Comparative functional analysis of human medium-chain dehydrogenases, short-chain dehydrogenases/reductases and aldo-keto reductases with retinoids

Retinoic acid biosynthesis in vertebrates occurs in two consecutive steps: the oxidation of retinol to retinaldehyde followed by the oxidation of retinaldehyde to retinoic acid. Enzymes of the MDR (medium-chain dehydrogenase/reductase), SDR (short-chain dehydrogenase/reductase) and AKR (aldo-keto reductase) superfamilies have been reported to catalyse the conversion between retinol and retinaldehyde. Estimation of the relative contribution of enzymes of each type was difficult since kinetics were performed with different methodologies, but SDRs would supposedly play a major role because of their low K(m) values, and because they were found to be active with retinol bound to CRBPI (cellular retinol binding protein type I). In the present study we employed detergent-free assays and HPLC-based methodology to characterize side-by-side the retinoid-converting activities of human MDR [ADH (alcohol dehydrogenase) 1B2 and ADH4), SDR (RoDH (retinol dehydrogenase)-4 and RDH11] and AKR (AKR1B1 and AKR1B10) enzymes. Our results demonstrate that none of the enzymes, including the SDR members, are active with CRBPI-bound retinoids, which questions the previously suggested role of CRBPI as a retinol supplier in the retinoic acid synthesis pathway. The members of all three superfamilies exhibit similar and low K(m) values for retinoids (0.12-1.1 microM), whilst they strongly differ in their kcat values, which range from 0.35 min(-1) for AKR1B1 to 302 min(-1) for ADH4. ADHs appear to be more effective retinol dehydrogenases than SDRs because of their higher kcat values, whereas RDH11 and AKR1B10 are efficient retinaldehyde reductases. Cell culture studies support a role for RoDH-4 as a retinol dehydrogenase and for AKR1B1 as a retinaldehyde reductase in vivo.     

Molecular docking for substrate identification: the short-chain dehydrogenases/reductases

Protein ligand docking has recently been investigated as a tool for protein function identification, with some success in identifying both known and unknown substrates of proteins. However, identifying a protein's substrate when cross-docking a large number of enzymes and their cognate ligands remains a challenge. To explore a more limited yet practically important and timely problem in more detail, we have used docking for identifying the substrates of a single protein family with remarkable substrate diversity, the short-chain dehydrogenases/reductases.We examine different protocols for identifying candidate substrates for 27 short-chain dehydrogenase/reductase proteins of known catalytic function. We present the results of docking > 900 metabolites from the human metabolome to each of these proteins together with their known cognate substrates and products, and we investigate the ability of docking to (a) reproduce a viable binding mode for the substrate and (b) to rank the substrate highly amongst the dataset of other metabolites. In addition, we examine whether our docking results provide information about the nature of the substrate, based on the best-scoring metabolites in the dataset. We compare two different docking methods and two alternative scoring functions for one of the docking methods, and we attempt to rationalise both successes and failures.Finally, we introduce a new protocol, whereby we dock only a set of representative structures (medoids) to each of the proteins, in the hope of characterising each binding site in terms of its ligand preferences, with a reduced computational cost. We compare the results from this protocol with our original docking experiments, and we find that although the rank of the representatives correlates well with the mean rank of the clusters to which they belong, a simple structure-based clustering is too naïve for the purpose of substrate identification. Many clusters comprise ligands with widely varying affinities for the same protein; hence important candidates can be missed if a single representative is used.     

Identification and characterization of retinoid-active short-chain dehydrogenases/reductases in Drosophila melanogaster

Background

In chordates, retinoid metabolism is an important target of short-chain dehydrogenases/reductases (SDRs). It is not known whether SDRs play a role in retinoid metabolism of protostomes, such as Drosophila melanogaster.

Methods

Drosophila genome was searched for genes encoding proteins with ∼ 50% identity to human retinol dehydrogenase 12 (RDH12). The corresponding proteins were expressed in Sf9 cells and biochemically characterized. Their phylogenetic relationships were analyzed using PHYLIP software.

Results

A total of six Drosophila SDR genes were identified. Five of these genes are clustered on chromosome 2 and one is located on chromosome X. The deduced proteins are 300 to 406 amino acids long and are associated with microsomal membranes. They recognize all-trans-retinaldehyde and all-trans-3-hydroxyretinaldehyde as substrates and prefer NADPH as a cofactor. Phylogenetically, Drosophila SDRs belong to the same branch of the SDR superfamily as human RDH12, indicating a common ancestry early in bilaterian evolution, before a protostome–deuterostome split.

