A single mutation in the castor Delta9-18:0-desaturase changes reaction partitioning from desaturation to oxidase chemistry

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 approximately 2 x 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-A 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.     

Revealing the catalytic potential of an acyl-ACP desaturase: tandem selective oxidation of saturated fatty acids

It is estimated that plants contain thousands of fatty acid structures, many of which arise by the action of membrane-bound desaturases and desaturase-like enzymes. The details of “unusual” e.g., hydroxyl or conjugated, fatty acid formation remain elusive, because these enzymes await structural characterization. However, soluble plant acyl-ACP (acyl carrier protein) desaturases have been studied in far greater detail but typically only catalyze desaturation (dehydrogenation) reactions. We describe a mutant of the castor acyl-ACP desaturase (T117R/G188L/D280K) that converts stearoyl-ACP into the allylic alcohol trans-isomer (E)-10-18:1-9-OH via a cis isomer (Z)-9-18:1 intermediate. The use of regiospecifically deuterated substrates shows that the conversion of (Z)-9-18:1 substrate to (E)-10-18:1-9-OH product proceeds via hydrogen abstraction at C-11 and highly regioselective hydroxylation (>97%) at C-9. ¹⁸O-labeling studies show that the hydroxyl oxygen in the reaction product is exclusively derived from molecular oxygen. The mutant enzyme converts (E)-9-18:1-ACP into two major products, (Z)-10-18:1-9-OH and the conjugated linolenic acid isomer, (E)-9-(Z)-11-18:2. The observed product profiles can be rationalized by differences in substrate binding as dictated by the curvature of substrate channel at the active site. That three amino acid substitutions, remote from the diiron active site, expand the range of reaction outcomes to mimic some of those associated with the membrane-bound desaturase family underscores the latent potential of O₂-dependent nonheme diiron enzymes to mediate a diversity of functionalization chemistry. In summary, this study contributes detailed mechanistic insights into factors that govern the highly selective production of unusual fatty acids.     

Redesign of soluble fatty acid desaturases from plants for altered substrate specificity and double bond position

Acyl-acyl carrier protein (ACP) desaturases introduce double bonds at specific positions in fatty acids of defined chain lengths and are one of the major determinants of the monounsaturated fatty acid composition of vegetable oils. Mutagenesis studies were conducted to determine the structural basis for the substrate and double bond positional specificities displayed by acyl-ACP desaturases. By replacement of specific amino acid residues in a Δ⁶-palmitoyl (16:0)-ACP desaturase with their equivalents from a Δ⁹-stearoyl (18:0)-ACP desaturase, mutant enzymes were identified that have altered fatty acid chain-length specificities or that can insert double bonds into either the Δ⁶ or Δ⁹ positions of 16:0- and 18:0-ACP. Most notably, by replacement of five amino acids (A181T/A200F/S205N/L206T/G207A), the Δ⁶-16:0-ACP desaturase was converted into an enzyme that functions principally as a Δ⁹-18:0-ACP desaturase. Many of the determinants of fatty acid chain-length specificity in these mutants are found in residues that line the substrate binding channel as revealed by x-ray crystallography of the Δ⁹-18:0-ACP desaturase. The crystallographic model of the active site is also consistent with the diverged activities associated with naturally occurring variant acyl-ACP desaturases. In addition, on the basis of the active-site model, a Δ⁹-18:0-ACP desaturase was converted into an enzyme with substrate preference for 16:0-ACP by replacement of two residues (L118F/P179I). These results demonstrate the ability to rationally modify acyl-ACP desaturase activities through site-directed mutagenesis and represent a first step toward the design of acyl-ACP desaturases for the production of novel monounsaturated fatty acids in transgenic oilseed crops.     

Stearoyl-acyl-carrier-protein desaturase from higher plants is structurally unrelated to the animal and fungal homologs.

