Unusual evolution of 11β- and 17β-hydroxysteroid and retinol dehydrogenases

11β-hydroxysteroid dehydrogenases regulate glucocorticoid concentrations and 17β-hydroxysteroid dehydrogenases regulate estrogen and androgen concentrations in mammals. Phylogenetic analysis of the sequences from two 11β-hydroxysteroid dehydrogenases and four mammalian 17β-hydroxysteroid dehydrogenases indicates unusual evolution in these enzymes. Type 1 11β- and 17β-hydroxysteroid dehydrogenases are on the same branch; Type 2 enzymes cluster on another branch with β-hydroxybutyrate dehydrogenase, 11-cis retinol dehydrogenase and retinol dehydrogenase; Type 3 17β-hydroxysteroid dehydrogenase is on a third branch; while the pig dehydrogenase clusters with a yeast multifunctional enzyme on a fourth branch. Pig 17β-hydroxysteroid dehydrogenase appears to have evolved independently from the other three 17β-hydroxysteroid dehydrogenase dehydrogenases; in which case, the evolution of 17β-hydroxysteroid dehydrogenase activity is an example of functional convergence. The phylogeny also suggests that independent evolution of specificity toward C11 substituents on glucocorticoids and C17 substituents on androgens and estrogens has occurred in Types 1 and 2 11β- and 17β-hydroxysteroid dehydrogenases.     

Sortase A as a novel molecular "stapler" for sequence-specific protein conjugation

The Sortase family of transpeptidase enzymes catalyzes sequence-specific ligation of proteins to the cell wall of Gram-positive bacteria. Here, we describe the application of recombinant Staphylococcus aureus Sortase A to attach a tagged model protein substrate (green fluorescent protein) to polystyrene beads chemically modified with either alkylamine or the in vivo Sortase A ligand, Gly-Gly-Gly, on their surfaces. Furthermore, we show that Sortase A can be used to sequence-specifically ligate eGFP to amino-terminated poly(ethylene glycol) and to generate protein oligomers and cyclized monomers using suitably tagged eGFP. We find that an alkylamine can substitute for the natural Gly3 substrate, which suggests the possibility of using the enzyme in materials applications. The highly specific and mild Sortase A-catalyzed reaction, based on small recognition tags unlikely to interfere with protein expression, thus represents a useful addition to the protein immobilization and modification tool kit.     

The binding mechanism,multiple binding modes,and allosteric regulation of Staphylococcus aureus Sortase A probed by molecular dynamics simulations

Sortase enzymes are vitally important for the virulence of gram‐positive bacteria as they play a key role in the attachment of surface proteins to the cell wall. These enzymes recognize a specific sorting sequence in proteins destined to be displayed on the surface of the bacteria and catalyze the transpeptidation reaction that links it to a cell wall precursor molecule. Because of their role in establishing pathogenicity, and in light of the recent rise of antibiotic‐resistant bacterial strains, sortase enzymes are novel drug targets. Here, we present a study of the prototypical sortase protein Staphylococcus aureus Sortase A (SrtA). Both conventional and accelerated molecular dynamics simulations of S. aureus SrtA in its apo state and when bound to an LPATG sorting signal (SS) were performed. Results support a binding mechanism that may be characterized as conformational selection followed by induced fit. Additionally, the SS was found to adopt multiple metastable states, thus resolving discrepancies between binding conformations in previously reported experimental structures. Finally, correlation analysis reveals that the SS actively affects allosteric pathways throughout the protein that connect the first and the second substrate binding sites, which are proposed to be located on opposing faces of the protein. Overall, these calculations shed new light on the role of dynamics in the binding mechanism and function of sortase enzymes.     

Eicosapentaenoic acid metabolism by cytochrome P450 enzymes of the CYP2C subfamily

CYP2C enzymes epoxidize arachidonic acid (AA) to metabolites involved in the regulation of vascular and renal function. We tested the hypothesis that eicosapentaenoic acid (EPA), a n-3 polyunsaturated fatty acid, may serve as an alternative substrate. Human CYP2C8 and CYP2C9, as well as rat CYP2C11 and CYP2C23, were co-expressed with NADPH-CYP reductase in a baculovirus/insect cell system. The recombinant enzymes showed high EPA and AA epoxygenase activities and the catalytic efficiencies were almost equal comparing the two substrates. The 17,18-double bond was the preferred site of EPA epoxidation by CYPs 2C8, 2C11, and 2C23. 17(R),18(S)-Epoxyeicosatetraenoic acid was produced with an optical purity of about 70% by CYPs 2C9, 2C11, and 2C23 whereas CYP2C8 showed the opposite enantioselectivity. These results demonstrate that EPA is an efficient substrate of CYP2C enzymes and suggest that n-3 PUFA-rich diets may shift the CYP2C-dependent generation of physiologically active eicosanoids from AA- to EPA-derived metabolites.     

