An imine reductase that captures reactive intermediates in the biosynthesis of the indolocarbazole reductasporine.

Imine reductases (IREDs) and reductive aminases have been used in the synthesis of chiral amine products for drug manufacturing; however, little is known about their biological contexts. Here we employ structural studies and site-directed mutagenesis to interrogate the mechanism of the IRED RedE from the biosynthetic pathway to the indolocarbazole natural product reductasporine. Cocrystal structures with the substrate-mimic arcyriaflavin A reveal an extended active site cleft capable of binding two indolocarbazole molecules. Site-directed mutagenesis of a conserved aspartate in the primary binding site reveals a new role for this residue in anchoring the substrate above the NADPH cofactor. Variants targeting the secondary binding site greatly reduce catalytic efficiency, while accumulating oxidized side-products. As indolocarbazole biosynthetic intermediates are susceptible to spontaneous oxidation, we propose the secondary site acts to protect against autooxidation, and the primary site drives catalysis through precise substrate orientation and desolvation effects. The structure of RedE with its extended active site can be the starting point as a new scaffold for engineering IREDs and reductive aminases to intercept large substrates relevant to industrial applications.

Evidence for cytochrome P450 3A4-mediated metabolic activation of SCO-267.

SCO-267 is a potent G-protein-coupled receptor 40 agonist that is undergoing clinical development for the treatment of type 2 diabetes mellitus. The current work was undertaken to investigate the bioactivation potential of SCO-267 in vitro and in vivo. Three SCO-267-derived glutathione (GSH) conjugates (M1-M3) were found both in rat and human liver microsomal incubations supplemented with GSH and nicotinamide adenine dinucleotide phosphate. Two GSH conjugates (M1-M2) together with two N-acetyl-cysteine conjugates (M4-M5) were detected in the bile of rats receiving SCO-267 at 10 mg/kg. The identified conjugates suggested the generation of quinone-imine and ortho-quinone intermediates. CYP3A4 was demonstrated to primarily catalyze the bioactivation of SCO-267. In addition, SCO-267 concentration-, time-, and NADPH-dependently inactivated CYP3A in human liver microsomes using testosterone as a probe substrate, along with KI and kinact values of 4.91 µM and 0.036 min-1 , respectively. Ketoconazole (a competitive inhibitor of CYP3A) displayed no significant protective effect on SCO-267-induced CYP3A inactivation. However, inclusion of GSH showed significant protection. These findings revealed that SCO-267 undergoes a facile CYP3A4-catalyzed bioactivation with the generation of quinone-imine and ortho-quinone intermediates, which were assumed to be involved in SCO-267 induced CYP3A inactivation. These findings provide further insight into the bioactivation pathways involved in the generation of reactive, potentially toxic metabolites of SCO-267. Further studies are needed to evaluate the influence of SCO-267 metabolism on the safety of this drug in vivo.

Asymmetric Synthesis of Sterically Hindered 1-Substituted Tetrahydro-ß-carbolines Enabled by Imine Reductase: Enzyme Discovery, Protein Engineering, and Reaction Development.

We report the discovery of a new imine reductase (IRED), named AtIRED, by genome mining. Site-saturation mutagenesis on AtIRED generated two single mutants M118'L and P120'G and the double mutant M118'L/P120'G with improved specific activity toward sterically hindered 1-substituted dihydro-ß-carbolines. The synthetic potential of these engineered IREDs was showcased by the preparative-scale synthesis of nine chiral 1-substituted tetrahydro-ß-carbolines (THßCs), including (S)-1-t-butyl-THßC and (S)-1-t-pentyl-THßC, in 30-87% isolated yields with excellent optical purities (98-99% ee).

Evidence for Metabolic Activation of Omeprazole In Vitro and In Vivo.

Omeprazole (OPZ) is a proton pump inhibitor commonly used for the treatment of gastric acid hypersecretion. Studies have revealed that use of OPZ can induce hepatotoxicity, but the mechanisms by which it induces liver injury are unclear. This study aimed to identify reactive metabolites of OPZ, determine the pathways of the metabolic activation, and define the correlation of the bioactivation with OPZ cytotoxicity. Quinone imine-derived glutathione (GSH), N-acetylcysteine (NAC), and cysteine (Cys) conjugates were detected in OPZ-fortified rat and human liver microsomal incubations captured with GSH, NAC, or Cys. The same GSH conjugates were detected in bile of rats and cultured liver primary cells after exposure to OPZ. Similarly, the same NAC conjugates were detected in urine of OPZ-treated rats. The resulting quinone imine was found to react with Cys residues of hepatic protein. CYP3A4 dominated the metabolic activation of OPZ. Exposure to OPZ resulted in decreased cell survival in cultured primary hepatocytes. Pretreatment with ketoconazole attenuated the susceptibility of hepatocytes to the cytotoxicity of OPZ.