Stearoyl-acyl-carrier-protein (ACP) desaturase (EC 1.14.99.6) was purified to homogeneity from avocado mesocarp, and monospecific polyclonal antibodies directed against the protein were used to isolate full-length cDNA clones from Ricinus communis (castor) seed and Cucumis sativus (cucumber). The nucleotide sequence of the castor clone pRCD1 revealed an open reading frame of 1.2 kilobases encoding a 396-amino acid protein of 45 kDa. The cucumber clone pCSD1 encoded a homologous 396-amino acid protein with 88% amino acid identity to the castor clone. Expression of pRCD1 in Saccharomyces cerevisiae resulted in the accumulation of a functional stearoyl-ACP desaturase, demonstrating that the introduction of this single gene product was sufficient to confer soluble desaturase activity to yeast. There was no detectable identity between the deduced amino acid sequences of the castor delta 9-stearoyl-ACP desaturase and either the delta 9-stearoyl-CoA desaturase from rat or yeast or the delta 12 desaturase from Synechocystis, suggesting that these enzymes may have evolved independently. However, there was a 48-residue region of 29% amino acid sequence identity between residues 53 and 101 of the castor desaturase and the proximal border of the dehydratase region of the fatty acid synthase from yeast. Stearoyl-ACP mRNA was present at substantially higher levels in developing seeds than in leaf and root tissue, suggesting that expression of the delta 9 desaturase is developmentally regulated.     

Stearoyl-acyl carrier protein delta 9 desaturase from Ricinus communis is a diiron-oxo protein.

A gene encoding stearoyl-acyl carrier protein delta 9 desaturase (EC 1.14.99.6) from castor was expressed in Escherichia coli. The purified catalytically active enzyme contained four atoms of iron per homodimer. The desaturase was studied in two oxidation states with Mössbauer spectroscopy in applied fields up to 6.0 T. These studies show conclusively that the oxidized enzyme contains two (identical) clusters consisting of a pair of antiferromagnetically coupled (J > 60 cm-1, H = JS1.S2) Fe3+ sites. The diferric cluster exhibited absorption bands from 300 to 355 nm; addition of azide elicited a charge transfer band at 450 nm. In the presence of dithionite, the clusters were reduced to the diferrous state. Addition of stearoyl-CoA and O2 returned the clusters to the diferric state. These properties are consistent with assigning the desaturase to the class of O2-activating proteins containing diiron-oxo clusters, most notably ribonucleotide reductase and methane monooxygenase hydroxylase. Comparison of the primary structures for these three catalytically diverse proteins revealed a conserved pair of the amino acid sequence -(Asp/Glu)-Glu-Xaa-Arg-His- separated by approximately 100 amino acids. Since each of these proteins can catalyze O2-dependent cleavage of unactivated C--H bonds, we propose that these amino acid sequences represent a biological motif used for the creation of reactive catalytic intermediates. Thus, eukaryotic fatty acid desaturation may proceed via enzymatic generation of a high-valent iron-oxo species derived from the diiron cluster.     

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

We demonstrate that naturally occurring C(14) and C(16)-specific acyl-acyl carrier protein (ACP) desaturases from plants can complement the unsaturated fatty acid (UFA) auxotrophy of an Escherichia coli fabA/fadR mutant. Under the same growth conditions, C(18)-specific delta(9)-stearoyl (18:0)-ACP desaturases are unable to complement the UFA auxotrophy. This difference most likely results from the presence of sufficient substrate pools of C(14) and C(16) acyl-ACPs but a relative lack of C(18) acyl-ACP pools in E. coli to support the activities of the plant fatty acid desaturase. Based on this, a substrate-dependent selection system was devised with the use of the E. coli UFA auxotroph to isolate mutants of the castor delta(9)-18:0-ACP desaturase that display enhanced specificity for C(14) and C(16) acyl-ACPs. Using this selection system, a number of desaturase variants with altered substrate specificities were isolated from pools of randomized mutants. These included several G188L mutant isolates, which displayed a 15-fold increase in specific activity with 16:0-ACP relative to the wild-type castor delta(9)-18:0-ACP desaturase. Expression of this mutant in Arabidopsis thaliana resulted in the accumulation of unusual monounsaturated fatty acids to amounts of >25% of the seed oil. The bacterial selection system described here thus provides a rapid means of isolating variant fatty acid desaturase activities for modification of seed oil composition.     