Histochemical distribution of delta5-3beta- and 17beta-hydroxysteroid dehydrogenases in hamster trophoblast.

The histochemical distribution of delta5-3beta- and 17beta-hydroxysteroid dehydrogenases was demonstrated in hamster trophoblast between Days 8 and 15 of pregnancy. The delta5-3beta-hydroxysteroid dehydrogenase activity in the ectoplacental trophoblast of 8-day embryos was demonstrated by use of delta5-pregnenolone and dehydroepiandrosterone as substrates; between Days 11 and 15, activity was demonstrated in the trophoblastic giant cells of the placenta and in the intra-arterial trophoblast cells when delta5-pregnenolone was the substrate. Between Days 11 and 15, 17beta-hydroxysteroid activity was present in the spongiotrophoblast, labyrinth, placental giant cells and intra-arterial trophoblast cells, as shown by use of testosterone and oestradiol as substrates. Both enzymes were demonstrated in ectopic trophoblast cells, indicating that these activities are autonomous.     

Human 17beta-hydroxysteroid dehydrogenases types 1, 2, and 3 catalyze bi-directional equilibrium reactions, rather than unidirectional metabolism, in HEK-293 cells

Human 17beta-hydroxysteroid dehydrogenases (17betaHSDs) catalyze the interconversion of weak and potent androgen and estrogen pairs. Although the reactions using purified enzymes can be driven in either direction, these enzymes appear to function unidirectionally in intact cells: only reductive reactions for 17betaHSD1 and 17beta HSD3 and only oxidative reactions for 17betaHSD2. We show that, after exhaustive incubations with either 17beta-hydroxy- or 17-ketosteroid, the medium for HEK-293 cells expressing 17betaHSD1 or 17betaHSD3 contains a 92:8 ratio of reduced:oxidized steroid. Similarly, 17betaHSD2 yields a >95:5 ratio of oxidized:reduced steroids for both androgens and estrogens. Dual-isotope kinetic measurements show that the rates of the forward and reverse reactions are identical at these functional equilibrium states in intact cells for all three 17betaHSD isoforms, and these rates are much faster than those estimated from single-isotope flux studies. Mutation L36D converts 17betaHSD1 to an oxidative enzyme in intact cells, reversing the equilibrium distribution of estradiol:estrone to 5:95; however, the rates of the forward and reverse reactions at equilibrium are equal and comparable to those of the wild-type enzymes. The co-expression of 17betaHSD2 paradoxically increases the potency of estrone in transactivation assays, demonstrating the physiological relevance of "backwards" metabolism to estradiol. We conclude that 17betaHSD types 1, 2, and 3 catalyze both oxidative and reductive reactions in HEK-293 cells at intrinsic rates that are much faster than those estimated from single-isotope studies. These 17betaHSD isoforms do not drive steroid flux in one direction but rather may achieve functional equilibria in intact cells, reflecting thermodynamically driven steroid distributions.     

Therapeutic effect of (Z)-3-(2,5-dimethoxyphenyl)-2-(4-methoxyphenyl) acrylonitrile (DMMA) against Staphylococcus aureus infection in a murine model

Sortase enzymes belong to a family of transpeptidases found in Gram-positive bacteria. Sortase is responsible for the reaction that anchors surface protein virulence factors to the peptidoglycan cell wall of the bacteria. The compound (Z)-3-(2,5-dimethoxyphenyl)-2-(4-methoxyphenyl) acrylonitrile (DMMA) has previously been reported as a novel sortase inhibitor in vitro, but the in vivo effects of DMMA have not been studied. Here, we evaluated the in vivo effects of DMMA against infection by wild-type and sortase A- and/or sortase B-deficient Staphylococcus aureus in Balb/c mice. With DMMA treatment, survival rates increased and kidney and joint infection rates decreased (p < 0.01) in a dose-dependent manner. The rate of kidney infection was significantly reduced in the mice treated with sortase A knock-out S. aureus (p < 0.01). These results indicate that by acting as a potent inhibitor of sortase A and moderate inhibitor of sortase B, DMMA can decrease kidney and joint infection rates and reduce mortality in mice infected with S. aureus. These findings suggest that DMMA is a promising therapeutic compound against Gram-positive bacteria.     