Kinetic and structural studies of the reaction of Escherichia coli dihydrodipicolinate synthase with (S)-2-bromopropionate.

Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step in the lysine-biosynthetic pathway converting pyruvate and L-aspartate-ß-semialdehyde to dihydrodipicolinate. Kinetic studies indicate that the pyruvate analog (S)-2-bromopropionate inactivates the enzyme in a pseudo-first-order process. An initial velocity pattern indicates that (S)-2-bromopropionate is a competitive inhibitor versus pyruvate, with an inhibition constant of about 8 mM. Crystals of DHDPS complexed with (S)-2-bromopropionate formed in a solution consisting of 50 mM HEPES pH 7.5, 18% polyethylene glycol 3350, 8 mM spermidine, 0.2 M sodium tartrate and 5.0 mg ml-1 DHDPS. The crystals diffracted to 2.15 Šresolution and belonged to space group P1. The crystal structure confirms the displacement of bromine and the formation of a covalent attachment between propionate and Lys161 at the active site of the enzyme. Lys161 is the active-site nucleophile that attacks the carbonyl C atom of pyruvate and subsequently generates an imine adduct in the first half-reaction of the ping-pong enzymatic reaction. A comparison of the crystal structures of DHDPS complexed with pyruvate or (S)-2-bromopropionate indicates the covalent adduct formed from (S)-2-bromopropionate leads to a rotation of about 180° of the ß-δ C atoms of Lys61 that aligns the covalently bound propionate fairly closely with the imine adduct formed with pyruvate.

miR-126-3p-loaded small extracellular vesicles secreted by urine-derived stem cells released from a phototriggered imine crosslink hydrogel could enhance vaginal epithelization after vaginoplasty.

BACKGROUND: Due to the large area and deep width of the artificial neovagina after vaginoplasty, it takes a considerable amount of time to achieve complete epithelization of the neovagina. Currently, the clinical therapies for vaginal epithelization after vaginoplasty are still dissatisfactory. Recent studies showed that small extracellular vesicles (sEVs) derived from stem cells could accelerate wound epithelization. The sustained release of sEVs from optimized hydrogels may be a promising strategy to accelerate vaginal epithelization after vaginoplasty. METHODS: The efficacy of phototriggered imine crosslink hydrogels (piGEL) containing sEVs derived from human urine-derived stem cells (hUSC-sEVs, piGEL-sEVs) on vaginal mucosa defects in rabbits was assessed by wound closure rates, histological analysis and immunofluorescence staining analysis. Cell counting kit-8, 5-ethynyl-2'-deoxyuridine and scratch wound assays were performed to assess the effects of hUSC-sEVs on the proliferation and migration ability of vaginal epithelial cells (VK2/E6E7). Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out to test the expression of epithelial differentiation markers in VK2 cells. Moreover, a microRNA (miRNA) microarray was used to find hUSC-sEVs-specific miRNAs that potentially affected the proliferation, migration and differentiation ability of VK2 cells. RESULTS: The in vitro release profile revealed that the piGEL could ensure sustained release of hUSC-sEVs. The in vivo results showed that piGEL-sEVs effectively promoted epithelization and angiogenesis of vaginal mucosa defects in rabbits. According to miRNA microarray and qRT-PCR results, miR-126-3p might be the crucial molecule among the various miRNAs contained in hUSC-sEVs. The data showed that hUSC-sEVs promoted the migration and differentiation of VK2 cells by delivering miR-126-3p to suppress the expression of Spred1 and PIK3R2, thereby activating the ERK1/2 and ATK signaling pathways. CONCLUSION: The results indicated that piGEL-sEVs could be a novel promising approach for enhancing the epithelization of the neovagina after vaginoplasty and provided useful data for understanding the underlying mechanism of the effect of hUSC-sEVs on epithelization.