Decoding allosteric regulation by the acyl carrier protein

Enzymes in multistep metabolic pathways utilize an array of regulatory mechanisms to maintain a delicate homeostasis [K. Magnuson, S. Jackowski, C. O. Rock, J. E. Cronan, Jr, Microbiol. Rev. 57, 522–542 (1993)]. Carrier proteins in particular play an essential role in shuttling substrates between appropriate enzymes in metabolic pathways. Although hypothesized [E. Płoskoń et al., Chem. Biol. 17, 776–785 (2010)], allosteric regulation of substrate delivery has never before been demonstrated for any acyl carrier protein (ACP)-dependent pathway. Studying these mechanisms has remained challenging due to the transient and dynamic nature of protein–protein interactions, the vast diversity of substrates, and substrate instability [K. Finzel, D. J. Lee, M. D. Burkart, ChemBioChem 16, 528–547 (2015)]. Here we demonstrate a unique communication mechanism between the ACP and partner enzymes using solution NMR spectroscopy and molecular dynamics to elucidate allostery that is dependent on fatty acid chain length. We demonstrate that partner enzymes can allosterically distinguish between chain lengths via protein–protein interactions as structural features of substrate sequestration are translated from within the ACP four-helical bundle to the protein surface, without the need for stochastic chain flipping. These results illuminate details of cargo communication by the ACP that can serve as a foundation for engineering carrier protein-dependent pathways for specific, desired products.

Maintaining steady-state homeostasis of complex cellular processes involves propagation of signals across interconnected networks of molecular assemblies, and the organization of enzymes within metabolic pathways offers a substantive example of such regulatory control (1). A thorough molecular understanding of these processes can benefit modifications of these pathways, particularly for bottom-up approaches to metabolic pathway engineering (2). Fatty acid biosynthesis (FAB) has served as an archetypical system for interconnected metabolic enzymes that encompasses a broad range of fundamental reactions essential for all domains of life (3). Here, a central acyl carrier protein (ACP) delivers the correct intermediate to one of multiple enzymes, not only providing substrates for the acetate pathways of primary and secondary metabolism but also serving to regulate the cell cycle and other global processes (4). Within fatty acid synthase (FAS), a series of enzymes catalyze the iterative condensation of two-carbon ketogenic units to a growing acyl chain, where each elongation is followed by stepwise reduction of the resulting β-ketone to a saturated fatty acid precursor (5). The ACP from Escherichia coli (AcpP) carries acyl chain lengths from 4 to 18 carbons, each of which transitions through four oxidation states, and each intermediate must be delivered to the appropriate enzyme within the 27 AcpP-binding proteins of the known interactome (6–8). How these interactions are organized cannot be easily explained by stochastic sampling of each ACP-tethered substrate by catalytic or regulatory proteins. Here we evaluate the functioning of an allosteric regulatory framework, guided by substrate identity, that regulates the protein–protein interactions required for enzyme activity within the bacterial FAS.Traditional models of protein allostery often refer to binding of effector molecules at distal binding sites which alter active site functionality (9). Rather than containing catalytic active sites, carrier proteins function upon binding to a partner enzyme, forming a protein–protein interaction. Upon binding, substrate delivery to each enzyme within FAS requires a large conformational change by the ACP, termed “chain flipping.” Here each acyl substrate, covalently tethered to a 4′-phosphopantetheine “arm” and sequestered within the ACP core, is translocated from the ACP pocket into the active site of the partner enzyme (10). Therefore, allostery described herein will refer to the classical definition which describes the phenomenon that indirect, specific protein sites are capable of modulating protein primary function (11). Here, the first site is within the ACP four-helical bundle, where cargos are sequestered, and the second site consists of the solvent exposed surface of this bundle protein, which interacts with catalytic partners.Within FAB, many partner enzymes have active sites which perform specific chain elongation or reductive chemistries and are promiscuous to fatty acid chain length. However, some enzymes demonstrate chain length specificities that control the formation of key pathway products (SI Appendix, Fig. S1) (12–17). In these instances, two conflicting and still unproven hypotheses have been proposed concerning ACP-dependent enzyme regulation. One involves stochastic sampling, where each enzyme randomly binds ACP and samples ACP-tethered content via chain flipping into the active site (18). Another involves enzyme regulation via allosteric communication with the acyl-chain bearing ACP regulating enzyme reactivity, and ultimately controlling pathway processivity, without the need for chain flipping (18–20). Here we uncover the fundamental role of allostery in the transfer of octanoic acid from AcpP to the enzyme LipB, which re-directs the fatty acid from FAB to lipoic acid biosynthesis, demonstrating that the structural changes imparted by substrate sequestration are leveraged to control enzyme activity without the need for substrate sampling by the active site.Characterization of ACPs bearing different chain lengths is inherently challenging to evaluate due to the lability of the 4′-phosphopantetheine thioester bond attachment of certain chain lengths. Leveraging a recently described methodology to enzymatically reverse acyl hydrolysis in situ (21), we acquired ¹H-¹⁵N heteronuclear single quantum coherence (HSQC) NMR spectra of every physiological acyl chain length attached to AcpP. We additionally performed molecular dynamics (MD) simulations that provide a complete view of how AcpP structure is affected by sequestration of each chain length. Using solution NMR HSQC titration experiments, we discovered that the length of the acyl chain is communicated through differences in AcpP–partner protein interactions with LipB and the cognate (C8-AcpP) and noncognate (C12-AcpP) chain lengths. With selective ¹³C labeling of the acyl chain termini, we observed that chain flipping occurs only when the specific cognate acyl chain is sequestered by AcpP. Next, we demonstrated that abrogation of this specific chain flipping could be achieved by mutation of a single surface residue discovered at the AcpP–LipB interface from MD simulations. These findings support the long-postulated hypothesis of allosteric control of ACP-dependent pathways by delineating each step in the process of acyl chain sequestration, partner-protein recognition, and chain flipping at the molecular level.     

