3-Oxoecdysteroid reductases in Manduca sexta midgut

The 80,000g supernatant o larval midgut homogenates of the tobacco hornworm, Manduca sexta, was fractionated by affinity chromatography on Blue Sepharose CL-6B and by anion exchange chromatography on Q Sepharose. Both methods resolved one major 3-oxoecdysteroid 3α-reductase and three major 3-oxoecdysteroid 3β-reductases. The 3β-reducates reacted only with BADPH as cosubstrate. The 3α-reductase was active with both NADPH and NADH, and the NADPH/NADH activity ratio increased with the NaCl concentration (0–0.5 M) in the incubation mixtures. The 3-α-reductase and one of the 3-β-reductases showed very similar chromatographic properties, and their isoelectric points were 5.2 and 5.8, respectively. © 1992 Wiley-Liss, Inc.     

Ecdysone oxidase and 3-oxoecdysteroid reductases in Manduca sexta: Developmental changes and tissue distribution

The activities of ecdysone oxidase (EO), 3-oxoecdysteroid 3α-reductase (3α-R), and 3-oxoecdysteroid 3β-reductase (3β-R) were determined for epidermis, hemolymph, and fat body of wandering fifth instar Manduca sexta larvae and for midguts of various developmental stages between 3 days after the last larval and 14 days after the pupal ecdysis. The larval midgut was the only organ showing substantial specific activities of EO and 3α-R, and both increased up to the seventh day after ecdysis. Hemolymph and fat body had only moderate to high 3β-R and low EO activites, and the epidermis did not contain significant activity of any of the enzymes. On the ninth day after the last larval ecdysis the larval midgut epithelium was replaced by a new pupal midgut epithelium. After this event only 3β-R was restored to high activities, whereas EO and 3α-R showed only low to marginal activities. It is concluded that only the larval midgut has a role in the inactivation of ecdysteroids by 3-epimerization. © 1993 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Conversion of human steroid 5β-reductase (AKR1D1) into 3β-hydroxysteroid dehydrogenase by single point mutation E120H: example of perfect enzyme engineering

Human aldo-keto reductase 1D1 (AKR1D1) and AKR1C enzymes are essential for bile acid biosynthesis and steroid hormone metabolism. AKR1D1 catalyzes the 5β-reduction of Δ(4)-3-ketosteroids, whereas AKR1C enzymes are hydroxysteroid dehydrogenases (HSDs). These enzymes share high sequence identity and catalyze 4-pro-(R)-hydride transfer from NADPH to an electrophilic carbon but differ in that one residue in the conserved AKR catalytic tetrad, His(120) (AKR1D1 numbering), is substituted by a glutamate in AKR1D1. We find that the AKR1D1 E120H mutant abolishes 5β-reductase activity and introduces HSD activity. However, the E120H mutant unexpectedly favors dihydrosteroids with the 5α-configuration and, unlike most of the AKR1C enzymes, shows a dominant stereochemical preference to act as a 3β-HSD as opposed to a 3α-HSD. The catalytic efficiency achieved for 3β-HSD activity is higher than that observed for any AKR to date. High resolution crystal structures of the E120H mutant in complex with epiandrosterone, 5β-dihydrotestosterone, and Δ(4)-androstene-3,17-dione elucidated the structural basis for this functional change. The glutamate-histidine substitution prevents a 3-ketosteroid from penetrating the active site so that hydride transfer is directed toward the C3 carbonyl group rather than the Δ(4)-double bond and confers 3β-HSD activity on the 5β-reductase. Structures indicate that stereospecificity of HSD activity is achieved because the steroid flips over to present its α-face to the A-face of NADPH. This is in contrast to the AKR1C enzymes, which can invert stereochemistry when the steroid swings across the binding pocket. These studies show how a single point mutation in AKR1D1 can introduce HSD activity with unexpected configurational and stereochemical preference.     

Are Bacteria the Major Producers of Extracellular Glycolytic Enzymes in Aquatic Environments?