Conclusions

Similarities in the substrate and cofactor specificities of Drosophila versus human SDRs suggest conservation of their function in retinoid metabolism throughout protostome and deuterostome phyla.

General significance

The discovery of Drosophila retinaldehyde reductases sheds new light on the conversion of β-carotene and zeaxantine to visual pigment and provides a better understanding of the evolutionary roots of retinoid-active SDRs.     

Theoretical calculations of the catalytic triad in short-chain alcohol dehydrogenases/reductases

Three highly conserved active site residues (Ser, Tyr, and Lys) of the family of short-chain alcohol dehydrogenases/reductases (SDRs) were demonstrated to be essential for catalytic activity and have been denoted the catalytic triad of SDRs. In this study computational methods were adopted to study the ionization properties of these amino acids in SDRs from Drosophila melanogaster and Drosophila lebanonensis. Three enzyme models, with different ionization scenarios of the catalytic triad that might be possible when inhibitors bind to the enzyme cofactor complex, were constructed. The binding of the two alcohol competitive inhibitors were studied using automatic docking by the Internal Coordinate Mechanics program, molecular dynamic (MD) simulations with the AMBER program package, calculation of the free energy of ligand binding by the linear interaction energy method, and the hydropathic interactions force field. The calculations indicated that deprotonated Tyr acts as a strong base in the binary enzyme-NAD⁺ complex. Molecular dynamic simulations for 5 ns confirmed that deprotonated Tyr is essential for anchoring and orientating the inhibitors at the active site, which might be a general trend for the family of SDRs. The findings here have implications for the development of therapeutically important SDR inhibitors.     

Halohydrin dehalogenases are structurally and mechanistically related to short-chain dehydrogenases/reductases

Halohydrin dehalogenases, also known as haloalcohol dehalogenases or halohydrin hydrogen-halide lyases, catalyze the nucleophilic displacement of a halogen by a vicinal hydroxyl function in halohydrins to yield epoxides. Three novel bacterial genes encoding halohydrin dehalogenases were cloned and expressed in Escherichia coli, and the enzymes were shown to display remarkable differences in substrate specificity. The halohydrin dehalogenase of Agrobacterium radiobacter strain AD1, designated HheC, was purified to homogeneity. The k(cat) and K(m) values of this 28-kDa protein with 1,3-dichloro-2-propanol were 37 s(-1) and 0.010 mM, respectively. A sequence homology search as well as secondary and tertiary structure predictions indicated that the halohydrin dehalogenases are structurally similar to proteins belonging to the family of short-chain dehydrogenases/reductases (SDRs). Moreover, catalytically important serine and tyrosine residues that are highly conserved in the SDR family are also present in HheC and other halohydrin dehalogenases. The third essential catalytic residue in the SDR family, a lysine, is replaced by an arginine in halohydrin dehalogenases. A site-directed mutagenesis study, with HheC as a model enzyme, supports a mechanism for halohydrin dehalogenases in which the conserved Tyr145 acts as a catalytic base and Ser132 is involved in substrate binding. The primary role of Arg149 may be lowering of the pK(a) of Tyr145, which abstracts a proton from the substrate hydroxyl group to increase its nucleophilicity for displacement of the neighboring halide. The proposed mechanism is fundamentally different from that of the well-studied hydrolytic dehalogenases, since it does not involve a covalent enzyme-substrate intermediate.     

Comparative genomic and phylogenetic analysis of short-chain dehydrogenases/reductases with dual retinol/sterol substrate specificity

Human short-chain dehydrogenases/reductases with dual retinol/sterol substrate specificity (RODH-like enzymes) are thought to contribute to the oxidation of retinol for retinoic acid biosynthesis and to the metabolism of androgenic and neuroactive 3alpha-hydroxysteroids. Here, we investigated the phylogeny and orthology of these proteins to understand better their origins and physiological roles. Phylogenetic and genomic analysis showed that two proteins (11-cis-RDH and RDHL) are highly conserved, and their orthologs can be identified in the lower taxa, such as amphibians and fish. Two other proteins (RODH-4 and 3alpha-HSD) are significantly less conserved. Orthologs for 3alpha-HSD are present in all mammals analyzed, whereas orthologs for RODH-4 can be identified in some mammalian species but not in others due to species-specific gene duplications. Understanding the evolution and divergence of RODH-like enzymes in various vertebrate species should facilitate further investigation of their in vivo functions using animal models.     

