determinants of fatty acid desaturase substrate specificity and bifunctionality could, thus, be narrowed down to a membrane-peripheral region close to the catalytic site defined by conserved histidine-rich motifs in the topology model. Metabolic processes are often controlled by the presence of highly specific enzymes. An interesting model to study enzyme specificity is a fatty acid desaturase, which introduces double bonds at specific positions into acyl chains (1Shanklin J. Cahoon E.B. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998; 49: 611-641Crossref PubMed Scopus (709) Google Scholar). Desaturase enzymes differ in substrate- and regioselectivity (2Heinz E. Lipid Metabolism in Plants.in: Moore Jr., T.S. CRC Press, London1993: 33-89Google Scholar). The strict substrate- and regiospecificities are of physiological relevance, because the major mechanism controlling the biophysical properties of a membrane, aside from changing its overall lipid class composition, is the modification of acyl chain length, position, and number of the double bonds in glycerolipids (3Somerville C. Browse J. Jaworski J.G. Ohlrogge J. Biochemistry & Molecular Biology of Plants.in: Buchanan B.B. Gruissem W. Jones R.L. American Society of Plant Physiologists, Rockville, MD. 2000: 456-527Google Scholar). Fatty acid desaturases may also control the acyl composition of seed storage lipids, which is of considerable economic interest. Despite the importance of desaturases, knowledge about the determinants of their enzymatic specificity is still in its infancy. The solution of the three-dimensional crystal structure of the soluble castor bean and ivy stearoyl-ACP 2The abbreviations used are:ACPacyl carrier proteinFAMEsfatty methyl estersGMMglucose minimal mediumORFopen reading framePCphosphatidylcholinePEphosphatidylethanolaminePIphosphatidylinositolPSphosphatidylserineERendoplasmic reticulumDMOX4,4-dimethyloxazoline 2The abbreviations used are:ACPacyl carrier proteinFAMEsfatty methyl estersGMMglucose minimal mediumORFopen reading framePCphosphatidylcholinePEphosphatidylethanolaminePIphosphatidylinositolPSphosphatidylserineERendoplasmic reticulumDMOX4,4-dimethyloxazoline-desaturases has brought some insight into the structure/function relationship involved in the determination of specificity of soluble desaturase enzymes (4Lindqvist Y. Huang W.J. Schneider G. Shanklin J. EMBO J. 1996; 15: 4081-4092Crossref PubMed Scopus (385) Google Scholar, 5Cahoon E.B. Lindqvist Y. Schneider G. Shanklin J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4872-4877Crossref PubMed Scopus (142) Google Scholar, 6Guy J.E. Whittle E. Kumaran D. Lindqvist Y. Shanklin J. J. Biol. Chem. 2007; 282: 19863-19871Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). However, the majority of desaturase enzymes reside within membranes, and up to now the principles of membrane-bound desaturase specificity are not understood. Moreover, structural information on membrane-bound desaturases is limited, so only hydropathy and topology analyses are available, predicting the formation of four transmembrane domains and either one or two membrane-peripheral protein domains (7Diaz A.R. Mansilla M.C. Vila A.J. de Mendoza D. J. Biol. Chem. 2002; 277: 48099-48106Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 8Stukey J.E. McDonough V.M. Martin C.E. J. Biol. Chem. 1990; 265: 20144-20149Abstract Full Text PDF PubMed Google Scholar, 9Man W.C. Miyazaki M. Chu K. Ntambi J.M. J. Biol. Chem. 2006; 281: 1251-1260Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Three highly conserved histidine-rich motifs are essential for catalysis (1Shanklin J. Cahoon E.B. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998; 49: 611-641Crossref PubMed Scopus (709) Google Scholar, 10Sperling P. Ternes P. Zank T.K. Heinz E. Prostaglandins Leukot. Essent. Fatty Acids. 2003; 68: 73-95Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar), and it was proposed that these histidine clusters coordinate two iron atoms in the active site (11Fox B.G. Lyle K.S. Rogge C.E. Acc. Chem. Res. 2004; 37: 421-429Crossref PubMed Scopus (141) Google Scholar). acyl carrier protein fatty methyl esters glucose minimal medium open reading frame phosphatidylcholine phosphatidylethanolamine phosphatidylinositol phosphatidylserine endoplasmic reticulum 4,4-dimethyloxazoline acyl carrier protein fatty methyl esters glucose minimal medium open reading frame phosphatidylcholine phosphatidylethanolamine phosphatidylinositol phosphatidylserine endoplasmic reticulum 4,4-dimethyloxazoline The clearly related genes for membrane-bound desaturases have developed a broad range of catalytic diversity of the encoded desaturase enzymes during evolution (12Shanklin J. Curr. Opin. Plant Biol. 2000; 3: 243-248Crossref PubMed Scopus (18) Google Scholar). Depending on the enzyme studied, introduction of a double bond can occur counting carbons from the carboxyl end or from the methyl terminus of the acyl chain. Other enzymes may use a pre-existing double bond as a reference point for subsequent desaturation (2Heinz E. Lipid Metabolism in Plants.in: Moore Jr., T.S. CRC Press, London1993: 33-89Google Scholar). Also the carbon chain length (2Heinz E. Lipid Metabolism in Plants.in: Moore Jr., T.S. CRC Press, London1993: 33-89Google Scholar) or the specific lipid head group of the glycerolipid to which the substrate fatty acid is esterified can be distinguished (13Heilmann I. Pidkowich M.S. Girke T. Shanklin J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 10266-10271Crossref PubMed Scopus (72) Google Scholar). One conceivable model how changes in regiospecificity may have developed in evolution is by gene duplication and random accumulation of mutations in the catalytic portion of the enzymes to produce first a broader and then a different specificity (10Sperling P. Ternes P. Zank T.K. Heinz E. Prostaglandins Leukot. Essent. Fatty Acids. 2003; 68: 73-95Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 12Shanklin J. Curr. Opin. Plant Biol. 2000; 3: 243-248Crossref PubMed Scopus (18) Google Scholar). Changes to an enzyme regiospecificity typically require between two and six specific alterations at key locations along the amino acid chain (6Guy J.E. Whittle E. Kumaran D. Lindqvist Y. Shanklin J. J. Biol. Chem. 2007; 282: 19863-19871Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 14Broadwater J.A. Whittle E. Shanklin J. J. Biol. Chem. 2002; 277: 15613-15620Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). According to the described model the first step toward a new specificity of an enzyme is the accumulation of mutations in the corresponding gene, which result in decreased enzyme specificity and lead to broader substrate acceptance and/or formation of multiple products (12Shanklin J. Curr. Opin. Plant Biol. 2000; 3: 243-248Crossref PubMed Scopus (18) Google Scholar). The simplest case of multi-functionality is represented by bifunctional enzymes. Examples include the oleate 12-hydroxylase/desaturase from the flowering plant Lesquerella fendleri (15Broun P. Boddupalli S. Somerville C. Plant J. 1998; 13: 201-210Crossref PubMed Scopus (139) Google Scholar), a Δ6 acetylenase/desaturase from the moss Ceratodon purpureus (16Sperling P. Lee M. Girke T. Zähringer U. Stymne S. Heinz E. Eur. J. Biochem. 2000; 267: 3801-3811Crossref PubMed Scopus (72) Google Scholar), a Δ5/6 desaturase from the fish Danio rerio (17Hastings N. Agaba M. Tocher D.R. Leaver M.J. Dick J.R. Sargent J.R. Teale A.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14304-14309Crossref PubMed Scopus (276) Google Scholar), an acyl-ACP desaturase from the flowering plant Hedera helix, which can synthesize 16- and 18-carbon monoene and diene products (18Whittle E. Cahoon E.B. Subrahmanyam S. Shanklin J. J. Biol. 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A. 2006; PubMed Scopus Google Scholar). a between the genes for the two domains for the substrate specificity and regioselectivity were and reside in a membrane-peripheral region in close to the active enzymes and enzymes were from of fatty acids as as were from were from Fatty acids were either from or and biochemical were as described J.R. J.A. K. in Molecular and and Scholar). of A. was in glucose minimal medium J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) at were in at in the and at were in at in the was by and of Desaturase were from from or was from The desaturase genes and were by the and a at of at at at followed by of at at and at and a step of at with and of the and of the The were into and as into the expression and of and Δ12/ω3 Desaturase of and desaturases were the and a with and of the and the of the The were into the and as into the expression with the a and and for of of was as an into the E. C. C. M. A. I. 2005; PubMed Scopus Google Scholar). The was by into the for to and expression the E. C. C. M. A. I. 2005; PubMed Scopus Google Scholar). The was into and into by Plant J. 1998; PubMed Google Scholar). were from individual to and by to from hydropathy analysis, the desaturases and were divided into nine domains, and and that were systematically exchanged between the two desaturase to determinants of enzymatic between and were by PubMed Scopus Google Scholar). a first domains were from an with in domains were from the respective desaturase domains were from and also in were with and a of at of at at followed by of at at and at and a step of at were in a subsequent and and the for the first the hybrid gene of the and desaturases in The hybrid genes were into the and as into the expression with the a used to hybrid genes of A. bifunctional and monofunctional Δ12 fatty acid in a new in and of were as described E. M. C. A. M. I. 2005; PubMed Scopus Google Scholar). of desaturases was by to the fatty acids were a of the was with from at a of were at for with to of Lipid analysis of was and of with acid or fatty fatty acid accumulation with expression between and with different expression not specific enzyme be only for of enzyme of were at in minimal medium these an of were to at were by at for and were with used for fatty acid The with the was used as a control in and Fatty the analysis of esterified fatty of and were in Fatty acids were with in or solution E. C. I. Eur. J. Biochem. 