Expression of a borage desaturase cDNA containing an N-terminal cytochrome b5 domain results in the accumulation of high levels of Δ6-desaturated fatty acids in transgenic tobacco

γ-Linolenic acid (GLA; C18:3 Δ^6,9,12) is a component of the seed oils of evening primrose (Oenothera spp.), borage (Borago officinalis L.), and some other plants. It is widely used as a dietary supplement and for treatment of various medical conditions. GLA is synthesized by a Δ⁶-fatty acid desaturase using linoleic acid (C18:2 Δ^9,12) as a substrate. To enable the production of GLA in conventional oilseeds, we have isolated a cDNA encoding the Δ⁶-fatty acid desaturase from developing seeds of borage and confirmed its function by expression in transgenic tobacco plants. Analysis of leaf lipids from a transformed plant demonstrated the accumulation of GLA and octadecatetraenoic acid (C18:4 Δ^6,9,12,15) to levels of 13.2% and 9.6% of the total fatty acids, respectively. The borage Δ⁶-fatty acid desaturase differs from other desaturase enzymes, characterized from higher plants previously, by the presence of an N-terminal domain related to cytochrome b₅.     

Vertebrate fatty acyl desaturase with Δ4 activity

Biosynthesis of the highly biologically active long-chain polyunsaturated fatty acids, arachidonic (ARA), eicosapentaenoic (EPA), and docosahexaenoic (DHA) acids, in vertebrates requires the introduction of up to three double bonds catalyzed by fatty acyl desaturases (Fad). Synthesis of ARA is achieved by Δ6 desaturation of 18∶2n - 6 to produce 18∶3n - 6 that is elongated to 20∶3n - 6 followed by Δ5 desaturation. Synthesis of EPA from 18∶3n - 3 requires the same enzymes and pathway as for ARA, but DHA synthesis reportedly requires two further elongations, a second Δ6 desaturation and a peroxisomal chain shortening step. This paper describes cDNAs, fad1 and fad2, isolated from the herbivorous, marine teleost fish (Siganus canaliculatus) with high similarity to mammalian Fad proteins. Functional characterization of the cDNAs by heterologous expression in the yeast Saccharomyces cerevisiae showed that Fad1 was a bifunctional Δ6/Δ5 Fad. Previously, functional dual specificity in vertebrates had been demonstrated for a zebrafish Danio rerio Fad and baboon Fad, so the present report suggests bifunctionality may be more widespread in vertebrates. However, Fad2 conferred on the yeast the ability to convert 22∶5n - 3 to DHA indicating that this S. canaliculatus gene encoded an enzyme having Δ4 Fad activity. This is a unique report of a Fad with Δ4 activity in any vertebrate species and indicates that there are two possible mechanisms for DHA biosynthesis, a direct route involving elongation of EPA to 22∶5n - 3 followed by Δ4 desaturation, as well as the more complicated pathway as described above.     