Short-chain dehydrogenases/reductases (SDRs).

Short-chain dehydrogenases/reductases (SDRs) are enzymes of great functional diversity. Even at sequence identities of typically only 15-30%, specific sequence motifs are detectable, reflecting common folding patterns. We have developed a functional assignment scheme based on these motifs and we find five families. Two of these families were known previously and are called 'classical' and 'extended' families, but they are now distinguished at a further level based on coenzyme specificities. This analysis gives seven subfamilies of classical SDRs and three subfamilies of extended SDRs. We find that NADP(H) is the preferred coenzyme among most classical SDRs, while NAD(H) is that preferred among most extended SDRs. Three families are novel entities, denoted 'intermediate', 'divergent' and 'complex', encompassing short-chain alcohol dehydrogenases, enoyl reductases and multifunctional enzymes, respectively. The assignment scheme was applied to the genomes of human, mouse, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana and Saccharomyces cerevisiae. In the animal genomes, the extended SDRs amount to around one quarter or less of the total number of SDRs, while in the A. thaliana and S. cerevisiae genomes, the extended members constitute about 40% of the SDR forms. The numbers of NAD(H)-dependent and NADP(H)-dependent SDRs are similar in human, mouse and plant, while the proportions of NAD(H)-dependent enzymes are much lower in fruit fly, worm and yeast. We show that, in spite of the great diversity of the SDR superfamily, the primary structure alone can be used for functional assignments and for predictions of coenzyme preference.     

A new member of the short-chain dehydrogenases/reductases superfamily: Purification, characterization and substrate specificity of a recombinant carbonyl reductase from Pichia stipitis

A novel short-chain dehydrogenases/reductases superfamily (SDRs) reductase (PsCR) from Pichia stipitis that produced ethyl (S)-4-chloro-3-hydroxybutanoate with greater than 99% enantiomeric excess, was purified to homogeneity using fractional ammonium sulfate precipitation followed by DEAE-Sepharose chromatography. The enzyme purified from recombinant Escherichia coli had a molecular mass of about 35 kDa on SDS–PAGE and only required NADPH as an electron donor. The K_m value of PsCR for ethyl 4-chloro-3-oxobutanoate was 4.9 mg/mL and the corresponding V_max was 337 μmol/mg protein/min. The catalytic efficiency value was the highest ever reported for reductases from yeasts. Moreover, PsCR exhibited a medium-range substrate spectrum toward various keto and aldehyde compounds, i.e., ethyl-3-oxobutanoate with a chlorine substitution at the 2 or 4-position, or α,β-diketones. In addition, the activity of the enzyme was strongly inhibited by SDS and β-mercaptoethanol, but not by ethylene diamine tetra acetic acid.     

Characteristics of short-chain alcohol dehydrogenases and related enzymes

Different short-chain dehydrogenases are distantly related, constituting a protein family now known from at least 20 separate enzymes characterized, but with extensive differences, especially in the C-terminal third of their sequences. Many of the first known members were prokaryotic, but recent additions include mammalian enzymes from placenta, liver and other tissues, including 15-hydroxyprostaglandin, 17 beta-hydroxysteroid and 11 beta-hydroxysteroid dehydrogenases. In addition, species variants, isozyme-like multiplicities and mutants have been reported for several of the structures. Alignments of the different enzymes reveal large homologous parts, with clustered similarities indicating regions of special functional/structural importance. Several of these derive from relationships within a common type of coenzyme-binding domain, but central-chain patterns of similarity go beyond this domain. Total residue identities between enzyme pairs are typically around 25%, but single forms deviate more or less (14-58%). Only six of the 250-odd residues are strictly conserved and seven more are conserved in all but single cases. Over one third of the conserved residues are glycine, showing the importance of conformational and spatial restrictions. Secondary structure predictions, residue distributions and hydrophilicity profiles outline a common, N-terminal coenzyme-binding domain similar to that of other dehydrogenases, and a C-terminal domain with unique segments and presumably individual functions in each case. Strictly conserved residues of possible functional interest are limited, essentially only three polar residues. Asp64, Tyr152 and Lys156 (in the numbering of Drosophila alcohol dehydrogenase), but no histidine or cysteine residue like in the completely different, classical medium-chain alcohol dehydrogenase family. Asp64 is in the suggested coenzyme-binding domain, whereas Tyr152 and Lys156 are close to the center of the protein chain, at a putative inter-domain, active-site segment. Consequently, the overall comparisons suggest the possibility of related mechanisms and domain properties for different members of the short-chain family.     