Metabolomic Evaluation of N-Acetyl-p-Benzoquinone Imine Protein Adduct Formation with Therapeutic Acetaminophen Administration: Sex-based Physiologic Differences.

BACKGROUND: Acetaminophen (APAP)-associated transaminase elevation, induced by N-acetyl-p-benzoquinone imine (NAPQI) protein adduction, remains an area of research interest. Distinct from known genetic, physiologic, and dosage associations dictating severity of hepatic injury, no known factors predict an absence of protein adduct formation at therapeutic APAP dosing. HYPOTHESIS: Sex-based physiology is predictive of APAP-induced protein adduct formation and differential metabolite expression at therapeutic doses. METHODS: This retrospective study interrogated serum samples collected for a prior study investigating fluctuations of alanine aminotransferase (ALT) over time with 4G daily APAP dosing for ≥ 16 days in subjects from Denver, Colorado. Subjects were grouped by adduct formation (n = 184) vs no adducts (n = 20). Samples were run on ultra-high-performance liquid chromatography mass spectrometry from study days 0, 7, 16, and 31. Significant metabolite expressions were identified using t-tests with false discovery rate correction (FDR), partial least squares discriminant, and ANOVA simultaneous comparison analyses. Demographic and clinical data were explored using t-tests with FDR (age, weight, BMI, ALT) and Chi-square (sex, ethnicity, race) analyses. RESULTS: In pre-treatment samples, relative quantitation caprylic acid was expressed ninefold higher and 6-carboxyhexanoate was expressed threefold lower in subjects who did not develop adducts. Lactate had greater expression in the no adducts group (p = 0.001). Using absolute quantitation, glutathione was expressed 2.6-fold greater among no adduct subjects. Odds of males developing NAPQI protein adducts at therapeutic APAP dosing were 5.91 times lower than females (95% CI = 2.3-14.9; p = 0.0001). CONCLUSION: Multiple metabolites were differentially expressed based on adduct group and sex. Metabolites were identified unique to adduct development independent of sex. At therapeutic APAP dosing, males were less likely to develop APAP protein adducts. Further research into lipid biosynthesis and metabolism may provide further insight into physiology associated with adduct production.

Mitochondrial versus microsomal bioactivation of paracetamol by human liver and kidney tissues.

Mitochondria appeared to be a major target for paracetamol (PAR)-induced hepatotoxicity. Studies suggested that microsomal CYPs catalyse bioactivation of PAR to N-acetyl-p-benzoquinone imine (NAPQI), which alkylates mitochondrial proteins, and causes transmission of death signal from mitochondria to nucleus. We hypothesised that local formation of NAPQI within mitochondria seems more likely compared to the translocation of NAPQI. We therefore tested whether the formation of NAPQI may be catalysed by mitochondrial CYPs. Cellular fractions were isolated from human liver and kidney to compare the metabolic capacities. Liver and kidney mitochondria are capable to generate NAPQI. Mitochondrial CYP2E1 and CYP3A4 activities were comparable to the microsomal counterparts in both organs. Previously reported higher kidney microsomal CYP2E1 activity in men compared women were observed in mitochondrial CYP2E1 as well in the present study. On the other hand, no correlation between kidney CYP2E1 activity and quantity of NAPQI formation, as well as no induction on mitochondrial permeability transition pore (mPTP) opening by PAR in kidney mitochondria strongly suggested a different toxicity mechanism in this organ.

An increase in surface hydrophobicity mediates chaperone activity in N-chlorinated RidA.

Under physiological conditions, Escherichia coli RidA is an enamine/imine deaminase, which promotes the release of ammonia from reactive enamine/imine intermediates. However, when modified by hypochlorous acid (HOCl), it turns into a potent chaperone-like holdase that can effectively protect E. coli's proteome during oxidative stress. However, it is unknown, which residues need to be chlorinated for activation. Here, we employ a combination of LC-MS/MS analysis, a chemo-proteomic approach, and a mutagenesis study to identify residues responsible for RidA's chaperone-like function. Through LC-MS/MS of digested RidAHOCl, we obtained direct evidence of the chlorination of one arginine residue. To overcome the instability of the N-chloramine modification, we established a chemoproteomic approach using 5-(dimethylamino) naphthalene-1-sulfinic acid (DANSO2H) as a probe to label N-chlorinated lysines. Using this probe, we were able to detect the N-chlorination of six additional lysine residues. Moreover, using a mutagenesis study to genetically probe the role of single arginine and lysine residues, we found that the removal of arginines R105 and/or R128 led to a substantial reduction of RidAHOCl's chaperone activity. These results, together with structural analysis, confirm that the chaperone activity of RidA is concomitant with the loss of positive charges on the protein surface, leading to an increased overall protein hydrophobicity. Molecular modelling of RidAHOCl and the rational design of a RidA variant that shows chaperone activity even in the absence of HOCl further supports our hypothesis. Our data provide a molecular mechanism for HOCl-mediated chaperone activity found in RidA and a growing number of other HOCl-activated chaperones.