In aquatic microbial ecology, it has been considered that most extracellular enzymes except phosphatases are of bacterial origin. We tested this paradigm by evaluating the relationship between bacterial cell number and the activity of three glycolytic enzymes from 17 fresh waters and also from a laboratory experiment. Our large sets of pooled data do not seem to support such a simple explanation, because we found only a weak correlation of bacterial number with activity of α‐glucosidase (r_s = 0.63), β‐glucosidase (r_s = 0.45), and β‐N‐acetylhexosaminidase (r_s = 0.44). We also tested relations of the enzymatic activities to potential sources of natural substrates: dissolved organic carbon (DOC) and phytoplankton (as chlorophyll a). Their correlations with the enzymatic activities tested were very weak or insignificant. On the other hand, we found evidence for distinct producers of extracellular enzymes by analysing enzyme kinetics. The kinetics usually did not follow the simple Michaelis‐Menten model but a more complex one, indicating a mixture of two enzymes with distinct affinity to a substrate. In combination with size fractionation, we could sometimes even distinguish three or more different enzymes. During diatom blooms, the diatom biomass tightly correlated with β‐N‐acetylhexosaminidase activity (>4 μm fraction). We also documented very tight relationships between activity of both glucosidases and dry weight of Daphnia longispina (r_s = 1.0 and 0.60 for α‐ and β‐glucosidases, respectively) in an alpine clear‐water lake. Our data and evidence from other studies indicate that extracellular glycosidic activities in aquatic ecosystems cannot generally be assigned only to bacteria. Also invertebrate animals and other eukaryotes (fungi, diatoms, protozoa etc.) should be considered as potentially very important enzyme producers. (© 2004 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

5α‐Androst‐16‐en‐3α‐ol β‐D‐Glucuronide,Precursor of 5α‐Androst‐16‐en‐3α‐ol in Human Sweat

5α‐Androst‐16‐en‐3α‐ol (α‐androstenol) is an important contributor to human axilla sweat odor. It is assumed that α‐andostenol is excreted from the apocrine glands via a H₂O‐soluble conjugate, and this precursor was formally characterized in this study for the first time in human sweat. The possible H₂O‐soluble precursors, sulfate and glucuronide derivatives, were synthesized as analytical standards, i.e., α‐androstenol, β‐androstenol sulfates, 5α‐androsta‐5,16‐dien‐3β‐ol (β‐androstadienol) sulfate, α‐androstenol β‐glucuronide, α‐androstenol α‐glucuronide, β‐androstadienol β‐glucuronide, and α‐androstenol β‐glucuronide furanose. The occurrence of α‐androstenol β‐glucuronide was established by ultra performance liquid chromatography (UPLC)/MS (heated electrospray ionization (HESI)) in negative‐ion mode in pooled human sweat, containing eccrine and apocrine secretions and collected from 25 female and 24 male underarms. Its concentration was of 79 ng/ml in female secretions and 241 ng/ml in male secretions. The release of α‐androstenol was observed after incubation of the sterile human sweat or α‐androstenol β‐glucuronide with a commercial glucuronidase enzyme, the urine‐isolated bacteria Streptococcus agalactiae, and the skin bacteria Staphylococcus warneri DSM 20316, Staphylococcus haemolyticus DSM 20263, and Propionibacterium acnes ATCC 6919, reported to have β‐glucuronidase activities. We demonstrated that if α‐ and β‐androstenols and androstadienol sulfates were present in human sweat, their concentrations would be too low to be considered as potential precursors of malodors; therefore, the H₂O‐soluble precursor of α‐androstenol in apocrine secretion should be a β‐glucuronide.     

Purification of 5β-reductase from hepatic cytosol fraction of chicken

From the cytosol fraction (supernatant fluid at 105,000 g) of chicken liver, 4-en-3-oxosteroid 5β-reductase (EC 1.3.1.23) was purified by ammonium sulfate precipitation, followed by Butyl Toyopearl, DEAE-Sepharose, Sephadex G-75 and hydroxylapatite column chromatographies. The enzyme activity was quantitated from amount of the 5β-reduced metabolites derived from [4-¹⁴C]testosterone. During the purification procedures, 17β-hydroxysteroid dehydrogenase which was present in the cytosol fraction was separated from 5β-reductase fraction by the Butyl Toyopearl column chromatography. By the DEAE-Sepharose column chromatography, 3α- and 3β-hydroxysteroid dehydrogenases were able to be removed from 5β-reductase fraction. The final enzyme preparation was apparently homogenous on SDS-polyacrylamide gel electrophoresis. Purification was about 13,600-fold from the hepatic cytosol. The molecular weight of this enzyme was estimated as 37,000 Da by SDS-polyacrylamide gel electrophoresis and also by Sephadex G-75 gel filtration. For 5β-reduction of 4-en-3-oxosteroids, such as testosterone, androstenedione and progesterone, NADPH was specifically required as cofactor. K_m of 5β-reductase for NADPH was estimated as 4.22 × 10⁻⁶M and for testosterone, 4.60 × 10⁻⁶M. The optimum pH of this enzyme ranged from pH 5.0 to 6.5 and other enzymic properties of the 5β-reductase were examined.     