Chinese hamster monomeric carbonyl reductases of the short-chain dehydrogenase/reductase superfamily

Chinese hamster monomeric carbonyl reductases (CHCRs) belong to the short-chain dehydrogenase/reductase (SDR) superfamily, which is a family of enzymes that metabolize many endogenous and xenobiotic compounds. We previously cloned three carbonyl reductase cDNAs-Chcr1, Chcr2, and Chcr3. By performing spectrophotometric analyses, we indicated that the enzymes CHCR1, CHCR2, and CHCR3 had similar specificities toward steroids; only CHCR3 did not show any reactivity with prostaglandins (PGs). In the present study, we investigated the characteristics of CHCRs in detail, that is, the differences in their expression patterns, physicochemical properties, and enzymatic activities. CHCR1 exhibited sex-dependent expression patterns. CHCRs showed multiple surface potentials in the zeta potential analysis and CHCR3 exhibited an isatin reductase activity with a high K(m) value. By the present HPLC-analysis, all the three enzymes exhibited PGF(2alpha) dehydrogenase activity and could oxidize PGF(2alpha) to PGE(2) and 15-keto-PGF(2alpha), i.e., the three enzymes exhibited 9- and 15-hydroxy PG dehydrogenase activities. Moreover, 15-keto-PGE(2) was detected in a comparatively higher amount in the dehydrogenase reaction products of CHCR2 than in those of CHCR1 and CHCR3, suggesting that CHCR2 can oxidize PGE(2) and/or 15-keto-PGF(2alpha) to 15-keto-PGE(2); however, these two PGs did not seem to be efficient substrates of CHCR1. Despite the differences in the dehydrogenase activities between CHCR1 and CHCR2, PGE(2) reductase activities of the two enzymes were similar, and PGF(2alpha) was predominantly produced from PGE(2) as a result of the PG 9-keto reductase activity. On the other hand, CHCR3 exhibited a reduced PGE(2) reductase activity. In conclusion, although the CHCRs share a high degree of sequence identity (>70%), they clearly differed in their enzymatic characteristics.     

Elements in the N-terminal signaling sequence that determine cytosolic topology of short-chain dehydrogenases/reductases. Studies with retinol dehydrogenase type 1 and cis-retinol/androgen dehydrogenase type 1

High affinity, retinoid-specific binding proteins chaperone retinoids to manage their transport and metabolism. Proposing mechanisms of retinoid transfer between these binding proteins and membrane-associated retinoid-metabolizing enzymes requires insight into enzyme topology. We therefore determined the topology of mouse retinol dehydrogenase type 1 (Rdh1) and cis-retinoid androgen dehydrogenase type 1 (Crad1) in the endoplasmic reticulum of intact mammalian cells. The properties of Rdh1 were compared with a chimera with a luminal signaling sequence (11beta-hydroxysteroid dehydrogenase (11beta-HSD1)(1-41)/Rdh1(23-317); the green fluorescent protein (GFP) fusion proteins Rdh1(1-22)/GFP, Crad1(1-22)/GFP, and 11beta-HSD1(1-41)/GFP; and signaling sequence charge difference mutants using confocal immunofluorescence, antibody access, proteinase K sensitivity, and deglycosylation assays. An N-terminal signaling sequence of 22 residues, consisting of a hydrophobic helix ending in a net positive charge, anchors Rdh1 and Crad1 in the endoplasmic reticulum facing the cytoplasm. Mutating arginine to glutamine in the signaling sequence did not affect topology. Inserting one or two arginine residues near the N terminus of the signaling sequence caused 28-95% inversion from cytoplasmic to luminal, depending on the net positive charge remaining at the C terminus of the signaling sequence; e.g. the mutant L3R,L5R,R16Q,R19Q,R21Q faced the lumen. Experiments with N- and C-terminal epitope-tagged Rdh1 and molecular modeling indicated that a hydrophobic helix-turn-helix near the C terminus of Rdh1 (residues 289-311) projects into the cytoplasm. These data provide insight into the features necessary to orient type III (reverse signal-anchor) proteins and demonstrate that Rdh1, Crad1, and other short-chain dehydrogenases/reductases, which share similar N-terminal signaling sequences such as human Rdh5 and mouse Rdh4, orient with their catalytic domains facing the cytoplasm.     