Development of an Efficient Extraction Method for Harvesting Gymnodimine-A from Large-Scale Cultures of Karenia selliformis.

Gymnodimine-A (GYM-A) is a fast-acting microalgal toxin and its production of certified materials requires an efficient harvesting technology from the large-scale cultures of toxigenic microalgae. In this study the recoveries of GYM-A were compared between several liquid-liquid extraction (LLE) treatments including solvents, ratios and stirring times to optimize the LLE technique for harvesting GYM-A from Karenia selliformis cultures, of which the dichloromethane was selected as the extractant and added to microalgal cultures at the ratio 55 mL L-1 (5.5%, v/v). The recovery of GYM-A obtained by the LLE technique was also compared with filtration and centrifugation methods. The stability of GYM-A in culture media were also tested under different pH conditions. Results showed that both the conventional filter filtration and centrifugation methods led to fragmentation of microalgal cells and loss of GYM-A in the harvesting processes. A total of 5.1 µg of GYM-A were obtained from 2 L of K. selliformis cultures with a satisfactory recovery of 88%. Interestingly, GYM-A obviously degraded in the culture media with the initial pH 8.2 and the adjusted pH of 7.0 after 7 days, but there was no obvious degradation in the acidic medium at pH 5.0. Therefore, the LLE method developed here permits the collection of large-volume cultures of K. selliformis and the high-efficiency extraction of GYM-A. This work provides a simple and valuable technique for harvesting toxins from large-scale cultures of GYM-producing microalgae.

Versatile selective evolutionary pressure using synthetic defect in universal metabolism.

The non-natural needs of industrial applications often require new or improved enzymes. The structures and properties of enzymes are difficult to predict or design de novo. Instead, semi-rational approaches mimicking evolution entail diversification of parent enzymes followed by evaluation of isolated variants. Artificial selection pressures coupling desired enzyme properties to cell growth could overcome this key bottleneck, but are usually narrow in scope. Here we show diverse enzymes using the ubiquitous cofactors nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) can substitute for defective NAD regeneration, representing a very broadly-applicable artificial selection. Inactivation of Escherichia coli genes required for anaerobic NAD regeneration causes a conditional growth defect. Cells are rescued by foreign enzymes connected to the metabolic network only via NAD or NADP, but only when their substrates are supplied. Using this principle, alcohol dehydrogenase, imine reductase and nitroreductase variants with desired selectivity modifications, and a high-performing isopropanol metabolic pathway, are isolated from libraries of millions of variants in single-round experiments with typical limited information to guide design.

Biocatalytic Asymmetric Cyclopropanations via Enzyme-Bound Iminium Ion Intermediates.

Cyclopropane rings are an important structural motif frequently found in many natural products and pharmaceuticals. Commonly, biocatalytic methodologies for the asymmetric synthesis of cyclopropanes rely on repurposed or artificial heme enzymes. Here, we engineered an unusual cofactor-independent cyclopropanation enzyme based on a promiscuous tautomerase for the enantioselective synthesis of various cyclopropanes via the nucleophilic addition of diethyl 2-chloromalonate to α,ß-unsaturated aldehydes. The engineered enzyme promotes formation of the two new carbon-carbon bonds with excellent stereocontrol over both stereocenters, affording the desired cyclopropanes with high diastereo- and enantiopurity (d.r. up to 25:1; e.r. up to 99:1). Our results highlight the usefulness of promiscuous enzymes for expanding the biocatalytic repertoire for non-natural reactions.

Pseudomonas aeruginosa adapts to octenidine via a combination of efflux and membrane remodelling.

