Glucosylation of Isoflavonoids in Engineered Escherichia coli

A glycosyltransferase, YjiC, from Bacillus licheniformis has been used for the modification of the commercially available isoflavonoids genistein, daidzein, biochanin A and formononetin. The in vitro glycosylation reaction, using UDP-α-D-glucose as a donor for the glucose moiety and aforementioned four acceptor molecules, showed the prominent glycosylation at 4′ and 7 hydroxyl groups, but not at the 5^th hydroxyl group of the A-ring, resulting in the production of genistein 4′-O-β-D-glucoside, genistein 7-O-β-D-glucoside (genistin), genistein 4′,7-O-β-D-diglucoside, biochanin A-7-O-β-D-glucoside (sissotrin), daidzein 4′-O-β-D-glucoside, daidzein 7-O-β-D-glucoside (daidzin), daidzein 4′, 7-O-β-D-diglucoside, and formononetin 7-O-β-D-glucoside (ononin). The structures of all the products were elucidated using high performance liquid chromatography-photo diode array and high resolution quadrupole time-of-flight electrospray ionization mass spectrometry (HR QTOFESI/MS) analysis, and were compared with commercially available standard compounds. Significantly higher bioconversion rates of all four isoflavonoids was observed in both in vitro as well as in vivo bioconversion reactions. The in vivo fermentation of the isoflavonoids by applying engineered E. coli BL21(DE3)/ΔpgiΔzwfΔushA overexpressing phosphoglucomutase (pgm) and glucose 1-phosphate uridyltransferase (galU), along with YjiC, found more than 60% average conversion of 200 μM of supplemented isoflavonoids, without any additional UDP-α-D-glucose added in fermentation medium, which could be very beneficial to large scale industrial production of isoflavonoid glucosides.     

An Endoglucanase, EglA, from the Hyperthermophilic Archaeon Pyrococcus furiosus Hydrolyzes β-1,4 Bonds in Mixed-Linkage (1→3),(1→4)-β-d-Glucans and Cellulose

The eglA gene, encoding a thermostable endoglucanase from the hyperthermophilic archaeon Pyrococcus furiosus, was cloned and expressed in Escherichia coli. The nucleotide sequence of the gene predicts a 319-amino-acid protein with a calculated molecular mass of 35.9 kDa. The endoglucanase has a 19-amino-acid signal peptide but not cellulose-binding domain. The P. furiosus endoglucanase has significant amino acid sequence similarities, including the conserved catalytic nucleophile and proton donor, with endoglucanases from glucosyl hydrolase family 12. The purified recombinant enzyme hydrolyzed β-1,4 but not β-1,3 glucosidic linkages and had the highest specific activity on cellopentaose (degree of polymerization [DP] = 5) and cellohexaose (DP = 6) oligosaccharides. To a lesser extent, EglA also hydrolyzed shorter cellodextrins (DP < 5) as well as the amorphous portions of polysaccharides which contain only β-1,4 bonds such as carboxymethyl cellulose, microcrystalline cellulose, Whatman paper, and cotton linter. The highest specific activity toward polysaccharides occurred with mixed-linkage β-glucans such as barley β-glucan and lichenan. Kinetics studies with cellooliogsaccharides and p-nitrophenyl-cellooligosaccharides indicated that the enzyme had three glucose binding subsites (−I, −II, and −III) for the nonreducing end and two glucose binding subsites (+I and +II) for the reducing end from the scissile glycosidic linkage. The enzyme had temperature and pH optima of 100°C and 6.0, respectively; a half-life of 40 h at 95°C; and a denaturing temperature of 112°C as determined by differential scanning calorimetry. The discovery of a thermostable enzyme with this substrate specificity has implications for both the evolution of enzymes involved in polysaccharide hydrolysis and the occurrence of growth substrates in hydrothermal vent environments.     

Hevea Linamarase-A Nonspecific beta-Glycosidase

In the leaf tissue of the cyanogenic plant Hevea brasiliensis, which contains large amounts of linamarin, there is no specific linamarase. In Hevea leaves only one β-glucosidase is detectable. It is responsible for the cleavage of all β-glucosides and β-galactosides occurring in Hevea leaf tissue, including the cyanogenic glucoside linamarin. Therefore, the enzyme is referred to as a β-glycosidase instead of the term β-glucosidase. This β-glycosidase has a broad substrate spectrum and occurs in multiple forms. These homo-oligomeric forms are interconvertible by dissociation-association processes. The monomer is a single protein of 64 kilodaltons.     