One-step synthesis of 12-ketoursodeoxycholic acid from dehydrocholic acid using a multienzymatic system

12-ketoursodeoxycholic acid (12-keto-UDCA) is a key intermediate for the synthesis of ursodeoxycholic acid (UDCA), an important therapeutic agent for non-surgical treatment of human cholesterol gallstones and various liver diseases. The goal of this study is to develop a new enzymatic route for the synthesis 12-keto-UDCA based on a combination of NADPH-dependent 7β-hydroxysteroid dehydrogenase (7β-HSDH, EC 1.1.1.201) and NADH-dependent 3α-hydroxysteroid dehydrogenase (3α-HSDH, EC 1.1.1.50). In the presence of NADPH and NADH, the combination of these enzymes has the capacity to reduce the 3-carbonyl- and 7-carbonyl-groups of dehydrocholic acid (DHCA), forming 12-keto-UDCA in a single step. For cofactor regeneration, an engineered formate dehydrogenase, which is able to regenerate NADPH and NADH simultaneously, was used. All three enzymes were overexpressed in an engineered expression host Escherichia coli BL21(DE3)Δ7α-HSDH devoid of 7α-hydroxysteroid dehydrogenase, an enzyme indigenous to E. coli, in order to avoid formation of the undesired by-product 12-chenodeoxycholic acid in the reaction mixture. The stability of enzymes and reaction conditions such as pH value and substrate concentration were evaluated. No significant loss of activity was observed after 5 days under reaction condition. Under the optimal condition (10 mM of DHCA and pH 6), 99 % formation of 12-keto-UDCA with 91 % yield was observed.     

The effect of progesterone analogues,naturally occurring steroids,and contraceptive progestins on hypothalamic and anterior pituitary Δ4-steroid (progesterone) 5α-reductase

The effects of a number of steroids on the conversion of progesterone to 5α-dihydroprogesterone by hypothalamic and pituitary progesterone 5α-reductase have been investigated. Using enzyme preparations from female rats and ³H-progesterone as substrate, 5α-reduced products (5α-dihydroprogesterone and 3α-hydroxy-5α-pregnan-20-one) were analyzed by reverse isotopic dilution analysis. The amount of total 5α-reduced products formed was compared in the presence and absence of the test steroid. Derivatives lacking the Δ⁴ and/or the 3-keto moiety were without effect. Corticosterone had no effect. 16β-Methylprogesterone inhibited progesterone 5α-reduction in both tissues by at least 65%, while the 2α-, 6α-, and 7α-methylated derivatives had lesser effects. 3-Oxo-4-pregnene-20β-carboxaldehyde and 21-fluoroprogesterone were potent inhibitors. 17-Hydroxyprogesterone was a competitive inhibitor (substrate) with Ki's of 0.27 μM (pituitary) and 0.29 μM (hypothalamus). Medroxyprogesterone exerted little inhibitory effect. Of the 19-norsteroids examined, only norethindrone appreciably inhibited the 5α-reduction. These results suggest that some natural Δ⁴-3-ketosteroids can modify enzymatic activity. Also, inhibitory analogues may be useful for studies on the role of this 5α-reduction of progesterone.     

The mobilization of ferritin iron by liver cytosol. A comparison of xanthine and NADH as reducing substrates.

