Substrate-dependent mutant complementation to select fatty acid desaturase variants for metabolic engineering of plant seed oils

Journal of Biological Chemistry

Whittle

Kumaran

et al. 2007

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The multifunctional acyl-acyl carrier protein (ACP) desaturase from Hedera helix (English ivy) catalyzes the ⌬ 4 desaturation of 16:0-ACP and the ⌬ 9 desaturation of 18:0-ACP and further desaturates ⌬ 9 -16:1 or ⌬ 9 -18:1 to the corresponding ⌬ 4,9 dienes. The crystal structure of the enzyme has been solved to 1.95 Å resolution, and both the iron-iron distance of ϳ3.2 Å and the presence of a -oxo bridge reveal this to be the only reported structure of a desaturase in the oxidized FeIII-FeIII form. Significant differences are seen between the oxidized active site and the reduced active site of the Ricinus communis (castor) desaturase; His 227 coordination to Fe2 is lost, and the side chain of Glu 224 , which bridges the two iron ions in the reduced structure, does not interact with either iron. Although carboxylate shifts have been observed on oxidation of other diiron proteins, this is the first example of the residue moving beyond the coordination range of both iron ions. Comparison of the ivy and castor structures reveal surface amino acids close to the annulus of the substrate-binding cavity and others lining the lower portion of the cavity that are potential determinants of their distinct substrate specificities. We propose a hypothesis that differences in side chain packing explains the apparent paradox that several residues lining the lower portion of the cavity in the ivy desaturase are bulkier than their equivalents in the castor enzyme despite the necessity for the ivy enzyme to accommodate three more carbons beyond the diiron site. Fatty acid acyl carrier protein (ACP)3 desaturases (EC 1.14.99.6) convert saturated fatty acyl-ACPs into their cis-monounsaturated equivalents in an oxygen-dependent reaction (1-6). ⌬ 9 -desaturation of 18:0-ACP 4 occurs ubiquitously in the plastids of plants and thus plays an important role in determining the fluidity of cell membranes (7). Plants can express distinct acyl ACP desaturases, e.g. Arabidopsis thaliana has a family of seven acyl-ACP desaturases that are differentially expressed in different tissues (8). A structurally related homolog with unknown function from Mycobacterium has also been described (9).Seed oils of various plants accumulate monounsaturated fatty acids with unusual chain lengths and/or double bond positions. Several ⌬ 9 desaturases that recognize 14-, 16-, and 18-carbon chain lengths (4, 10 -12) have been described, and others with ⌬ 6 and ⌬ 9 regiospecificities have also been reported (13,14). The biosynthetic origin of unusual fatty acids is not always straightforward; for instance, Coriandrum sativum L. (coriander) and Hedera helix (English ivy) accumulate petroselinic acid (18:1⌬ 6 ) that originates via a ⌬ 4 desaturation of 16:0-ACP followed by 2-carbon elongation to 18:1⌬ 6 (15). The genes encoding these ⌬ 4 -16:0-ACP desaturase enzymes have been isolated (15,16), and their amino acid sequences are homologous to that of the castor ⌬ 9 -18:0-ACP desaturase for which the crystal structure has been determined (17,18). This implies that ac...

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

The Crystal Structure of the Ivy Δ4-16:0-ACP Desaturase Reveals Structural Details of the Oxidized Active Site and Potential Determinants of Regioselectivity

Journal of Biological Chemistry

Whittle

Kumaran

et al. 2007

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“…Mutant Asp280Ala was further mutagenized to convert Ala284 to either an Asp or Lys. Fatty acid desaturase reactions were performed by incubation of the desaturase with 14∶0-ACP substrate in the presence of recombinant spinach ACP-I (32) as previously described (31,33). Fatty acid methyl esters (FAMEs) were prepared by addition of 2 mL of 1% NaOCH 3 in methanol and incubation for 60 min at 50°C, after which the mixture was acidified with glacial acetic acid and the FAMEs extracted into hexane.…”

Section: Methodsmentioning

confidence: 99%

Remote control of regioselectivity in acyl-acyl carrier protein-desaturases

Proc. Natl. Acad. Sci. U.S.A.