Pseudomonas aeruginosa is an opportunistic pathogen capable of stably adapting to the antiseptic octenidine by an unknown mechanism. Here we characterise this adaptation, both in the laboratory and a simulated clinical setting, and identify a novel antiseptic resistance mechanism. In both settings, 2 to 4-fold increase in octenidine tolerance was associated with stable mutations and a specific 12 base pair deletion in a putative Tet-repressor family gene (smvR), associated with a constitutive increase in expression of the Major Facilitator Superfamily (MFS) efflux pump SmvA. Adaptation to higher octenidine concentrations led to additional stable mutations, most frequently in phosphatidylserine synthase pssA and occasionally in phosphatidylglycerophosphate synthase pgsA genes, resulting in octenidine tolerance 16- to 256-fold higher than parental strains. Metabolic changes were consistent with mitigation of oxidative stress and altered plasma membrane composition and order. Mutations in SmvAR and phospholipid synthases enable higher level, synergistic tolerance of octenidine.

Asymmetric Synthesis of N-Substituted α-Amino Esters from α-Ketoesters via Imine Reductase-Catalyzed Reductive Amination.

N-Substituted α-amino esters are widely used as chiral intermediates in a range of pharmaceuticals. Here we report the enantioselective biocatalyic synthesis of N-substituted α-amino esters through the direct reductive coupling of α-ketoesters and amines employing sequence diverse metagenomic imine reductases (IREDs). Both enantiomers of N-substituted α-amino esters were obtained with high conversion and excellent enantioselectivity under mild reaction conditions. In addition >20 different preparative scale transformations were performed highlighting the scalability of this system.

Navigating the Unnatural Reaction Space: Directed Evolution of Heme Proteins for Selective Carbene and Nitrene Transfer.

Despite the astonishing diversity of naturally occurring biocatalytic processes, enzymes do not catalyze many of the transformations favored by synthetic chemists. Either nature does not care about the specific products, or if she does, she has adopted a different synthetic strategy. In many cases, the appropriate reagents used by synthetic chemists are not readily accessible to biological systems. Here, we discuss our efforts to expand the catalytic repertoire of enzymes to encompass powerful reactions previously known only in small-molecule catalysis: formation and transfer of reactive carbene and nitrene intermediates leading to a broad range of products, including products with bonds not known in biology. In light of the structural similarity of iron carbene (Fe═C(R1)(R2)) and iron nitrene (Fe═NR) to the iron oxo (Fe═O) intermediate involved in cytochrome P450-catalyzed oxidation, we have used synthetic carbene and nitrene precursors that biological systems have not encountered and repurposed P450s to catalyze reactions that are not known in the natural world. The resulting protein catalysts are fully genetically encoded and function in intact microbial cells or cell-free lysates, where their performance can be improved and optimized by directed evolution. By leveraging the catalytic promiscuity of P450 enzymes, we evolved a range of carbene and nitrene transferases exhibiting excellent activity toward these new-to-nature reactions. Since our initial report in 2012, a number of other heme proteins including myoglobins, protoglobins, and cytochromes c have also been found and engineered to promote unnatural carbene and nitrene transfer. Due to the altered active-site environments, these heme proteins often displayed complementary activities and selectivities to P450s.Using wild-type and engineered heme proteins, we and others have described a range of selective carbene transfer reactions, including cyclopropanation, cyclopropenation, Si-H insertion, B-H insertion, and C-H insertion. Similarly, a variety of asymmetric nitrene transfer processes including aziridination, sulfide imidation, C-H amidation, and, most recently, C-H amination have been demonstrated. The scopes of these biocatalytic carbene and nitrene transfer reactions are often complementary to the state-of-the-art processes based on small-molecule transition-metal catalysts, making engineered biocatalysts a valuable addition to the synthetic chemist's toolbox. Moreover, enabled by the exquisite regio- and stereocontrol imposed by the enzyme catalyst, this biocatalytic platform provides an exciting opportunity to address challenging problems in modern synthetic chemistry and selective catalysis, including ones that have eluded synthetic chemists for decades.

Unleashing the Power of Biocatalysts.

Biocatalysis is a continuously expanding subfield in chemical biology. Herein, I describe two categories of biocatalysts, the LEGO-brick-like and game-console-like type, both of which can streamline the synthetic routes to therapeutics. A multi-disciplinary approach to expand the biocatalytic toolkit will open up opportunities to develop new therapeutics.