2002; PubMed Scopus Google Scholar). Fatty acid methyl esters were then by the determination and of the of double bond positions in fatty fatty acid methyl esters were into their 4,4-dimethyloxazoline as described 1998; PubMed Scopus Google Scholar) and according to 1998; PubMed Scopus Google Scholar, the the analysis of fatty were from by with and of at of was and were with of Lipid and and of by were as described E. M. C. A. M. I. 2005; PubMed Scopus Google Scholar). of the from the of each was with and by the of the the of fatty acids was by the in of by of were and the for at of acid was to the and the was with and W.J. J. Biochem. Physiol. 37: PubMed Scopus Google Scholar). The resulting was and in of of each was with to to of was to the of each to fatty acids into their corresponding methyl for at and of acid was to were The resulting were in and by analysis was with an with an with a was used as a carrier were at The was for at at and for Fatty acids were according to analysis was an to an with a was used as a carrier were at The was for at at and for The was to an of an of and a of of A. and new desaturases from we the A. J.E. C. J.R. S. Lee M. J. J. C. M. J. S. S. S. C. C. D. S. J. K. A. M. M. T. T. K. M. W.C. M. M. M. P. 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However, only can the substrate of the desaturases encoded by the of and the open reading of both were into the expression the and The were in the S. a was also with the corresponding expression of in yeast, new fatty acid products were fatty acids were as and by analysis of their 4,4-dimethyloxazoline not the desaturation with fatty acids was for the acid was up to of fatty acids an desaturation of was only up to which to an of of the result from of was up to of the fatty of fatty acids in were to fatty Fatty acid analysis of resulted in the of fatty and the fatty acids The of and bifunctional activity of which double first at the Δ12 and then at the V.M. T. 2003; Google Scholar), which described as a strict by characterization in in with D. Proc. Natl. Acad. Sci. U. S. A. 2006; PubMed Scopus Google Scholar). to the fatty acid products the enzyme up to of fatty acids in analysis of substrate and were with fatty acids and the of the was acids acid acid or acid were as and into the corresponding acids expression of the bifunctional resulted also in the of of from The of for different fatty acids are in fatty acid products were by with and their were by analysis of their corresponding not The that 18-carbon fatty acids to the corresponding followed in by fatty these as a processive bifunctional Δ12/ω3 desaturase of a double bond into acid in the first step and acid from specificity of acids fatty of each fatty acid was as of esterified fatty the of number of and the is of each fatty acid was as of esterified fatty the of number of and the is of each fatty acid was as of esterified fatty the of number of and the is of each fatty acid was as of esterified fatty the of number of and the is of each fatty acid was as of esterified fatty the of number of and the is of each fatty acid was as of esterified fatty the of number of and the is in a new of substrate to by acids not to the acids not to the acids not to the Fatty acids not to the in a new Based on the the enzyme can be categorized with a group of bifunctional desaturases described D. Proc. Natl. Acad. Sci. U. S. A. 2006; PubMed Scopus Google Scholar). clearly that with the introduction of the double bond at the is that at the Δ12 position, and that is a substrate for the Δ12 desaturase of bifunctional enzymes was D. Proc. Natl. Acad. Sci. U. S. A. 2006; PubMed Scopus Google Scholar), not by and or of and of fatty acid was of fatty acids by was was first to and to the was to has Δ12 specificity for the desaturation of with that the desaturation at the position, as from the of acid to and of acid to acid that the bifunctional Δ12/ω3 are with the analyses by D. Proc. Natl. Acad. Sci. U. S. A. 2006; PubMed Scopus Google Scholar), which the with bifunctional desaturases from However, with from a G. 2005; Google Scholar), in which as as a bifunctional desaturase from N. were as The substrate by a close relationship of with the bifunctional desaturase from D. Proc. Natl. Acad. Sci. U. S. A. 2006; PubMed Scopus Google desaturation of fatty acids also similarities to a bifunctional desaturase from I. D. J.A. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar). of and Lipid lipid substrate of end desaturases from different have previously P. J. C. W. U. Heinz E. Plant Physiol. 