Evidence for a tungsten-stimulated aldehyde dehydrogenase activity of Desulfovibrio simplex that oxidizes aliphatic and aromatic aldehydes with flavins as coenzymes

The aldehyde dehydrogenase activity of the sulfate-reducing bacterium Desulfovibrio simplex strain DSM 4141 was characterized in cell-free extracts. Oxygen-sensitive, constitutive aldehyde dehydrogenase activity was found in cells grown on l(+)-lactate, hydrogen, or vanillin with sulfate as the electron acceptor. A 1.83- to 2.6-fold higher specific activity was obtained in cells grown in media supplemented with 1 μM WO₄ ^2–. The aldehyde dehydrogenase in cell-free extracts catalyzed the oxidation of aliphatic (K _m < 20 μM) and aromatic aldehydes (K _m < 0.32 mM) using methyl viologen as the electron acceptor. Flavins (FMN and FAD) were also active and are proposed to be the natural cofactors, while no activity was obtained with NAD⁺ or NADP⁺. ¹⁸⁵WO₄ ^2– was incorporated in vivo into D. simplex; it was found exclusively in the soluble fraction (≥ 98%). Anionic-exchange chromatography demonstrated coelution of ¹⁸⁵W with two distinct peaks, the first one containing hydrogenase and formate dehydrogenase activities, and the second one aldehyde dehydrogenase activity. Received: 7 February 1997 / Accepted: 6 June 1997     

Oxidation of lactaldehyde by cytosolic aldehyde dehydrogenase and inhibition of cytosolic and mitochondrial aldehyde dehydrogenase by metabolites

An enzyme fraction which oxidizes lactaldehyde to lactic acid has been purified from goat liver. This enzyme was found to be identical with the cytosolic aldehyde dehydrogenase. Lactaldehyde was found to be primarily oxidized by this enzyme. Almost 90% of the total lactaldehyde-oxidizing activity is located in the cytosol. Methylglyoxal and glyceraldehyde 3-phosphate were found to be strong competitive inhibitors of this enzyme. Aldehyde dehydrogenase from goat liver mitochondria has also been partially purified and found to be strongly inhibited by these metabolites. The inhibitory effects of these metabolites on both these enzymes are highly pH dependent. The inhibitory effects of both the metabolites have been found to be stronger for the cytosolic enzyme at pH values higher than the physiological pH. For the mitochondrial enzyme, the inhibition with methylglyoxal was more pronounced at higher pH values, whereas stronger inhibition was observed with glyceraldehyde 3-phosphate at physiological pH.     

Haloenol lactones as inactivators and substrates of aldehyde dehydrogenase

Human aldehyde dehydrogenase (EC 1.2.1.3) isozymes E1 and E2 were irreversibly inactivated by stoichiometric concentrations of the haloenol lactones 3-isopropyl-6(E)-bromomethylene tetrahydro-pyran-2-one and 3-phenyl-6(E)-bromomethylene tetrahydro-pyran-2-one. No inactivation occurred with the corresponding nonhalogenated enol lactones. Both the dehydrogenase and esterase activities were abolished. Activity was not regained on dialysis or treatment with 2-mercaptoethanol. The inactivation was subject to substrate protection: NAD afforded protection which increased in the presence of the aldehyde-substrate competitive inhibitor chloral. Saturation kinetics gave positivey-axis intercepts, allowing the determination of binding constants. Inactivation stiochiometry determined with¹⁴C-labeled 3-(1-naphthyl)-6(E)-iodomethylene tetrahydropyran-2-one was found to correspond to the active-site number. The nonhalogenated lactone, 3-(1-naphthyl)-6(E)-methylene tetrahydropyran-1-one was shown to be a substrate for aldehyde dehydrogenase via its esterase function. Inactivation and enzymatic hydrolysis occurred within a similar time frame. Opening of the lactone ring to form enzyme-acyl intermediate with active site cysteine appears to be a necessary prerequisite to inactivation, since halogen in the lactone ring is nonreactive. Thus, the inactivation of aldehyde dehydrogenase by haloenol lactones is mechanism-based. Inactivation by haloenol lactones occurs in a manner analogous to that of chymotrypsin with which aldehyde dehydrogenase shares esterase activity and binding of haloenol lactones at the active site.     