Whittle

Moche

et al. 2011

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Regiospecific desaturation of long-chain saturated fatty acids has been described as approaching the limits of the discriminatory power of enzymes because the substrate entirely lacks distinguishing features close to the site of dehydrogenation. To identify the elusive mechanism underlying regioselectivity, we have determined two crystal structures of the archetypal Δ9 desaturase from castor in complex with acyl carrier protein (ACP), which show the bound ACP ideally situated to position C9 and C10 of the acyl chain adjacent to the diiron active site for Δ9 desaturation. Analysis of the structures and modeling of the complex between the highly homologous ivy Δ4 desaturase and ACP, identified a residue located at the entrance to the binding cavity, Asp280 in the castor desaturase (Lys275 in the ivy desaturase), which is strictly conserved within Δ9 and Δ4 enzymes but differs between them. We hypothesized that interaction between Lys275 and the phosphate of the pantetheine, seen in the ivy model, is key to positioning C4 and C5 adjacent to the diiron center for Δ4 desaturation. Mutating castor Asp280 to Lys resulted in a major shift from Δ9 to Δ4 desaturation. Thus, interaction between desaturase sidechain 280 and phospho-serine 38 of ACP, approximately 27 Å from the site of double-bond formation, predisposes ACP binding that favors either Δ9 or Δ4 desaturation via repulsion (acidic side chain) or attraction (positively charged side chain), respectively. Understanding the mechanism underlying remote control of regioselectivity provides the foundation for reengineering desaturase enzymes to create designer chemical feedstocks that would provide alternatives to those currently obtained from petrochemicals.diiron enzyme | enzyme redesign | lipid metabolism | enzyme mechanism A cyl-acyl carrier protein (ACP) desaturases are a family of diiron center containing soluble enzymes that introduce double bonds into acyl-ACPs in an oxygen-dependent reaction. In plants, acyl-ACP desaturases play a key role in biosynthesis of monounsaturated fatty acids, acting as the primary determinant for the composition of unsaturated fatty acids in plant membranes and seed oils. A striking feature of the reaction catalyzed by these enzymes is that it is performed with extremely high stereo-and regioselectivity, despite the fact that the substrates are composed of essentially equivalent methylene chains that completely lack distinguishing landmarks close to the site of desaturation. As Nobel Prize winner Konrad Bloch observed in 1969, "The stereospecific removal of hydrogen in the formation of oleate, although predictable on principle grounds would seem to approach the limits of the discriminatory power of enzymes" (1).We have a long-standing interest in identifying the bases of this discriminatory power of desaturases. Acyl-ACP desaturases are Δ counters-i.e., they insert a double bond a certain number of carbons from the carboxyl end of the fatty acid (2). A detailed understanding of how this counting occurs could enable reengineering of ...

“…The resulting enriched desaturase was concentrated with the use of an Amicon PM30 ultrafilter (Milliport, Framingham, MA) and subjected to HPLC size-exclusion chromatography with the use of a preparative G-3000SW (Toso Haas, Montgomeryville, PA) developed with 20 mM Hepes͞70 mM NaCl (pH 7.0). Castor ⌬ 9 -18:0-ACP desaturase variants were assayed with [1-14 C]18:0-ACP substrate with the use of recombinant spinach ACP-I (17). Methyl esters of fatty acids were analyzed by argentation TLC, and radioactivity in products was quantified as previously described (18).…”

Section: Methodsmentioning

confidence: 99%

A single mutation in the castor Δ ⁹ -18:0-desaturase changes reaction partitioning from desaturation to oxidase chemistry

Proc. Natl. Acad. Sci. U.S.A.

Abreu²,

Moche³

et al. 2006

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Sequence analysis of the diiron cluster-containing soluble desaturases suggests they are unrelated to other diiron enzymes; however, structural alignment of the core four-helix bundle of desaturases to other diiron enzymes reveals a conserved iron binding motif with similar spacing in all enzymes of this structural class, implying a common evolutionary ancestry. Detailed structural comparison of the castor desaturase with that of a peroxidase, rubrerythrin, shows remarkable conservation of both identity and geometry of residues surrounding the diiron center, with the exception of residue 199. Position 199 is occupied by a threonine in the castor desaturase, but the equivalent position in rubrerythrin contains a glutamic acid. We previously hypothesized that a carboxylate in this location facilitates oxidase chemistry in rubrerythrin by the close apposition of a residue capable of facilitating proton transfer to the activated oxygen (in a hydrophobic cavity adjacent to the diiron center based on the crystal structure of the oxygen-binding mimic azide). Here we report that desaturase mutant T199D binds substrate but its desaturase activity decreases by Ϸ2 ؋ 10 3 -fold. However, it shows a >31-fold increase in peroxide-dependent oxidase activity with respect to WT desaturase, as monitored by single-turnover stopped-flow spectrometry. A 2.65-Å crystal structure of T199D reveals active-site geometry remarkably similar to that of rubrerythrin, consistent with its enhanced function as an oxidase enzyme. That a single amino acid substitution can switch reactivity from desaturation to oxidation provides experimental support for the hypothesis that the desaturase evolved from an ancestral oxidase enzyme.binuclear ͉ diiron ͉ enzyme N onheme diiron-containing four-helix-bundle proteins possess the ability to functionalize unactivated C-H groups and mediate a diversity of chemical reactions including oxidation, hydroxylation, desaturation, and epoxidation (1, 2). A wealth of mechanistic information is available from various diironcontaining proteins including methane monooxygenases, ⌬ 9 desaturases, ribonucleotide reductases, rubrerythrins, alternate oxidases, ferritins, and bacterioferritins (1-3).The diiron-containing proteins are highly divergent in their amino acid sequences, with identities typically falling below that necessary for conventional phylogenetic analysis. However, when the analysis is restricted to the four helices that coordinate the diiron active site, the amino acid identity rises to 16-31% (4). A shared diiron-binding motif within the conserved four-helix bundle is involved in oxygen chemistry. The reactions have been described as occurring in two phases, an oxygen activation phase followed by reaction phases (1). Oxygen activation likely placed strong evolutionary constraints on the organization of the diiron center, whereas the reaction phases exhibit great diversity of functional outcome. In addition to their individual catalytic reactions, rubrerythrin, methane monooxygenase, ribonucleotide reductas...