Ultrafiltration,a useful method for isolation of intermediates in native chemical ligation exemplified with the total synthesis of Sortase AΔN59

In this paper, ultrafiltration was employed to facilitate the isolation of intermediates in native chemical ligation. Depending on the molecular weight cutoff of the membrane used, molecules with different sizes could be purified, separated, or concentrated by the ultrafiltration process. Total chemical synthesis of the polypeptide chain of the enzyme Sortase A_ΔN59 was used as an example of the application of ultrafiltration in chemical protein synthesis. Sortase A is a ligase that catalyzes transpeptidation reactions between proteins that have C‐terminal LPXTG recognition sequence and Gly5‐ on the peptidoglycan of bacterial cell walls [3]. Ultrafiltration technique facilitated synthesis of Sortase A_ΔN59 and was a promising tool in isolation of intermediates in native chemical ligation. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Androgen and estrogen treatments alter steady state messengers RNA (mRNA) levels of testicular steroidogenic enzymes in the rainbow trout, Oncorhynchus mykiss

Recent investigations have shown that estrogens have profound inhibitory effects on steroidogenic enzyme gene expressions before and after testicular differentiation in the rainbow trout, Oncorhynchus mykiss. This present study bring new data on juvenile rainbow trout treated with estrogens and androgens. Following a 8 days oral treatment of juvenile male with 17alpha-ethynyl-estradiol (EE2, 20 mg/kg diet) or 11beta-hydroxyandrostenedione (11betaOHDelta4, 10 mg/kg diet), we observed a fast and marked decrease of steady-state mRNA levels for 3betaHSD, P450scc, P450c17, and P450c11 enzymes in the testis. After completion of these treatments, mRNA levels of these enzymes remained low in EE2 treated males whereas in 11betaOHDelta4 treated males they recovered their initial levels in 8 days. This demonstrate that both androgen and estrogen treatments have profound effects on testicular steroidogenesis by decreasing steroid enzymes steady-state mRNA. After in vitro incubation of testicular explants with 17beta-estradiol (E2, 600 ng/ml of medium), we also observed a decrease of mRNA levels for 3betaHSD and P450c11. This suggest that estrogens effects could be triggered, at least to some extend, directly on the testis. We also investigated the hypothesis of a negative feedback of steroids on follicle stimulating hormone (FSH) secretion, but FSH plasmatic levels in treated fish did not showed any significant decrease. This demonstrate that FSH is not implied in this steroids inhibition of steroidogenic enzymes gene expression.     

A broad-host-range vibriophage, KVP40, isolated from sea water.

A broad-host-range vibriophage, KVP40, was isolated from sea water by using Vibrio parahaemolyticus 1010 (EB101) as the indicator host. The host range of KVP40 extended over at least 8 Vibrio and 1 Photobacterium species. KVP40 was a large tailed phage containing double-stranded DNA and belonged to Ackermann's morphotype A2. KVP40 DNA was cleaved by 11 different type II restriction endonucleases including EcoRI and HindIII, but not by 17 other enzymes including BamHI, KpnI and SalI.     

Characteristics of human types 1, 2 and 3 17β-hydroxysteroid dehydrogenase activities: Oxidation/reduction and inhibition

Following transfection of types 1, 2 and 3 17β-hydroxysteroid dehydrogenase (17β-HSD) cDNAs into transformed embryonal kidney (293) cells, we have characterized the selective directional and inhibitory characteristics of these activities. While homogenates of transfected cells could catalyze interconversion of the substrate and product, in agreement with the general belief on the activity of these enzymes, the same activities measured in intact cells, in order to better reflect the physiological conditions, showed an unidirectional reaction. Types 1 and 3 17β-HSD catalyzed the reduction of estrone to estradiol and 4-androstenedione to testosterone, respectively, while type 2 17β-HSD catalyzed the oxidative transformation of both testosterone and 17β-estradiol to 4-androstenedione and estrone, respectively. In addition, types 1, 2 and 3 17β-HSD activities showed different pH optima. While types 1 and 3 showed pH optimum values centered at around 5 and 6, respectively, type 2 17β-HSD activity, which preferentially catalyzes the oxidation reaction, has higher activity at an alkaline pH (8–10). Differences in the optimum incubation temperatures were also observed: type 1 17β-HSD shows a relatively high temperature tolerance (55°C). In contrast, type 2 and 3 functioned best at 37°C. Types 1, 2 and 3 17β-HSD activities could be also differentiated by their sensitivity toward various specific inhibitors: type 1 was potently inhibited by an estradiol derivative containing a bromo/or iodopropyl group at position 16. On the other hand a derivative of estrone containing a spiro-γ-lactone at position 17 showed a potent inhibitory effect on type 2 17β-HSD, whereas type 3 was strongly inhibited by 1,4-androstadiene-1,6,17-trione.     