The ferritin Fe2 site at the diiron catalytic center controls the reaction with O2 in the rapid mineralization pathway

Oxidoreduction in ferritin protein nanocages occurs at sites that bind two Fe(II) substrate ions and O₂, releasing Fe(III)₂–O products, the biomineral precursors. Diferric peroxo intermediates form in ferritins and in the related diiron cofactor oxygenases. Cofactor iron is retained at diiron sites throughout catalysis, contrasting with ferritin. Four of the 6 active site residues are the same in ferritins and diiron oxygenases; ferritin-specific Gln¹³⁷ and variable Asp/Ser/Ala¹⁴⁰ substitute for Glu and His, respectively, in diiron cofactor active sites. To understand the selective functions of diiron substrate and diiron cofactor active site residues, we compared oxidoreductase activity in ferritin with diiron cofactor residues, Gln¹³⁷ → Glu and Asp¹⁴⁰ → His, to ferritin with natural diiron substrate site variations, Asp¹⁴⁰, Ser¹⁴⁰, or Ala¹⁴⁰. In Gln¹³⁷ → Glu ferritin, diferric peroxo intermediates were undetectable; an altered Fe(III)–O product formed, ΔA₃₅₀ = 50% of wild type. In Asp¹⁴⁰ → His ferritin, diferric peroxo intermediates were also undetectable, and Fe(II) oxidation rates decreased 40-fold. Ferritin with Asp¹⁴⁰, Ser¹⁴⁰, or Ala¹⁴⁰ formed diferric peroxo intermediates with variable kinetic stabilities and rates: t_1/2 varied 1- to 10-fold; k_cat varied approximately 2- to 3-fold. Thus, relatively small differences in diiron protein catalytic sites determine whether, and for how long, diferric peroxo intermediates form, and whether the Fe–active site bonds persist throughout the reaction cycle (diiron cofactors) or break to release Fe(III)₂–O products (diiron substrates). The results and the coding similarities for cofactor and substrate site residues—e.g., Glu/Gln and His/Asp pairs share 2 of 3 nucleotides—illustrate the potential simplicity of evolving active sites for diiron cofactors or diiron substrates.     

Divergent evolution of extreme production of variant plant monounsaturated fatty acids

Metabolic extremes provide opportunities to understand enzymatic and metabolic plasticity and biotechnological tools for novel biomaterial production. We discovered that seed oils of many Thunbergia species contain up to 92% of the unusual monounsaturated petroselinic acid (18:1Δ6), one of the highest reported levels for a single fatty acid in plants. Supporting the biosynthetic origin of petroselinic acid, we identified a Δ6-stearoyl-acyl carrier protein (18:0-ACP) desaturase from Thunbergia laurifolia, closely related to a previously identified Δ6-palmitoyl-ACP desaturase that produces sapienic acid (16:1Δ6)-rich oils in Thunbergia alata seeds. Guided by a T. laurifolia desaturase crystal structure obtained in this study, enzyme mutagenesis identified key amino acids for functional divergence of Δ6 desaturases from the archetypal Δ9-18:0-ACP desaturase and mutations that result in nonnative enzyme regiospecificity. Furthermore, we demonstrate the utility of the T. laurifolia desaturase for the production of unusual monounsaturated fatty acids in engineered plant and bacterial hosts. Through stepwise metabolic engineering, we provide evidence that divergent evolution of extreme petroselinic acid and sapienic acid production arises from biosynthetic and metabolic functional specialization and enhanced expression of specific enzymes to accommodate metabolism of atypical substrates.