Considerable evidence suggests that the release of iron from ferritin is a reductive process. A role in this process has been proposed for two hepatic enzymes, namely xanthine oxidoreductase and an NADH oxidoreductase. The abilities of xanthine and NADH to serve as a source of reducing power for the enzyme-mediated release of ferritin iron (ferrireductase activity) were compared with turkey liver and rat liver homogenates. The maximal velocity (Vmax.) for the reaction with NADH was 50 times greater than with xanthine; however, the substrate concentration required to achieve half-maximal velocity (Km) was 1000 times less with xanthine than with NADH. NADPH could be substituted for NADH with little loss in activity. Dicoumarol did not inhibit the reaction with NADH or NADPH, demonstrating that the ferrireductase activity with those substrates was not the result of the liver enzyme 'DT-diaphorase' [NAD(P)H dehydrogenase (quinone)]. A flavin nucleotide was required for ferrireductase activity with rat and turkey liver cytosol when xanthine, NADH or NADPH was used as the reducing substrate. FMN yielded twice the activity with NADH or NADPH, whereas FAD was twice as effective with xanthine as substrate. Kinetic comparisons, differences in lability and partial chromatographic resolution of the ferrireductase activities with the two types of reducing substrates strongly indicate that the ferrireductase activities with xanthine and NADH are catalysed by separate enzyme systems contained in liver cytosol. Complete inhibition by allopurinol of the ferrireductase activity endogenous to undialysed liver cytosol preparations and the ability of xanthine to restore equivalent activity to dialysed preparations indicate that the source of reducing power for the endogenous activity is xanthine. These studies suggest that xanthine, NADH or NADPH can serve as a source of reducing power for the enzyme-mediated reduction of ferritin iron, with a flavin nucleotide serving as the shuttle of electrons from the enzymes to the ferritin iron.     

The identification and characterization of the c19o3 steroid metabolites of 5α-androstane-3β, 17β-diol produced by the canine prostate: 5α-androstane-3β, 6α, 17β-triol and 5α-androstane-3β, 7α, 17β-triol

This study has identified the polar metabolites of 5α-androstane-3β, 17β-diol(3β-diol) produced by the canine prostate. The major metabolite is 5α-androstane-3β, 7α, 17β-triol (7α-triol) accounting for approximately 80% of the total polar metabolites of 3β-diol. The remaining 20% is accounted for exclusively by another triol, 5α-androstane-3β, 6α, 17β-triol(6α-triol). This study has also characterized two enzymatic hydroxylases responsible for respective triol formation: 5α-androstane-3β, 17β-diol 6α-hydroxylase (6α-hydroxylase) and 5α-androstane-3β, 17β-diol 7α-hydroxylase (7α-hydroxylase). Both of these irreversible hydroxylases are located in the particulate fraction of the prostate and can utilize either NADH or NADPH as cofactor. Several $\text{in vitro}$ steroid inhibitors of these hydroxylases were identified including cholesterol, estradiol and diethylstilbestrol. Neither of the hydroxylases were found to be decreased by castration (3 months) when expressed as activity/DNA. Using a variety of C₁₉ androstane substrates, 6α- and 7α-triol were found to be major components of the total 3β-hydroxy-5α-androstane metabolites produced by the canine prostate.     

A chemiluminescent immunoassay for the direct measurement of urinary 5α-androstane-3α, 17β-diol-glucuronide in urine

We described a chemiluminescent immunoassay (CIA) for 5α-androstane-3α, 17β-diol-glucuronide (3α-diol-G) in human diluted urine. This method allowed the direct measurement in 1μl of urine avoiding the hydrolysis and extraction steps for sample pretreatment commonly used in routine methods. The hapten 3α-diol-G was synthesized by a Koenigs–Knorr reaction. The immunogenic complex, 3α-diol-G conjugated to bovine serum albumin (BSA), was employed to induce the formation of specific antibodies in New Zealand rabbits. In addition, the required chemiluminescent (CL) tracer was prepared. The characteristics of the antibody was determined as regard to specificity and sensitivity and the precision of the assay methods established. In 22 hirsute women affected by policystic ovarian syndrome we found 3α-diol-G values significantly (p < 0.01) higher (146.28 ± 73.77μg/g of creatinine; mean ± SD) than those observed in normal women (72.1 ± 32.58 μg/g of creatinine; mean ± SD).     