The measurement of 4-androstene-3, 11, 17-trione (11-oxo-androstenedione) by radioimmunoassay in human plasma

A radioimmunoassay (RIA) method is described for the determination of 4-androstene-3, 11, 17-trione (11-oxo-androstenedione) in human plasma. 4-androstene-3, 11, 17-trione 3-(0-carboxymethyl) oxime-bovine serum albumin conjugate was used to generate highly specific antiserum in rabbits. Cross reactivities of several other steroids with the antiserum were less than 4%. [1,2-3H] 4-androstene-3, 11, 17-trione was synthesized from [1,2-3H] 17 alpha, 21-dihydroxy-4-pregnene-3, 11, 20-trione. The intra- and interassay variation was 7.3% and 9.8%, respectively. The mean serum 4-androstene-3, 11, 17-trione level for healthy young subjects was 2.37 +/- 0.56 nM (X +/- SD) in males and 3.16 +/- 0.43 nM in females at 8 a.m. During the night, there was a marked decrease in serum level, giving at 11 p.m. 0.87 +/- 0.33 and 1.15 +/- 0.52 nM, respectively. During ACTH stimulation tests, 4-androstene-3, 11, 17-trione increased from 1.81 +/- 0.58 to 2.32 +/- 0.69 nM, while in dexamethasone suppression tests a decrease from 3.20 +/- 0.03 nM was seen. In contrast, HCG administration on 3 consecutive days did not influence plasma concentrations of 4-androstene-3, 11, 17-trione.     

The stereochemistry of trans-4-hydroxynonenal-derived exocyclic 1,N2-2'-deoxyguanosine adducts modulates formation of interstrand cross-links in the 5'-CpG-3' sequence

The trans-4-hydroxynonenal (HNE)-derived exocyclic 1, N(2)-dG adduct with (6S,8R,11S) stereochemistry forms interstrand N(2)-dG-N(2)-dG cross-links in the 5'-CpG-3' DNA sequence context, but the corresponding adduct possessing (6R,8S,11R) stereochemistry does not. Both exist primarily as diastereomeric cyclic hemiacetals when placed into duplex DNA [Huang, H., Wang, H., Qi, N., Kozekova, A., Rizzo, C. J., and Stone, M. P. (2008) J. Am. Chem. Soc. 130, 10898-10906]. To explore the structural basis for this difference, the HNE-derived diastereomeric (6S,8R,11S) and (6R,8S,11R) cyclic hemiacetals were examined with respect to conformation when incorporated into 5'-d(GCTAGC XAGTCC)-3' x 5'-d(GGACTCGCTAGC)-3', containing the 5'-CpX-3' sequence [X = (6S,8R,11S)- or (6R,8S,11R)-HNE-dG]. At neutral pH, both adducts exhibited minimal structural perturbations to the DNA duplex that were localized to the site of the adduction at X(7) x C(18) and its neighboring base pair, A(8) x T(17). Both the (6S,8R,11S) and (6R,8S,11R) cyclic hemiacetals were located within the minor groove of the duplex. However, the respective orientations of the two cyclic hemiacetals within the minor groove were dependent upon (6S) versus (6R) stereochemistry. The (6S,8R,11S) cyclic hemiacetal was oriented in the 5'-direction, while the (6R,8S,11R) cyclic hemiacetal was oriented in the 3'-direction. These cyclic hemiacetals effectively mask the reactive aldehydes necessary for initiation of interstrand cross-link formation. From the refined structures of the two cyclic hemiacetals, the conformations of the corresponding diastereomeric aldehydes were predicted, using molecular mechanics calculations. Potential energy minimizations of the duplexes containing the two diastereomeric aldehydes predicted that the (6S,8R,11S) aldehyde was oriented in the 5'-direction while the (6R,8S,11R) aldehyde was oriented in the 3'-direction. These stereochemical differences in orientation suggest a kinetic basis that explains, in part, why the (6S,8R,11S) stereoisomer forms interchain cross-links in the 5'-CpG-3' sequence whereas the (6R,8S,11R) stereoisomer does not.     