Seed oils are characterized by diverse fatty acid (FA) structures (1–3). Many of these deviate from typical C16 (e.g., palmitic acid, 16:0) and C18 (e.g., oleic acid, 18:1Δ9cis) FA that occur widely in seed oils, including major commercial vegetable oils. Structurally variant FAs, often referred to as “unusual” FAs, can have chain lengths other than 16 or 18 carbon atoms, double bonds in nontypical positions or cis-trans configurations, or carbon-chain modifications, such as hydroxyl or epoxy groups (4). The study of unusual FA biochemistry has increased understanding of enzyme structural determinants of substrate binding and reaction outcomes (4). These studies have also revealed how variations in biosynthetic and metabolic pathways allow for high levels of unusual FA biosynthesis and accumulation and have also facilitated research on the evolution of biochemical diversity in plants. Unusual FAs can occur in selected species or arise throughout a genus or family, and their accumulation may be associated with compensatory mutations in enzymes beyond the initial biosynthetic enzyme (5). Moreover, genes for unusual FA biosynthesis and metabolism can be useful for biotechnological research in plants and microbes to develop vegetable oils with enhanced nutritional or industrial value (6).In a variety of taxa, metabolic variations result in “extreme” unusual FA accumulation to ≥90% of seed oil, which can account for ≤50% of seed weight (2). Examples of extreme unusual FA production include medium chain-length FAs (C8–C14) in seeds of Cuphea species and ricinoleic acid, a hydroxylated C18 monounsaturated FA in castor seeds (Ricinus communis) (7, 8). Studies of FA metabolism in Cuphea have uncovered structurally variant FatB acyl-acyl carrier protein (ACP) thioesterases and acyltransferases associated with the biosynthesis and accumulation of medium chain-length FAs. These variant thioesterases have provided primary sequences for uncovering information about amino acid residues that control their substrate recognition properties (9–11). Studies of FA metabolism in castor seeds led to the discovery that structural variations in FAD2 genes for Δ12-oleic acid desaturases can lead to variant FA modifications, including insertion of hydroxyl and epoxy groups, triple bonds, and conjugated double bonds (12–14). FA biosynthetic and metabolic genes associated with these pathways have also been used to develop new oil functionalities in existing oilseed crops.Studies of unusual monounsaturated FAs have provided a wealth of biochemical information and biotechnological utility (15). Oleic acid, the most widely occurring monounsaturated FA in plants, is synthesized by the soluble Δ9-stearoyl-ACP desaturase (16). Structural variants of this enzyme result in a number of unusual FAs, including palmitoleic acid (16:1Δ9), sapienic acid (16:1Δ6), and petroselinic acid (18:1Δ6) (15). Studies of variant acyl-ACP desaturases in combination with three-dimensional (3D) structural data (17, 18) have revealed amino acid residues that control FA chain length- and regio-specificities of this enzyme class (19). This information has facilitated biotechnological development of oilseeds with high levels of unusual monounsaturated FAs using rationally designed acyl-ACP desaturases (20). In the case of petroselinic acid, a biosynthetic pathway has been deduced that involves not only a variant Δ4-16:0-ACP desaturase but also a coevolved variant β-ketoacyl-ACP synthase I and acyl-ACP thioesterase that together generate petroselinic acid at ≤85% of the total FAs of Apiaceae and Araliaceae seeds (21–24). This highlights that unusual FA biosynthesis can result from the initial evolution of a specialized biosynthetic enzyme, followed by evolution of additional enzymes that facilitate unusual FA production and accumulation.As part of our larger efforts to characterize the evolution of unusual FA biosynthesis in plants, we examined the FA composition of seeds in diverse Thunbergia species. It has been previously shown that Thunbergia alata seeds accumulate sapienic acid (16:1Δ6cis) to ∼85% of total FAs via the activity of a Δ6-16:0-ACP desaturase (25, 26). As reported here, we discovered that seeds of several Thunbergia species accumulate petroselinic acid instead of sapienic acid. In these seeds, petroselinic acid accumulates >90% of total FAs, which is among the highest naturally occurring levels of monounsaturated FA in plant seed oils. We provide biochemical, structural, and genetic evidence to explain the divergent evolution of increased sapienic and petroselinic acid production in Thunbergia. Furthermore, we show that the petroselinic acid biosynthetic pathway in Thunbergia is distinct from that in Apiaceae and Araliaceae (21) and can guide biotechnological production of unusual monounsaturated FAs in engineered crops and microbes.     

Engineered biosynthesis of bacterial aromatic polyketides in Escherichia coli

Bacterial aromatic polyketides are important therapeutic compounds including front line antibiotics and anticancer drugs. It is one of the last remaining major classes of natural products of which the biosynthesis has not been reconstituted in the genetically superior host Escherichia coli. Here, we demonstrate the engineered biosynthesis of bacterial aromatic polyketides in E. coli by using a dissected and reassembled fungal polyketide synthase (PKS). The minimal PKS of the megasynthase PKS4 from Gibberella fujikuroi was extracted by using two approaches. The first approach yielded a stand-alone Ketosynthase (KS)_malonyl-CoA:ACP transferase (MAT) didomain and an acyl-carrier protein (ACP) domain, whereas the second approach yielded a compact PKS (PKS_WJ) that consists of KS, MAT, and ACP on a single polypeptide. Both minimal PKSs produced nonfungal polyketides cyclized via different regioselectivity, whereas the fungal-specific C2-C7 cyclization mode was not observed. The kinetic properties of the two minimal PKSs were characterized to confirm both PKSs can synthesize polyketides with similar efficiency as the parent PKS4 megasynthase. Both minimal PKSs interacted effectively with exogenous polyketide cyclases as demonstrated by the synthesis of predominantly PK8 3 or NonaSEK4 6 in the presence of a C9-C14 or a C7-C12 cyclase, respectively. When PKS_WJ and downstream tailoring enzymes were expressed in E. coli, the expected nonaketide anthraquinone SEK26 was recovered in good titer. High-cell density fermentation was performed to demonstrate the scale-up potential of the in vivo platform for the biosynthesis of bacterial polyketides. Using engineered fungal PKSs can therefore be a general approach toward the heterologous biosynthesis of bacterial aromatic polyketides in E. coli.     