Gel-Electrophoretic Separation, Detection, and Characterization of Plant and Bacterial UDP-Glucose Glucosyltransferases

We have developed procedures for detection and characterization of UDP-glucose: glucosyltransferases following electrophoretic separation in nondenaturing polyacrylamide gels. Using digitonin-solubilized membrane protein preparations from a variety of plants and two cellulose-producing bacteria, activity can be demonstrated for several UDP-glucose:β-glucan synthases with an in situ assay following gel electrophoresis. These enzymes can be characterized within the gels with respect to effector requirements and products produced, and several advantages of this assay over solution assays are demonstrated. For example, the clear dependence of plant UDP-glucose:(1→3)-β-glucan synthase on both Ca²⁺ and a β-linked glucoside is shown; bacterial cellulose synthases show direct stimulation within the gel by guanyl oligonucleotide, and the Acetobacter xylinum enzyme appears more stable in the gel assay than in solution assay.     

Characterization of a Novel β-Galactosidase from Bifidobacterium adolescentis DSM 20083 Active towards Transgalactooligosaccharides

This paper reports on the effects of both reducing and nonreducing transgalactooligosaccharides (TOS) comprising 2 to 8 residues on the growth of Bifidobacterium adolescentis DSM 20083 and on the production of a novel β-galactosidase (β-Gal II). In cells grown on TOS, in addition to the lactose-degrading β-Gal (β-Gal I), another β-Gal (β-Gal II) was detected and it showed activity towards TOS but not towards lactose. β-Gal II activity was at least 20-fold higher when cells were grown on TOS than when cells were grown on galactose, glucose, and lactose. Subsequently, the enzyme was purified from the cell extract of TOS-grown B. adolescentis by anion-exchange chromatography, adsorption chromatography, and size-exclusion chromatography. β-Gal II has apparent molecular masses of 350 and 89 kDa as judged by size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively, indicating that the enzyme is active in vivo as a tetramer. β-Gal II had an optimal activity at pH 6 and was not active below pH 5. Its optimum temperature was 35°C. The enzyme showed highest V_max values towards galactooligosaccharides with a low degree of polymerization. This result is in agreement with the observation that during fermentation of TOS, the di- and trisaccharides were fermented first. β-Gal II was active towards β-galactosyl residues that were 1→4, 1→6, 1→3, and 1↔1 linked, signifying its role in the metabolism of galactooligosaccharides by B. adolescentis.     

THE SYNTHESIS AND TURNOVER OF RAT LIVER PEROXISOMES : IV. Biochemical Pathway of Catalase Synthesis

Early events in the biosynthesis of liver catalase were studied on female rats receiving [³H]leucine or [³H]δ-aminolevulinic acid or a mixture of [³H]leucine with [¹⁴C]δ-aminolevulinic acid by intraportal injection. Catalase antigen was selectively separated from homogenates by immunoprecipitation, both without and after partial purification of the enzyme. Label from both precursors appeared first in immunoprecipitable material which was lost upon purification of catalase; the label subsequently became associated with material indistinguishable from catalase. Kinetic analysis of the results indicates that the nonpurifiable material identified by early labeling consists of two distinct biosynthetic intermediates, the first lacking heme and representing about 1.6% of the total catalase content or 13 µg/g liver, the second containing heme and representing about 0.5% of the total catalase content or 4 µg/g liver. The first intermediate migrates at the same rate as catalase upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and therefore has a monomeric molecular weight of about 60,000.     

Production and Characterization of Amylase from Calvatia gigantea

α-Amylase (EC 3.2.1.1) was excreted by Calvatia gigantea in liquid growth media containing different sources of starch. Among the factors affecting enzyme production in shake flasks were the type and the concentration of starch and the nitrogen source supplied. Optimum cultural conditions for maximum enzyme production were: soluble starch concentration, 5%; inoculum size, 3.75 × 10⁵ conidia per ml; 5-day cultivation time at 28 to 30°C. The observed maximum yield of 81.3 U of saccharifying enzyme activity per ml of growth medium was the highest ever reported in the literature for submerged cultures. Partially purified enzyme functioned optimally at pH 4.5 to 5.5 and 53 to 58°C. The activation energy of enzymic hydrolysis of starch in the range of 20 to 40°C was 8,125 cal/mol (ca. 3.41 × 10⁴ J). The apparent K_m value of the enzyme at 25°C was 7.68 × 10⁻⁴ g/ml. Some of the properties of the enzyme under investigation were similar to those of α-amylases excreted from molds producing large amounts of the enzyme.     