Functional Analysis of the AKR4C Subfamily of Arabidopsis thaliana: Model Structures,Substrate Specificity,Acrolein Toxicity,and Responses to Light and [CO2]

In Arabidopsis thaliana, the aldo-keto reductase (AKR) family includes four enzymes (The AKR4C subfamily: AKR4C8, AKR4C9, AKR4C10, and AKR4C11). AKR4C8 and AKR4C9 might detoxify sugar-derived reactive carbonyls (RCs). We analyzed AKR4C10 and AKR4C11, and compared the enzymatic functions of the four enzymes. Modeling of protein structures based on the known structure of AKR4C9 found an (α/β)₈-barrel motif in all four enzymes. Loop structures (A, B, and C) which determine substrate specificity, differed among the four. Both AKR4C10 and AKR4C11 reduced methylglyoxal. AKR4C10 reduced triose phosphates, dihydroxyacetone phosphate (DHAP), and glyceraldehydes 3-phosphate (GAP), the most efficiently of all the AKR4Cs. Acrolein, a lipid-derived RC, inactivated the four enzymes to different degrees. Expression of the AKR4C genes was induced under high-[CO₂] and high light, when photosynthesis was enhanced and photosynthates accumulated in the cells. These results suggest that the AKR4C subfamily contributes to the detoxification of sugar-derived RCs in plants.     

Synthesis, spectral analysis and in vitro microbiological evaluation of novel ethyl 4-(naphthalen-2-yl)-2-oxo-6-arylcyclohex-3-enecarboxylates and 4,5-dihydro-6-(napthalen-2-yl)-4-aryl-2H-indazol-3-ols

A series of ethyl 4-(naphthalen-2-yl)-2-oxo-6-arylcyclohex-3-enecarboxylates 8-14 and 4,5-dihydro-6-(naphthalen-2-yl)-4-aryl-2H-indazol-3-ols 15-21 were synthesised and characterised by their spectroscopic data. In vitro microbiological evaluations were carried out for all the newly synthesised compounds 8-21 against clinically isolated bacterial and fungal strains. Compounds 9, 12 and 20 against Staphylococcus aureus, 10, 12, 20 against β-haemolytic streptococcus, 11, 17 against Bacillus subtilis, 12, 16 and 20 against Vibreo cholerae, 13, 16 against Escherichia coli, 13, 16, 18, 19 against Salmonella typhii, 12, 18 against Shigella flexneri, 10 against Salmonella typhii, 10, 13, 17, 18 against Aspergillus flavus, 12, 17, 21 against Aspergillus niger, 12, 15, 17, 18, 20 against Mucor, Rhizopus and Microsporeum gypsuem exhibit potent antimicrobial activity.     

Molecular Modelling reveals the inhibition mechanism and structure–activity relationship of curcumin and its analogues to Staphylococcal aureus Sortase A

Previous studies found that the activity of Sortase A, a bacterial surface protein from Staphylococcus aureus, was inhibited by curcumin and its analogues. To explore this inhibitory mechanism, Sortase A and its inhibitors in complex systems were studied by molecular docking, molecular modelling, binding energy decomposition calculation and steered molecular dynamics simulations. Energy decomposition analysis indicated that PRO-163, LEU-169, GLN-172, ILE-182 and ILE-199 are key residues in Sortase A-inhibitor complexes. Furthermore, interactions between the methoxyl group on the benzene ring in the conjugated molecule (curcumin, demethoxycurcumin, bisdemethoxycurcumin) and VAL-168, LEU-169 and GLN-172 induce the inhibitory activity based on the energy decomposition and distance analyses between the whole residues and inhibitors. However, because of its coiled structure, the non-conjugated molecule, tetrahydrocurcumin, with key residues in the binding sites of Sortase A, interacted weakly with SrtA, leading to the loss of inhibitory activity. Based on these results, the methoxyl group on the benzene ring in the conjugated molecule largely influenced the inhibitory activity of the Sortase A inhibitors.     