An oleate 12-hydroxylase from Ricinus communis L. is a fatty acyl desaturase homolog.

Recent spectroscopic evidence implicating a binuclear iron site at the reaction center of fatty acyl desaturases suggested to us that certain fatty acyl hydroxylases may share significant amino acid sequence similarity with desaturases. To test this theory, we prepared a cDNA library from developing endosperm of the castor-oil plant (Ricinus communis L.) and obtained partial nucleotide sequences for 468 anonymous clones that were not expressed at high levels in leaves, a tissue deficient in 12-hydroxyoleic acid. This resulted in the identification of several cDNA clones encoding a polypeptide of 387 amino acids with a predicted molecular weight of 44,407 and with approximately 67% sequence homology to microsomal oleate desaturase from Arabidopsis. Expression of a full-length clone under control of the cauliflower mosaic virus 35S promoter in transgenic tobacco resulted in the accumulation of low levels of 12-hydroxyoleic acid in seeds, indicating that the clone encodes the castor oleate hydroxylase. These results suggest that fatty acyl desaturases and hydroxylases share similar reaction mechanisms and provide an example of enzyme evolution.     

Maternal low protein diet in rats programmes fatty acid desaturase activities in the offspring

Summary Numerous studies show an association between poor fetal growth and adult insulin resistance. Recent studies have shown relation between the long chain polyunsaturated fatty acid composition of skeletal muscle membranes and insulin sensitivity. More detailed analysis has indicated that the activity of Δ5 desaturase is inversely correlated to insulin resistance. The amount of docosahexaenoic acid (C22:6n3) is also thought to play a part in determining insulin sensitivity. The purpose of this study was to test the hypothesis that early growth retardation in the rat, as a result of maternal protein restriction, would lead to alterations in desaturase activities similar to those observed in human insulin resistance. There were no differences in phospholipid fatty acid composition in liver or muscle from control and low protein rats. In both muscle and liver the ratio of docosahexaenoic acid to docosapentaenoic acid was, however, reduced in low protein offspring. Direct measurement of Δ5 desaturase activity in hepatic microsomes showed a reduction (p < 0.03) in the low protein offspring which was negatively corrrelated (r = – 0.855) with fasting plasma insulin. No correlation was observed in controls. These results show that it is possible to programme the activity of key enzymes involved in the desaturation of long chain polyunsaturated fatty acids. This is possibly a mechanism linking fetal growth retardation to insulin resistance. [Diabetologia (1998) 41: 1337–1342] Received: 25 March 1998 and in final revised form: 17 June 1998     

Molecular recognition between ketosynthase and acyl carrier protein domains of the 6-deoxyerythronolide B synthase

Every polyketide synthase module has an acyl carrier protein (ACP) and a ketosynthase (KS) domain that collaborate to catalyze chain elongation. The same ACP then engages the KS domain of the next module to facilitate chain transfer. Understanding the mechanism for this orderly progress of the growing polyketide chain represents a fundamental challenge in assembly line enzymology. Using both experimental and computational approaches, the molecular basis for KS-ACP interactions in the 6-deoxyerythronolide B synthase has been decoded. Surprisingly, KS-ACP recognition is controlled at different interfaces during chain elongation versus chain transfer. In fact, chain elongation is controlled at a docking site remote from the catalytic center. Not only do our findings reveal a new principle in the modular control of polyketide antibiotic biosynthesis, they also provide a rationale for the mandatory homodimeric structure of polyketide synthases, in contrast to the monomeric nonribosomal peptide synthetases.     