Acylated flavonol glucosides of Pinus contorta needles

From fresh Pinus contorta Doug (Coastal) needles four flavonol acylated glucosides and 6-methyl-kaempferol 3-β-D-glucoside were isolated. The three monoacylated glucosides were kaempferol-3-β-D(6-O-p- coumaryl)glucoside, isorhamnetin-3-β-D-(6-O-acetyl)glucoside, quercetin-3-β-D-(p-coumaryl)glucoside and the diacyl compound was kaempferol-3-β-D-(di-p-coumaryl)glucoside.     

Roles of MPBQ-MT in Promoting α/γ-Tocopherol Production and Photosynthesis under High Light in Lettuce

2-methyl-6-phytyl-1, 4-benzoquinol methyltransferase (MPBQ-MT) is a vital enzyme catalyzing a key methylation step in both α/γ-tocopherol and plastoquinone biosynthetic pathway. In this study, the gene encoding MPBQ-MT was isolated from lettuce (Lactuca sativa) by rapid amplification of cDNA ends (RACE), named LsMT. Overexpression of LsMT in lettuce brought about a significant increase of α- and γ-tocopherol contents with a reduction of phylloquinone (vitamin K1) content, suggesting a competition for a common substrate phytyl diphosphate (PDP) between the two biosynthetic pathways. Besides, overexpression of LsMT significantly increased plastoquinone (PQ) level. The increase of tocopherol and plastoquinone levels by LsMT overexpression conduced to the improvement of plants’ tolerance and photosynthesis under high light stress, by directing excessive light energy toward photosynthetic production rather than toward generation of more photooxidative damage. These findings suggest that the role and function of MPBQ-MT can be further explored for enhancing vitamin E value, strengthening photosynthesis and phototolerance under high light in plants.     

Recognition of (Sesc) for Easy Identification of Staphylococcus Epidermidis and Molecular and Phenotypic Study of Β-Lactam Resistance in Staphylococcus Epidermidis Isolates in Isfahan

Background:Not only is it crucial to rapidly detect Staphylococcus epidermidis (S. epidermidis) isolates from a broad range of bacteria, but recognizing resistance agents can greatly improve current diagnostic and therapeutic strategies.Methods:The current cross-sectional study investigated 120 clinical isolates from a nosocomial S. epidermidis infection. The isolates were identified using common biochemical tests, and specific S. epidermidis surface protein C (SesC) primers were used to confirm the presence of S. epidermidis. PCR and special primers were used to detect the β-lactamase gene (blaZ). Methicillin resistance was measured using the agar screening method and antibiotic susceptibility was measured by disk diffusion. Results:100 samples were characterized as S. epidermidis using a phenotypic and genotypic methods. From the 100 specimens examined, 80% contained blaZ. According to agar screening, 60% of isolates were methicillin-resistant. S. epidermidis isolates demonstrated the highest resistance to penicillin (93%) and the highest sensitivity to cefazolin (39%).Conclusion:The increased resistance to β-lactam antibiotics in S. epidermidis isolates is alarming, and certain precautions should be taken by healthcare systems to continuously monitor the antimicrobial pattern of S. epidermidis, so that an appropriate drug treatment can be established.Key Words: Antibiotic resistance, β-lactam, Staphylococcus epidermidis     

Novel Maltotriose-Hydrolyzing Thermoacidophilic Type III Pullulan Hydrolase from Thermococcus kodakarensis