The role of Streptococcus pneumoniae sortase A in colonisation and pathogenesis

Sortase enzymes are found throughout Gram-positive bacteria and are responsible for the covalent attachment of specific proteins to the cell wall. Through the anchoring of these cell wall proteins, sortase enzymes are important in the ability of several Gram-positive pathogens to cause disease. Previously, deletion of srtA from Streptococcus pneumoniae (the pneumococcus) was shown to disturb the localisation of surface proteins, and decrease bacterial adherence to human pharyngeal cells in vitro. Here we present data demonstrating, for the first time, a role for srtA as a pneumococcal fitness factor in experimental models of pneumonia and bacteraemia. In addition, srtA contributed to nasopharyngeal colonisation in vivo. Furthermore, we find that the contribution of srtA varied between two pneumococcal strains. We show that the known role of srtA in adherence in vitro is dependent on capsule expression, the role of SrtA in adherence to human cells only being apparent in the absence of the pneumococcal capsule. The srtA gene was detected by PCR in all 82 clinical isolates examined and sequencing of the gene from 20 strains showed srtA to be highly conserved. The ubiquitous distribution of srtA was in contrast to the other known pneumococcal sortase genes, srtB, C and D, which were found in only 14 of the 82 tested strains (17%).     

Covalent attachment of proteins to solid supports and surfaces via Sortase-mediated ligation

Background

There is growing interest in the attachment of proteins to solid supports for the development of supported catalysts, affinity matrices, and micro devices as well as for the development of planar and bead based protein arrays for multiplexed assays of protein concentration, interactions, and activity. A critical requirement for these applications is the generation of a stable linkage between the solid support and the immobilized, but still functional, protein.

Methodology

Solid supports including crosslinked polymer beads, beaded agarose, and planar glass surfaces, were modified to present an oligoglycine motif to solution. A range of proteins were ligated to the various surfaces using the Sortase A enzyme of S. aureus. Reactions were carried out in aqueous buffer conditions at room temperature for times between one and twelve hours.

Conclusions

The Sortase A transpeptidase of S. aureus provides a general, robust, and gentle approach to the selective covalent immobilization of proteins on three very different solid supports. The proteins remain functional and accessible to solution. Sortase mediated ligation is therefore a straightforward methodology for the preparation of solid supported enzymes and bead based assays, as well as the modification of planar surfaces for microanalytical devices and protein arrays.     

Primary structure, regioselectivity, and evolution of the membrane-bound fatty acid desaturases of Claviceps purpurea

Two cDNAs with sequence similarity to fatty acid desaturase genes were isolated from the phytopathogenic fungus, Claviceps purpurea. The predicted amino acid sequences of the corresponding genes, named CpDes12 and CpDesX, share 87% identity. Phylogenetic analysis indicates that CpDes12 and CpDesX arose by gene duplication of an ancestral Delta(12)-desaturase gene after the divergence of Nectriaceae and Clavicipitaceae. Functional expression of CpDes12 and CpDesX in yeast (Saccharomyces cerevisiae) indicated that CpDes12 is primarily a "Delta(12)"-desaturase, whereas CpDesX is a novel desaturase catalyzing "Delta(12)," "Delta(15)," and "omega(3)" types of desaturation with omega(3) activity predominating. CpDesX sequentially desaturates both 16:1-9c and 18:1-9c to give 16:3-9c,12c,15c and 18:3-9c,12c,15c, respectively. In addition, it could also act as an omega(3)-desaturase converting omega(6)-polyunsaturates 18:3-6c,9c,12c, 20:3-8c,11c,14c, and 20:4-5c,8c,11c,14c to their omega(3) counterparts 18:4-6c,9c,12c,15c, 20:4-8c,11c,14c,17c, and 20:5-5c,8c,11c,14c,17c, respectively. By using reciprocal site-directed mutagenesis, we demonstrated that two residues (isoleucine at 152 and alanine at 206) are critical in defining the catalytic specificity of these enzymes and the C-terminal amino acid sequence (residues 302-477) was also found to be important. These data provide insights into the nature of regioselectivity in membrane-bound fatty acid desaturases and the relevant structural determinants. The authors suggest that the regios-electivity of such enzymes may be best understood by considering the relative importance of more than one regioselective preference. In this view, CpDesX is designated as anu + 3(omega(3)) desaturase, which primarily references an existing double bond (nu + 3 regioselectivity) and secondarily shows preference for omega(3) desaturation.