Cloning, expression, purification, and characterization of AmphiTip30, a member of short-chain dehydrogenases/reductases family from the amphioxus Branchiostoma belcheri tsingtauense

In this paper we describe the cloning, expression and identification study of the TIP30 gene from amphioxus (Branchiostoma belcheri). The amphioxus TIP30 cDNA is comprised of 1499 bp and is translated in one open-reading frame to give a protein of 237 amino acids, with a predicted 23 amino acids signal peptide, a 147 bp 5'-UTR and a 638 bp 3'-UTR. A multiple alignment of TIP30 from amphioxus with other known TIP30 sequences shows the conservation of most amino acid residues involved in the peculiar structural domains found within TIP30's. Phylogenetic analysis places AmphiTIP30 at the base of the phylogenetic tree, suggesting that AmphiTIP30 is the archetype of the vertebrate TIP30 genes. We express the amphioxus TIP30 gene in Escherichia coli. driven by T7 promoter. The recombinant amphioxus TIP30 protein was purified by HisTrap affinity column. Subsequently, the binding constant and enzyme activity was mensurated. Western blot and immunohistochemistry analysis confirmed that amphioxus has a native molecular mass of approximately 26 kDa, and TIP30 was strongly expressed in ovary. Finally, the initial function of TIP30 is discussed.     

Porcine carbonyl reductase. structural basis for a functional monomer in short chain dehydrogenases/reductases

Porcine testicular carbonyl reductase (PTCR) belongs to the short chain dehydrogenases/reductases (SDR) superfamily and catalyzes the NADPH-dependent reduction of ketones on steroids and prostaglandins. The enzyme shares nearly 85% sequence identity with the NADPH-dependent human 15-hydroxyprostaglandin dehydrogenase/carbonyl reductase. The tertiary structure of the enzyme at 2.3 A reveals a fold characteristic of the SDR superfamily that uses a Tyr-Lys-Ser triad as catalytic residues, but exhibits neither the functional homotetramer nor the homodimer that distinguish all SDRs. It is the first known monomeric structure in the SDR superfamily. In PTCR, which is also active as a monomer, a 41-residue insertion immediately before the catalytic Tyr describes an all-helix subdomain that packs against interfacial helices, eliminating the four-helix bundle interface conserved in the superfamily. An additional anti-parallel strand in the PTCR structure also blocks the other strand-mediated interface. These novel structural features provide the basis for the scaffolding of one catalytic site within a single molecule of the enzyme.     

Role of short-chain hydroxyacyl CoA dehydrogenases in SCHAD deficiency

Short-chain hydroxyacyl CoA dehydrogenase deficiency is an ill-defined, severe pediatric disorder of mitochondrial fatty acid β-oxidation of short-chain hydroxyacyl CoAs. To understand the relative contributions of the two known short-chain hydroxyacyl CoA dehydrogenases (HADH) tissue biopsies of six distinct family individuals were analyzed and kinetic parameters were compared. Steady-state kinetic constants for HADH 1 and HADH 2 suggest that type 1 is the major enzyme involved in mitochondrial β-oxidation of short-chain hydroxyacyl-CoAs. Two patients are heterozygous carriers of a HADH 1 polymorphism, whereas no mutation is detected in the HADH 2 gene of all patients. The data suggest that protein interactions rather than HADH mutations are responsible for the disease phenotype. Pull-down experiments of recombinant HADH 1 and 2 with human mitochondrial extracts reveal two proteins interacting with HADH 1, one of which was identified as glutamate dehydrogenase. This association provides a possible link between fatty acid metabolism and the hyperinsulinism/hyperammonia syndrome.     

Critical role of histidine residues in cyclohexanone monooxygenase expression, cofactor binding and catalysis

Cyclohexanone monooxygenase (CMO) is a member of the flavin monooxygenase superfamily of enzymes that catalyze both nucleophilic and electrophilic reactions involving a common C4a hydroperoxide intermediate. To begin to probe structure-function relationships for these enzymes, we investigated the roles of histidine residues in CMO derived from Acinetobacter NCIB 9871, with particular emphasis on the wholly conserved residue, His163 (H163). CMO activity was readily inactivated by diethyl pyrocarbonate (DEPC), a selective chemical modifier of histidine residues. Each of the seven histidines in CMO was then individually mutated to glutamine and the mutants expressed and purified from Escherichia coli. Only the H59Q mutant failed to express at significant levels. The H96Q enzyme was found to have a greatly reduced flavin adenine dinucleotide (FAD) content, indicative of compromised cofactor retention. The only significant effect on kcat occurred with the H163Q mutant, which exhibited an approximately 10-fold lower turnover of the prototypical substrate, cyclohexanone. This was accompanied by a doubling in the Km [NADPH] compared to the wild-type enzyme, suggesting that the functional decrement in H163Q is probably not solely a reflection of impaired NADPH binding. These data establish a critical role for H163 in CMO catalysis and prompt the hypothesis that this conserved residue plays a similarly important functional role across the flavin monooxygenase family of enzymes.     