A novel thermoacidophilic pullulan-hydrolyzing enzyme (PUL) from hyperthermophilic archaeon Thermococcus kodakarensis (TK-PUL) that efficiently hydrolyzes starch under industrial conditions in the absence of any additional metal ions was cloned and characterized. TK-PUL possessed both pullulanase and α-amylase activities. The highest activities were observed at 95 to 100°C. Although the enzyme was active over a broad pH range (3.0 to 8.5), the pH optima for both activities were 3.5 in acetate buffer and 4.2 in citrate buffer. TK-PUL was stable for several hours at 90°C. Its half-life at 100°C was 45 min when incubated either at pH 6.5 or 8.5. The K_m value toward pullulan was 2 mg ml⁻¹, with a V_max of 109 U mg⁻¹. Metal ions were not required for the activity and stability of recombinant TK-PUL. The enzyme was able to hydrolyze both α-1,6 and α-1,4 glycosidic linkages in pullulan. The most preferred substrate, after pullulan, was γ-cyclodextrin, which is a novel feature for this type of enzyme. Additionally, the enzyme hydrolyzed a variety of polysaccharides, including starch, glycogen, dextrin, amylose, amylopectin, and cyclodextrins (α, β, and γ), mainly into maltose. A unique feature of TK-PUL was the ability to hydrolyze maltotriose into maltose and glucose.     

Core Bioactive Components Promoting Blood Circulation in the Traditional Chinese Medicine Compound Xueshuantong Capsule (CXC) Based on the Relevance Analysis between Chemical HPLC Fingerprint and In Vivo Biological Effects

Compound xueshuantong capsule (CXC) is an oral traditional Chinese herbal formula (CHF) comprised of Panax notoginseng (PN), Radix astragali (RA), Salvia miltiorrhizae (SM), and Radix scrophulariaceae (RS). The present investigation was designed to explore the core bioactive components promoting blood circulation in CXC using high-performance liquid chromatography (HPLC) and animal studies. CXC samples were prepared with different proportions of the 4 herbs according to a four-factor, nine-level uniform design. CXC samples were assessed with HPLC, which identified 21 components. For the animal experiments, rats were soaked in ice water during the time interval between two adrenaline hydrochloride injections to reduce blood circulation. We assessed whole-blood viscosity (WBV), erythrocyte aggregation and red corpuscle electrophoresis indices (EAI and RCEI, respectively), plasma viscosity (PV), maximum platelet aggregation rate (MPAR), activated partial thromboplastin time (APTT), and prothrombin time (PT). Based on the hypothesis that CXC sample effects varied with differences in components, we performed grey relational analysis (GRA), principal component analysis (PCA), ridge regression (RR), and radial basis function (RBF) to evaluate the contribution of each identified component. Our results indicate that panaxytriol, ginsenoside Rb₁, angoroside C, protocatechualdehyde, ginsenoside Rd, and calycosin-7-O-β-D-glucoside are the core bioactive components, and that they might play different roles in the alleviation of circulation dysfunction. Panaxytriol and ginsenoside Rb₁ had close relevance to red blood cell (RBC) aggregation, angoroside C was related to platelet aggregation, protocatechualdehyde was involved in intrinsic clotting activity, ginsenoside Rd affected RBC deformability and plasma proteins, and calycosin-7-O-β-D-glucoside influenced extrinsic clotting activity. This study indicates that angoroside C, calycosin-7-O-β-D-glucoside, panaxytriol, and protocatechualdehyde may have novel therapeutic uses.     

Exploiting Natural Variation of Secondary Metabolism Identifies a Gene Controlling the Glycosylation Diversity of Dihydroxybenzoic Acids in Arabidopsis thaliana

Plant secondary metabolism is an active research area because of the unique and important roles the specialized metabolites have in the interaction of plants with their biotic and abiotic environment, the diversity and complexity of the compounds and their importance to human medicine. Thousands of natural accessions of Arabidopsis thaliana characterized with increasing genomic precision are available, providing new opportunities to explore the biochemical and genetic mechanisms affecting variation in secondary metabolism within this model species. In this study, we focused on four aromatic metabolites that were differentially accumulated among 96 Arabidopsis natural accessions as revealed by leaf metabolic profiling. Using UV, mass spectrometry, and NMR data, we identified these four compounds as different dihydroxybenzoic acid (DHBA) glycosides, namely 2,5-dihydroxybenzoic acid (gentisic acid) 5-O-β-D-glucoside, 2,3-dihydroxybenzoic acid 3-O-β-D-glucoside, 2,5-dihydroxybenzoic acid 5-O-β-D-xyloside, and 2,3-dihydroxybenzoic acid 3-O-β-D-xyloside. Quantitative trait locus (QTL) mapping using recombinant inbred lines generated from C24 and Col-0 revealed a major-effect QTL controlling the relative proportion of xylosides vs. glucosides. Association mapping identified markers linked to a gene encoding a UDP glycosyltransferase gene. Analysis of Transfer DNA (T-DNA) knockout lines verified that this gene is required for DHBA xylosylation in planta and recombinant protein was able to xylosylate DHBA in vitro. This study demonstrates that exploiting natural variation of secondary metabolism is a powerful approach for gene function discovery.     