Protective effect of aldehyde dehydrogenase 2 against rat corneal dysfunction caused by streptozotocin-induced type I diabetes

Aldehyde dehydrogenase 2 plays a pivotal role in detoxifying aldehydes, and our previous study revealed that aldehyde dehydrogenase 2 could alleviate diabetic retinopathy-associated damage. We aimed to characterize the potential role of aldehyde dehydrogenase 2 in diabetic keratopathy. Twenty-four rats with streptozotocin-induced (60 mg/kg, single intraperitoneal injection) type 1 diabetes mellitus (T1DM) were divided the T1DM group and the T1DM + Alda1 (an activator of aldehyde dehydrogenase 2) group (5 mg/kg/d, intraperitoneal injection, 1/2/3 months), while an additional 12 healthy rats served as the control group. Corneal morphology was examined in vivo and in vitro at one, two, and three months after T1DM induction. Additionally, serum inflammatory factors were measured by ELISA, and the expression of corneal vascular endothelial growth factor A (VEGF-A) and aldehyde dehydrogenase 2 was measured by immunofluorescence staining. Corneal cell death was evaluated by terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) staining. Slit lamp analysis showed that the area of corneal epithelial cell injury in the T1DM + Alda1 group was significantly smaller than that in the T1DM group at one and two months after T1DM induction (all P < 0.05). OCT analysis and HE staining showed that the central corneal thickness (indication of corneal edema) and the epithelial keratinization level in the T1DM + Alda1 group was evidently decreased compared with those in the T1DM group (all P < 0.05). The serum inflammatory factors interleukin-1 and interleukin-6 were significantly upregulated in the T1DM group compared with the T1DM + Alda1 group at three months after T1DM induction (all P < 0.05), while there were no differences in SOD or TNF-α levels among all groups. Furthermore, corneal VEGF-A expression and corneal cell death in the T1DM + Alda1 group were dramatically reduced compared to those in the T1DM group (all P < 0.05). In conclusion, the aldehyde dehydrogenase 2 agonist Alda1 attenuated rat corneal dysfunction induced by T1DM by alleviating corneal edema, decreasing corneal cell death, and downregulating corneal VEGF-A expression.     

Pesticide induced up-regulation of esterase and aldehyde dehydrogenase in indigenous Bacillus spp.

During bacterial growth an increased mRNA level is usually linked with higher rate of metabolism related to biodegradation of an unusual compound. In this study, quantitative gene expression data derived from mRNA level reveal the presence of pesticide degrading genes in 6 bacterial isolates showing biodegradation of cypermethrin (SG2, SG4), sulfosulfuron (SA2, Sulfo3), and fipronil (FA3, FA4). A correlation existed between the level of esterase coding mRNA and mineralization of cypermethrin in SG2 and SG4. Similarly the level of EST coding mRNA increased with biodegradation of fipronil and sulfosulfuron in FA3, FA4, SA2, and Sulfo3. Expression of est gene was observed in all the bacterial strains, but their level of expression was different. Bacterial strains SG4 and Sulfo3 showed higher level of est gene expression as compared to SG2, FA3, FA4, and SA2 and was in the range of approximately 30- to 60-fold, respectively, in comparison to control. Expression of genes for aldehyde dehydrogenase was observed in SG4 and Sulfo3. We report co expression of aldh (1000?bp) and est (～550?bp) genes at the same time of pesticide induction/biodegradation.     

The N-terminal portion of mature aldehyde dehydrogenase affects protein folding and assembly

Human liver cytosolic (ALDH1) and mitochondrial (ALDH2) aldehyde dehydrogenases are both encoded in the nucleus and synthesized in the cytosol. ALDH1 must fold in the cytosol, but ALDH2 is first synthesized as a precursor and must remain unfolded during import into mitochondria. The two mature forms share high identity (68%) at the protein sequence level except for the first 21 residues (14%); their tertiary structures were found to be essentially identical. ALDH1 folded faster in vitro than ALDH2 and could assemble to tetramers while ALDH2 remained as monomers. Import assay was used as a tool to study the folding status of ALDH1 and ALDH2. pALDH1 was made by fusing the presequence of precursor ALDH2 to the N-terminal end of ALDH1. Its import was reduced about 10-fold compared to the precursor ALDH2. The exchange of the N-terminal 21 residues from the mature portion altered import, folding, and assembly of precursor ALDH1 and precursor ALDH2. More of chimeric ALDH1 precursor was imported into mitochondria compared to its parent precursor ALDH1. The import of chimeric ALDH2 precursor, the counterpart of chimeric ALDH1 precursor, was reduced compared to its parent precursor ALDH2. Mature ALDH1 proved to be more stable against urea denaturation than ALDH2. Urea unfolding improved the import of precursor ALDH1 and the chimeric precursors but not precursor ALDH2, consistent with ALDH1 and the chimeric ALDHs being more stable than ALDH2. The N-terminal segment of the mature protein, and not the presequence, makes a major contribution to the folding, assembly, and stability of the precursor and may play a role in folding and hence the translocation of the precursor into mitochondria.     