Switching desaturase enzyme specificity by alternate subcellular targeting

The functionality, substrate specificity, and regiospecificity of enzymes typically evolve by the accumulation of mutations in the catalytic portion of the enzyme until new properties arise. However, emerging evidence suggests enzyme functionality can also be influenced by metabolic context. When the plastidial Arabidopsis 16:0Delta7 desaturase FAD5 (ADS3) was retargeted to the cytoplasm, regiospecificity shifted 70-fold, Delta7 to Delta9. Conversely, retargeting of two related cytoplasmic 16:0Delta9 Arabidopsis desaturases (ADS1 and ADS2) to the plastid, shifted regiospecificity approximately 25-fold, Delta9 to Delta7. All three desaturases exhibited Delta9 regiospecificity when expressed in yeast, with desaturated products found predominantly on phosphatidylcholine. Coexpression of each enzyme with cucumber monogalactosyldiacylglycerol (MGDG) synthase in yeast conferred Delta7 desaturation, with 16:1Delta7 accumulating specifically on the plastidial lipid MGDG. Positional analysis is consistent with ADS desaturation of 16:0 on MGDG. The lipid headgroup acts as a molecular switch for desaturase regiospecificity. FAD5 Delta7 regiospecificity is thus attributable to plastidial retargeting of the enzyme by addition of a transit peptide to a cytoplasmic Delta9 desaturase rather than the numerous sequence differences within the catalytic portion of ADS enzymes. The MGDG-dependent desaturase activity enabled plants to synthesize 16:1Delta7 and its abundant metabolite, 16:3Delta(7,10,13). Bioinformatics analysis of the Arabidopsis genome identified 239 protein families that contain members predicted to reside in different subcellular compartments, suggesting alternative targeting is widespread. Alternative targeting of bifunctional or multifunctional enzymes can exploit eukaryotic subcellular organization to create metabolic diversity by permitting isozymes to interact with different substrates and thus create different products in alternate compartments.     

Arginine residues at internal positions in a protein are always charged

Many functionally essential ionizable groups are buried in the hydrophobic interior of proteins. A systematic study of Lys, Asp, and Glu residues at 25 internal positions in staphylococcal nuclease showed that their pK_a values can be highly anomalous, some shifted by as many as 5.7 pH units relative to normal pK_a values in water. Here we show that, in contrast, Arg residues at the same internal positions exhibit no detectable shifts in pK_a; they are all charged at pH ≤ 10. Twenty-three of these 25 variants with Arg are folded at both pH 7 and 10. The mean decrease in thermodynamic stability from substitution with Arg was 6.2 kcal/mol at this pH, comparable to that for substitution with Lys, Asp, or Glu at pH 7. The physical basis behind the remarkable ability of Arg residues to remain protonated in environments otherwise incompatible with charges is suggested by crystal structures of three variants showing how the guanidinium moiety of the Arg side chain is effectively neutralized through multiple hydrogen bonds to protein polar atoms and to site-bound water molecules. The length of the Arg side chain, and slight deformations of the protein, facilitate placement of the guanidinium moieties near polar groups or bulk water. This unique capacity of Arg side chains to retain their charge in dehydrated environments likely contributes toward the important functional roles of internal Arg residues in situations where a charge is needed in the interior of a protein, in a lipid bilayer, or in similarly hydrophobic environments.     

Unexpected tautomeric equilibria of the carbanion-enamine intermediate in pyruvate oxidase highlight unrecognized chemical versatility of thiamin

Thiamin diphosphate, the vitamin B1 coenzyme, plays critical roles in fundamental metabolic pathways that require acyl carbanion equivalents. Studies on chemical models and enzymes had suggested that these carbanions are resonance-stabilized as enamines. A crystal structure of this intermediate in pyruvate oxidase at 1.1 Å resolution now challenges this paradigm by revealing that the enamine does not accumulate. Instead, the intermediate samples between the ketone and the carbanion both interlocked in a tautomeric equilibrium. Formation of the keto tautomer is associated with a loss of aromaticity of the cofactor. The alternate confinement of electrons to neighboring atoms rather than π-conjugation seems to be of importance for the enzyme-catalyzed, redox-coupled acyl transfer to phosphate, which requires a dramatic inversion of polarity of the reacting substrate carbon in two subsequent catalytic steps. The ability to oscillate between a nucleophilic (carbanion) and an electrophilic (ketone) substrate center highlights a hitherto unrecognized versatility of the thiamin cofactor. It remains to be studied whether formation of the keto tautomer is a general feature of all thiamin enzymes, as it could provide for stable storage of the carbanion state, or whether this feature represents a specific trait of thiamin oxidases. In addition, the protonation state of the two-electron reduced flavin cofactor can be fully assigned, demonstrating the power of high-resolution cryocrystallography for elucidation of enzymatic mechanisms.