Light dependence of catalase synthesis and degradation in leaves and the influence of interfering stress conditions

The enzyme catalase (EC 1.11.1.6) is light sensitive and subject to a rapid turnover in light, similar to the D1 reaction center protein of photosystem II. After 3 h of preadaptation to darkness or to different light intensities (90 and 520 μmol m⁻² s⁻¹ photosynthetic photon flux density), sections of rye leaves (Secale cereale L.) were labeled for 4 h with l-[³⁵S]methionine. From leaf extracts, catalase was immunoprecipitated with an antiserum prepared against the purified enzyme from rye leaves. Both incorporation into catalase and degradation of the enzyme polypeptide during a subsequent 16-h chase period increased with light intensity. At a photon flux density of 520 μmol m⁻² s⁻¹, the apparent half-time of catalase in rye leaves was 3 to 4 h, whereas that of the D1 protein was much shorter, about 1.5 h. Exposure to stress conditions, such as 0.6 m NaCl or a heat-shock temperature of 40°C, greatly suppressed both total protein synthesis and incorporation of the label into catalase and into the D1 protein. Immunoblotting assays indicated that in light, but not in darkness, steady-state levels of catalase and of the D1 protein strongly declined during treatments with salt, heat shock, or translation inhibitors that block repair synthesis. Because of the common property of rapid photodegradation and the resulting dependence on continuous repair, declines in catalase as well as of the D1 protein represent specific and sensitive indicators for stress conditions that suppress the translational activities of leaves.     

Does Short-Term Vitamin C Reduce Cardiovascular Risk in Type 2 Diabetes?

ObjectiveVitamin C is a powerful antioxidant that is potentially useful in the prevention of atherosclerosis in diabetic individuals. However, the mechanism(s) of vitamin C’s anti-atherosclerotic effects in vivo are unresolved, and clinical trials in nondiabetic individuals have yielded conflicting results. Therefore, we performed 32 studies in a randomized, crossover, dose-response trial in 8 volunteers with type 2 diabetes to determine the effects of vitamin C on serum vitamin C levels, lipids, inflammation, and coagulation.MethodsWell-controlled, type 2 volunteers received, in randomized order for 2-week periods, each of the following: 1) no supplemental vitamin C, 2) low-dose vitamin C (250 mg/day), 3) medium-dose vitamin C (500 mg/ day), and 4) high-dose vitamin C (1,000 mg/day). A high-caloric content lunch was ingested during each study arm to enhance oxidative stress. Serum vitamin C levels and atherosclerotic risk factors including lipids and markers of oxidative stress, inflammation, and hypercoagulation were determined.ResultsSerum vitamin C levels increased significantly at all dosages. In addition, the high-caloric content meal resulted in acute elevations of glucose, insulin, and triglycerides for several hours postmeal. However, no significant effect of vitamin C was observed on lipid parameters or any of the surrogate markers of oxidative stress, inflammation, or hypercoagulability.ConclusionOur study suggests that if vitamin C does have anti-atherosclerotic effects in diabetes, it does not exert them through the traditional pathways identifiable by established surrogate markers of cardiovascular risk. (Endocr Pract. 2013;19:785-791)     

Maternal Education and Micro-Geographic Disparities in Nutritional Status among School-Aged Children in Rural Northwestern China

Objectives

Prior evidence suggests geographic disparities in the effect of maternal education on child nutritional status between countries, between regions and between urban and rural areas. We postulated its effect would also vary by micro-geographic locations (indicated by mountain areas, plain areas and the edge areas) in a Chinese minority area.

Methods

A cross-sectional study was conducted with a multistage random sample of 1474 school children aged 5-12 years in Guyuan, China. Child nutritional status was measured by height-for-age z scores (HAZ). Linear mixed models were used to examine its association with place of residence and maternal education.