2003; PubMed Scopus Google on and Δ12 desaturases are the lipid and specificity of the two desaturases from A. were from either or lipid were by and their fatty acid composition was of and were tested by with a and the The for a for substrate a at the an of for both positions in with an of in or of in the desaturation for fatty acids in was that for or The of to may a for the in and with of at the up to about at the was for the of to acid is the substrate of may an of substrate with acyl of fatty acids on in fatty are the of and the is are as fatty acid in the not are the of and the is are as fatty acid in the not in a new to a for acyl chains esterified to with a of of the esterified fatty followed by and The was clearly in with up to of esterified to the of with that of in the of the lipid of substrate have described for the bifunctional desaturase from A. I. D. J.A. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar), to some similarities of the of fatty acids on in fatty are as of fatty acid in the are the of six not are as of fatty acid in the are the of six not in a new on Fatty of with of the bifunctional enzyme can be in the of plant seed composition, the of the bifunctional enzyme was tested in the seed composition of of a was with that of from with the The of and products in from the and control were and the respective are in The in was in comparison to the control changing the from to up to of the enzyme in seed fatty acid composition of in a new The for are for a bifunctional Δ12/ω3 desaturase from that was in and the from to up to 18 D. Proc. Natl. Acad. Sci. U. S. A. 2006; PubMed Scopus Google Scholar), the overall fatty acid of is different in comparison to that of and the may be to Moreover, the to was in was to bifunctional of of enzyme or the of Δ12 desaturase activity However, because of can be to it may be that at of the was also the of and that the enzyme in the characterization of the A. and enzymes and from by it is that a number of may have developed and a of fatty acid desaturase genes that are in enzymes in their The close of the two proposed by D. Proc. Natl. Acad. Sci. U. S. A. 2006; PubMed Scopus Google Scholar) represented by and in A. an gene of a gene for an monofunctional Δ12 desaturase, which on the desaturase gene and for the accumulation of mutations to broader substrate specificity as a first step in the of a gene for a new enzyme of Desaturase Based on and processive activity of the bifunctional desaturase may provide a in the of and of and in bifunctional Δ12/ω3 desaturases may chain fatty acid toward of fatty the determinants of fatty acid desaturase bifunctionality and were in the The of two related fatty acid desaturase enzymes with different catalytic properties the to determinants of substrate specificity and regioselectivity. was to protein domains of the Δ12/ω3 bifunctional desaturase, to bifunctionality in the frame of the monofunctional Δ12 desaturase, Based on structure S. 2001; PubMed Scopus Google Scholar) and J. Res. 2003; PubMed Scopus Google Scholar), we the of and corresponding to the and domains previously for membrane-bound fatty acid desaturases (7Diaz A.R. Mansilla M.C. Vila A.J. de Mendoza D. J. Biol. Chem. 2002; 277: 48099-48106Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 8Stukey J.E. McDonough V.M. Martin C.E. J. Biol. Chem. 1990; 265: 20144-20149Abstract Full Text PDF PubMed Google Scholar, 9Man W.C. Miyazaki M. Chu K. Ntambi J.M. J. Biol. Chem. 2006; 281: 1251-1260Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, D. J. Biol. Chem. 267: Full Text PDF PubMed Google Scholar). to studies, the sequences of and were divided into the domains as is to the two transmembrane by a is of the subsequent the first histidine to the catalytic the first the and the histidine the the the of transmembrane by a and is of the terminus the histidine of Desaturase of and domains of the monofunctional Δ12 desaturase, were systematically exchanged for their respective corresponding matches of the bifunctional desaturase. The hybrid genes were according to their parental followed by a the from the bifunctional desaturase expression of the hybrid in it was tested the hybrid enzymes were still and the of the hybrid enzymes were from that of the parental enzyme. a control were with the were for new fatty acid products to the expression of the hybrid desaturase hybrid were active and the regioselectivity of their parental desaturase, with accumulation accumulation may be to the use of a different expression of is also that of the in activity is a of of the hybrid domains of However, most of the hybrid not bifunctional activity thus, two of the nine and bifunctional Δ12/ω3 activity thus, as in The domains and of the enzyme amino acids and were as determinants to bifunctionality in the monofunctional desaturase enzyme. a region to the active site of the desaturase the topology model in the and the histidine domains, amino and amino are either to the active site or are involved in the active of the desaturase enzyme. result to the that both domains are involved in and the fatty acid substrate to the active The that domains for bifunctionality are to each to the of a both domains, and hybrid enzyme was and desaturation is to amino acids that the bifunctionality of group of acid products to the expression of the hybrid desaturase not the of is not the of is in a new and for and for reading of the