Mitochondrial protein adduct and superoxide generation are prerequisites for early activation of c-jun N-terminal kinase within the cytosol after an acetaminophen overdose in mice.

Acetaminophen (APAP) overdose is the most common cause of acute liver failure in the United States and formation of APAP-protein adducts, mitochondrial oxidant stress and activation of the mitogen activated protein (MAP) kinase c-jun N-terminal kinase (JNK) are critical for APAP-induced cell death. However, direct evidence linking these mechanistic features are lacking and were investigated by examining the early temporal course of these changes in mice after 300 mg/kg APAP. Protein adducts were detectable in the liver (0.05-0.1 nmol/mg protein) by 15 and 30 min after APAP, which increased (>500 %) selectively in mitochondria by 60 min. Cytosolic JNK activation was only evident at 60 min, and was significantly attenuated by scavenging superoxide specifically in the cytosol by TEMPO treatment. Treatment of mouse hepatocytes with APAP revealed mitochondrial superoxide generation within 15 min, accompanied by hydrogen peroxide production without change in mitochondrial respiratory function. The oxidant stress preceded JNK activation and its mitochondrial translocation. Inhibitor studies identified the putative source of mitochondrial superoxide as complex III, which released superoxide towards the intermembrane space after APAP resulting in activation of JNK in the cytosol. Our studies provide direct evidence of mechanisms involved in mitochondrial superoxide generation after NAPQI-adduct formation and its activation of the MAP kinase cascade in the cytosol, which are critical features of APAP hepatotoxicity.

Screening and characterization of a diverse panel of metagenomic imine reductases for biocatalytic reductive amination.

Finding faster and simpler ways to screen protein sequence space to enable the identification of new biocatalysts for asymmetric synthesis remains both a challenge and a rate-limiting step in enzyme discovery. Biocatalytic strategies for the synthesis of chiral amines are increasingly attractive and include enzymatic asymmetric reductive amination, which offers an efficient route to many of these high-value compounds. Here we report the discovery of over 300 new imine reductases and the production of a large (384 enzymes) and sequence-diverse panel of imine reductases available for screening. We also report the development of a facile high-throughput screen to interrogate their activity. Through this approach we identified imine reductase biocatalysts capable of accepting structurally demanding ketones and amines, which include the preparative synthesis of N-substituted ß-amino ester derivatives via a dynamic kinetic resolution process, with excellent yields and stereochemical purities.

Engineering of Thermostable ß-Hydroxyacid Dehydrogenase for the Asymmetric Reduction of Imines.

The ß-hydroxyacid dehydrogenase from Thermocrinus albus (Ta-ßHAD), which catalyzes the NADP+ -dependent oxidation of ß-hydroxyacids, was engineered to accept imines as substrates. The catalytic activity of the proton-donor variant K189D was further increased by the introduction of two nonpolar flanking residues (N192 L, N193 L). Engineering the putative alternative proton donor (D258S) and the gate-keeping residue (F250 A) led to a switched substrate specificity as compared to the single and triple variants. The two most active Ta-ßHAD variants were applied to biocatalytic asymmetric reductions of imines at elevated temperatures and enabled enhanced product formation at a reaction temperature of 50 °C.

Structural Characterization of an S-enantioselective Imine Reductase from Mycobacterium Smegmatis.

NADPH-dependent imine reductases (IREDs) are enzymes capable of enantioselectively reducing imines to chiral secondary amines, which represent important building blocks in the chemical and pharmaceutical industry. Since their discovery in 2011, many previously unknown IREDs have been identified, biochemically and structurally characterized and categorized into families. However, the catalytic mechanism and guiding principles for substrate specificity and stereoselectivity remain disputed. Herein, we describe the crystal structure of S-IRED-Ms from Mycobacterium smegmatis together with its cofactor NADPH. S-IRED-Ms belongs to the S-enantioselective superfamily 3 (SFam3) and is the first IRED from SFam3 to be structurally described. The data presented provide further evidence for the overall high degree of structural conservation between different IREDs of various superfamilies. We discuss the role of Asp170 in catalysis and the importance of hydrophobic amino acids in the active site for stereospecificity. Moreover, a separate entrance to the active site, potentially functioning according to a gatekeeping mechanism regulating access and, therefore, substrate specificity is described.