The crystal structure of progesterone 5beta-reductase from Digitalis lanata defines a novel class of short chain dehydrogenases/reductases

Progesterone 5beta-reductase (5beta-POR) catalyzes the stereospecific reduction of progesterone to 5beta-pregnane-3,20-dione and is a key enzyme in the biosynthetic pathway of cardenolides in Digitalis (foxglove) plants. Sequence considerations suggested that 5beta-POR is a member of the short chain dehydrogenase/reductase (SDR) family of proteins but at the same time revealed that the sequence motifs that in standard SDRs contain the catalytically important residues are missing. Here we present crystal structures of 5beta-POR from Digitalis lanata in complex with NADP(+) at 2.3A and without cofactor bound at 2.4A resolution together with a model of a ternary complex consisting of 5beta-POR, NADP(+), and progesterone. Indeed, 5beta-POR displays the fold of an extended SDR. The architecture of the active site is, however, unprecedented because none of the standard catalytic residues are structurally conserved. A tyrosine (Tyr-179) and a lysine residue (Lys-147) are present in the active site, but they are displayed from novel positions and are part of novel sequence motifs. Mutating Tyr-179 to either alanine or phenylalanine completely abolishes the enzymatic activity. We propose that the distinct topology reflects the fact that 5beta-POR reduces a conjugated double bond in a steroid substrate via a 1-4 addition mechanism and that this requires a repositioning of the catalytically important residues. Our observation that the sequence motifs that line the active site are conserved in a number of bacterial and plant enzymes of yet unknown function leads us to the proposition that 5beta-POR defines a novel class of SDRs.     

A new family of D-2-hydroxyacid dehydrogenases that comprises D-mandelate dehydrogenases and 2-ketopantoate reductases

The gene for the D-mandelate dehydrogenase (D-ManDH) of Enterococcus faecalis IAM10071 was isolated by means of an activity staining procedure and PCR and expressed in Escherichia coli cells. The recombinant enzyme exhibited high catalytic activity toward various 2-ketoacid substrates with bulky hydrophobic side chains, particularly C3-branched substrates such as benzoylformate and 2-ketoisovalerate, and strict coenzyme specificity for NADH and NAD(+). It showed marked sequence similarity with known NADP-dependent 2-ketopantoate reductases (KPR). These results indicate that together with KPR, D-ManDH constitutes a new family of D-2-hydroxyacid dehydrogenases that act on C3-branched 2-ketoacid substrates with various specificities for coenzymes and substrates.     

The functional divergence of short-chain dehydrogenases involved in tropinone reduction

Tropane alkaloids typically occur in the Solanaceae and are also found in Cochlearia officinalis, a member of the Brassicaceae. Tropinone reductases are key enzymes of tropane alkaloid metabolism. Two different tropinone reductases form one stereoisomeric product each, either tropine for esterified alkaloids or pseudotropine that is converted to calystegines. A cDNA sequence with similarity to known tropinone reductases (TR) was cloned from C. officinalis. The protein was expressed in Escherichia coli, and found to catalyze the reduction of tropinone. The enzyme is a member of the short-chain dehydrogenase enzyme family and shows broad substrate specificity. Several synthetic ketones were accepted as substrates, with higher affinity and faster enzymatic turnover than observed for tropinone. C. officinalis TR produced both the isomeric alcohols tropine and pseudotropine from tropinone using NADPH + H(+) as co-substrate. Tropinone reductases of the Solanaceae, in contrast, are strictly stereospecific and form one tropane alcohol only. The Arabidopsis thaliana homologue of C. officinalis TR showed high sequence similarity, but did not reduce tropinone. A tyrosine residue was identified in the active site of C. officinalis TR that appeared responsible for binding and orientation of tropinone. Mutagenesis of the tyrosine residue yielded an active reductase, but with complete loss of TR activity. Thus C. officinalis TR presents an example of an enzyme with relaxed substrate specificity, like short-chain dehydrogenases, that provides favorable preconditions for the evolution of novel functions in biosynthetic sequences.