Modified level of miR‐376a is associated with Parkinson's disease

Parkinson's disease (PD) is a frequent progressive neurodegenerative disorder. Impaired mitochondrial function is a major feature of sporadic PD. Some susceptibility or causative genes detected in PD are strongly associated with mitochondrial dysfunction including PGC1α, TFAM and GSK3β. microRNAs (miRNAs) are non‐coding RNAs whose altered levels are proven in disparate PD models and human brains. Therefore, the aim of this study was to detect modulations of miRs upstream of PGC1α, TFAM and GSK3β in association with PD onset and progress. In this study, a total of 33 PD subjects and 25 healthy volunteers were recruited. Candidate miRNA (miR‐376a) was selected through target prediction tools and literature survey. Chronic and acute in vitro PD models were created by MPP⁺‐intoxicated SHSY5Y cells. The levels of miR‐376a and aforementioned genes were assessed by RT‐qPCR. The expression of target genes was decreased in chronic model while there were dramatically up‐regulated levels of those genes in acute model of PD. miR‐376a was strongly altered in both acute and chronic PD models as well as PBMCs of PD patients. Our results also showed overexpression of PGC1α, and TFAM in PBMCs is inversely correlated with down‐regulation of miR‐376a, suggesting that miR‐376a possibly has an impact on PD pathogenesis through regulation of these genes which are involved in mitochondrial function. miR‐376a expression in PD‐derived PBMCs was also correlated with disease severity and may serve as a potential biomarker for PD diagnosis. This is the first study showing altered levels of miR‐376a in PD models and PBMCs, suggesting the probable role of this miRNA in PD pathogenesis. The present study also proposed TFAM and PGC1α as target genes of miR‐376a for the first time, through which it possibly can exert its impact on PD pathogenesis.     

3β,20α-羟基甾体脱氢酶的动力学性质

3β,20α-羟基甾体脱氢酶(3β,20α-Hydroxysteroid dehydrogenase,3β,20α-HSD)是从胎羊血中分离得到的。分子量为35kD。该酶以NADPH为辅酶,有两种底物。以孕酮为底物时,Km=30.8μmol/L,Vmax=0.7nmol min~(-1)(nmol enzyme)~(-1);以5α-二氢睾酮(5α-Dihydrotestosterone,5α-DHT)为底物时,Km=74μmol/L,Vmax=1.3nmol min~(-1)(nmol enzyme)~(-1)。5α-DHT竞争性抑制20α-还原活性,Ki=102μmol/L。16α-溴代乙酰氧基(16α-Bromo acetoxyprogesterone,16α-BAP)是3β,20α-HSD不可逆竞争性抑制剂,t_(1/2)=75min。对3β和20α还原活性的抑制常数Ki分别为23μmol/L和58μmol/L。     

Characterization and expression of Cd8 molecules in mandarin fish Siniperca chuatsi

The full‐length complementary DNA (cDNA) sequences encoding cd8α and cd8β molecules were sequenced and characterized from mandarin fish Siniperca chuatsi. Conserved motifs and residues were found to be present in derived peptides of the Cd8 molecules. For example, WXR motif, DXGXYXC motif, and four cysteine residues were present in the extracellular region of the Cd8 protein. Threonine, serine and proline residues involved in multiple O‐linked glycosylation events were located in the membrane proximal hinge region. The common CPH motif in the cytoplasmic tail was detected similar to other teleost Cd8 molecules. Different from those in mammals, S. chuatsi Cd8 sequences have many extra cysteine residues (C149 in Cd8α sequence and C46, C51 and C158 in Cd8β sequence), which also exist in other teleost Cd8 molecules. Real‐time polymerase chain reaction (RT‐PCR) and Western blot analyses revealed that the thymus had the highest expression of cd8 messenger (m)RNA and protein. After stimulated with phytohaemagglutinin, polyriboinsine‐polyribocyaidylic acid and concanavalin A (ConA), the expression level of cd8 mRNA increased significantly in head‐kidney lymphocytes at 4 and 8 h, but decreased to normal level at 12 h. Similarly, stimulation with ConA in vivo also led to an increase in the cd8 mRNA level in the spleen. Immunohistochemistry analysis demonstrated that Cd8α‐positive cells can be detected in the thymus, spleen and intestine by using polyclonal anti‐Cd8α antibody.     

5α-reduction of an anti-androgen TSAA-291, 16β-ethyl-17β-hydroxy-4-estren-3-one,by nuclear 5α-reductase in rat prostates