Results

Micro-geographic disparities in child nutritional status and the level of socioeconomic composition were found. Children living in mountain areas had poorer nutritional status, even after adjusting for demographic (plain versus mountain, β = 0.16, P = 0.033; edge versus mountain, β = 0.29, P = 0.002) and socioeconomic factors (plain versus mountain, β = 0.12, P = 0.137; edge versus mountain, β = 0.25, P = 0.009). The disparities significantly widened with increasing years of mothers’ schooling (maternal education*plain versus mountain: β = 0.06, P = 0.007; maternal education*edge versus mountain: β = 0.07, P = 0.005). Moreover, the association between maternal education and child nutrition was negative (β = -0.03, P = 0.056) in mountain areas but positive in plain areas (β = 0.02, P = 0.094) or in the edge areas (β = 0.04, P = 0.055).

Conclusions

Micro-geographic disparities in child nutritional status increase with increasing level of maternal education and the effect of maternal education varies by micro-geographic locations, which exacerbates child health inequity. Educating rural girls alone is not sufficient; improving unfavorable conditions in mountain areas might make such investments more effective in promoting child health. Nutrition programs targeting to the least educated groups in plain and in edge areas would be critical to their cost-effectiveness.     

Cell-Wall-Bound Proteinase of Lactobacillus delbrueckii subsp. lactis ACA-DC 178: Characterization and Specificity for β-Casein

Lactobacillus delbrueckii subsp. lactis ACA-DC 178, which was isolated from Greek Kasseri cheese, produces a cell-wall-bound proteinase. The proteinase was removed from the cell envelope by washing the cells with a Ca²⁺-free buffer. The crude proteinase extract shows its highest activity at pH 6.0 and 40°C. It is inhibited by phenylmethylsulfonyl fluoride, showing that the enzyme is a serine-type proteinase. Considering the substrate specificity, the enzyme is similar to the lactococcal P_I-type proteinases, since it hydrolyzes β-casein mainly and α- and κ-caseins to a much lesser extent. The cell-wall-bound proteinase from L. delbrueckii subsp. lactis ACA-DC 178 liberates four main peptides from β-casein, which have been identified.     

Characterization of an Allylic/Benzyl Alcohol Dehydrogenase from Yokenella sp. Strain WZY002, an Organism Potentially Useful for the Synthesis of α,β-Unsaturated Alcohols from Allylic Aldehydes and Ketones

A novel whole-cell biocatalyst with high allylic alcohol-oxidizing activities was screened and identified as Yokenella sp. WZY002, which chemoselectively reduced the C=O bond of allylic aldehydes/ketones to the corresponding α,β-unsaturated alcohols at 30°C and pH 8.0. The strain also had the capacity of stereoselectively reducing aromatic ketones to (S)-enantioselective alcohols. The enzyme responsible for the predominant allylic/benzyl alcohol dehydrogenase activity was purified to homogeneity and designated YsADH (alcohol dehydrogenase from Yokenella sp.), which had a calculated subunit molecular mass of 36,411 Da. The gene encoding YsADH was subsequently expressed in Escherichia coli, and the purified recombinant YsADH protein was characterized. The enzyme strictly required NADP(H) as a coenzyme and was putatively zinc dependent. The optimal pH and temperature for crotonaldehyde reduction were pH 6.5 and 65°C, whereas those for crotyl alcohol oxidation were pH 8.0 and 55°C. The enzyme showed moderate thermostability, with a half-life of 6.2 h at 55°C. It was robust in the presence of organic solvents and retained 87.5% of the initial activity after 24 h of incubation with 20% (vol/vol) dimethyl sulfoxide. The enzyme preferentially catalyzed allylic/benzyl aldehydes as the substrate in the reduction of aldehydes/ketones and yielded the highest activity of 427 U mg⁻¹ for benzaldehyde reduction, while the alcohol oxidation reaction demonstrated the maximum activity of 79.9 U mg⁻¹ using crotyl alcohol as the substrate. Moreover, kinetic parameters of the enzyme showed lower K_m values and higher catalytic efficiency for crotonaldehyde/benzaldehyde and NADPH than for crotyl alcohol/benzyl alcohol and NADP⁺, suggesting the nature of being an aldehyde reductase.