Inhibition of 5α-reduction of testosterone by an anti-androgen TSAA-291 (16β-ethyl-17β-hydroxy-4-estren-3-one) was studied in rat ventral prostates and the metabolic conversion of ³H-TSAA-291 was examined both $\text{in vitro}$ and $\text{in vivo}$. In the $\text{in vitro}$ experiment using nuclear 5α-reductase of the prostate, 5α-dihydrotestosterone formation from ³H-testosterone was inhibited in a competitive manner by the anti-androgen. In the $\text{in vitro}$ experiment using ³H-TSAA-291, 5α-reduction of the anti-androgen occurred. One, 2 and 4 hr after an intravenous administration of 140 μCi/rat of ³H-TSAA-291 to castrated rats, the unchanged TSAA-291 accumulated in higher amounts in the ventral prostate than in the plasma, skeletal muscle and levator ani muscle, thereby indicating the selective uptake of the anti-androgen by the androgen target organ. No appreciable amounts of the 5α-reduced metabolite of TSAA-291 were detected in the prostate, thus suggesting that TSAA-291 itself may be responsible for the anti-androgenic properties. The inhibitory potency on the 5α-reductase activity of several other 16β-substituted androstane and estrane analogues was also examined.     

Steroidal 5α-reductase inhibitors using 4-androstenedione as substrate

The aim of this study was to determine the capacity of some progesterone derivatives, to inhibit the conversion of labeled androstenedione ([(3)H] 4-dione) to [(3)H]dihydrotestosterone ([(3)H]DHT) in prostate nuclear membrane fractions, where the 5α-reductase activity is present. The enzyme 5α-reductase catalyzes the 5α-reduction of 4-dione whereas the 17β-hydroxysteroid dehydrogenase catalyzes the transformation of 4-dione to testosterone or 5α-dione to dihydrotestosterone (DHT). Moreover, we also investigated the role of unlabeled 5α-dione in these pathways. In order to determine the inhibitory effect of different concentrations of the progesterone derivatives in the conversion of [(3)H] 4-dione to [(3)H]DHT, homogenates of human prostate were incubated with [(3)H] 4-dione, NADPH and increasing concentrations of non-labeled 5α-dione. The incubating mixture was extracted and purified using thin layer chromatography. The fraction of the chromatogram corresponding to the standard of DHT was separated and the radioactivity determined. The results showed that the presence of [(3)H] 4-dione plus unlabelled 5α-dione produced similar levels of DHT as compared to [(3)H] 4-dione. On the other hand, the results indicated that 17α-hydroxypregn-4-ene-3,20-dione 5 and 4-bromo-17α-hydroxypregn-4-ene-3,20-dione 7b, were the most potent steroids to inhibit the conversion of [(3)H] 4-dione to [(3)H]DHT, showing IC(50) values of 2 and 1.6?nM, respectively.     

Structural determinants in bacterial 2‐keto‐3‐deoxy‐D‐gluconate dehydrogenase KduD for dual‐coenzyme specificity

Short‐chain dehydrogenase/reductase (SDR) is distributed in many organisms, from bacteria to humans, and has significant roles in metabolism of carbohydrates, lipids, amino acids, and other biomolecules. An important intermediate in acidic polysaccharide metabolism is 2‐keto‐3‐deoxy‐d ‐gluconate (KDG). Recently, two short and long loops in Sphingomonas KDG‐producing SDR enzymes (NADPH‐dependent A1‐R and NADH‐dependent A1‐R′) involved in alginate metabolism were shown to be crucial for NADPH or NADH coenzyme specificity. Two SDR family enzymes—KduD from Pectobacterium carotovorum (PcaKduD) and DhuD from Streptococcus pyogenes (SpyDhuD)—prefer NADH as coenzyme, although only PcaKduD can utilize both NADPH and NADH. Both enzymes reduce 2,5‐diketo‐3‐deoxy‐d ‐gluconate to produce KDG. Tertiary and quaternary structures of SpyDhuD and PcaKduD and its complex with NADH were determined at high resolution (approximately 1.6 Å) by X‐ray crystallography. Both PcaKduD and SpyDhuD consist of a three‐layered structure, α/β/α, with a coenzyme‐binding site in the Rossmann fold; similar to enzymes A1‐R and A1‐R′, both arrange the two short and long loops close to the coenzyme‐binding site. The primary structures of the two loops in PcaKduD and SpyDhuD were similar to those in A1‐R′ but not A1‐R. Charge neutrality and moderate space at the binding site of the nucleoside ribose 2′ coenzyme region were determined to be structurally crucial for dual‐coenzyme specificity in PcaKduD by structural comparison of the NADH‐ and NADPH‐specific SDR enzymes. The corresponding site in SpyDhuD was negatively charged and spatially shallow. This is the first reported study on structural determinants in SDR family KduD related to dual‐coenzyme specificity. Proteins 2016; 84:934–947. © 2016 Wiley Periodicals, Inc.