Methylglyoxal as substrate and inhibitor of human aldehyde dehydrogenase: Comparison of kinetic properties among the three isozymes

Methylglyoxal was demonstrated to be a substrate for the isozymes E1, E2 and E3 of human aldehyde dehydrogenase. Pyruvate was the product from the oxidation of methylglyoxal by the three isozymes. At pH 7.4 and 25^oC, the major and minor components of the E3 isozyme catalyzed the reaction with V_max of 1.1 and 0.8 μmol NADH min⁻¹ mg⁻¹ protein, respectively, compared to 0.067 and 0.060 μmol NADH min⁻¹ mg⁻¹ protein for the E1 and E2 isozymes, respectively. The E2 isozyme had a K_m for methylglyoxal of 8.6 μM, the lowest compared to 46 μM for E1 and 586 and 552 μM for the major and minor components of the E3 isozyme, respectively. Both components of the E3 isozyme showed substrate inhibition by methylglyoxal, with K_i values of 2.0 mM for the major component and 12 mM for the minor component at pH 9.0. Substrate inhibition by methylglyoxal was not observed with the E1 and E2 isozymes. Methylglyoxal strongly inhibited the glycolaldehyde activity of the E1 and E2 isozymes. Mixed-type models of inhibition were employed as an approach to calculate the inhibition constants, 44 and 10.6 μM for E1 and E2 isozymes, respectively.     

Inactivation of aldehyde dehydrogenase: a key factor for engineering 1,3-propanediol production by Klebsiella pneumoniae

Production of 1,3-propanediol (1,3-PD) from glycerol by Klebsiella pneumoniae is restrained by ethanol formation. The first step in the formation of ethanol from acetyl-CoA is catalyzed by aldehyde dehydrogenase (ALDH), an enzyme that competes with 1,3-PD oxidoreductase for the cofactor NADH. This study aimed to improve the production of 1,3-PD by engineering the ethanol formation pathway. An inactivation mutation of the aldA gene encoding ALDH in K. pneumoniae YMU2 was generated by insertion of a tetracycline resistance marker. Inactivation of ALDH resulted in a nearly abolished ethanol formation but a significantly improved 1,3-PD production. Metabolic flux analysis revealed that a pronounced redistribution of intracellular metabolic flux occurred. The final titer, the productivity of 1,3-PD and the yield of 1,3-PD relative to glycerol of the mutant strain reached 927.6 mmol L(-1), 14.05 mmol L(-1)h(-1) and 0.699 mol mol(-1), respectively, which were much higher than those of the parent strain. In addition, the specific 1,3-PD-producing capability (1,3-PD produced per gram of cells) of the mutant strain was 2-fold that of the parent strain due to a lower growth yield of the mutant. By increasing NADH availability, this study demonstrates an important metabolic engineering approach to improve the efficiency of oxidoreduction-coupled bioprocesses.     

Rat sperm galactosyl receptor: Purification and identification by polyclonal antibodies raised against multiple antigen peptides

We have previously reported the purification of rat testis galactosyl receptor, an equivalent to the Ca²⁺-dependent (C-type) minor variant of rat hepatic lectin-2/3 (RHL-2/3). We now report the purification of galactosyl receptor from rat sperm and its immunolocalization in the intact rat testis and sperm by polyclonal antibodies prepared using multiple antigen peptides (MAP) as immunogens. Two MAP antigens (designated 27-mer and 28-mer), corresponding to amino acid sequences of the carbohydrate-recognition domain (galactose) and adjacent Ca²⁺-binding sites of RHL-2/3, were used for immunization. Anti-RHL-2/3, anti-p27, and anti-p28 sera crossreacted with rat hepatocyte RHL-2/3 and its rat testis and sperm equivalent, galactosyl receptor, purified by chromatofocusing followed by galactose-Hydropore-EP affinity chromatography. Neither anti-p27 nor anti-p28 sera crossreacted with the major hepatocyte variant, RHL-1. A RHL-1-equivalent was not detected in rat testis and sperm. Immunofluorescence studies demonstrated that anti-p27 and anti-p28 sera recognize galactosyl receptor sites at the Sertoli cell-spermatogenic cell interface and on the dorsal surfacae of the sperm head, overlying the acrosome. The characteristic crescent-shaped immunoreactive pattern in sperm was lost after induction of the acrosome reaction. Further studies should determine whether antisera to MAP antigens 27-mer and 28-mer, corresponding to specific protein motifs, can serve as immunological probes for examining cell-cell interaction events during spermatogenesis and at fertilization. © 1995 Wiley-Liss, Inc.     

Microbial oxidation of methane and methanol: Purification and properties of a heme-containing aldehyde dehydrogenase from Methylomonas methylovora

Procedures for the purification of an aldehyde dehydrogenase from extracts of the obligate methylotroph, Methylomonas methylovora are described. The purified enzyme is homogeneous as judged from polyacrylamide gel electrophoresis. In the presence of an artificial electron acceptor (phenazine methosulfate), the purified enzyme catalyzes the oxidation of straight chain aldehydes (C₁-C₁₀ tested), aromatic aldehydes (benzaldehyde, salicylaldehyde), glyoxylate, and glyceraldehyde. Biological electron acceptors such as NAD⁺, NADP⁺, FAD, FMN, pyridoxal phosphate, and cytochrome c cannot act as electron carriers. The activity of the enzyme is inhibited by sulfhydryl agents [p-chloromercuribenzoate, N-ethylmaleimide and 5,5-dithiobis (2-nitrobenzoic acid)], cuprous chloride, and ferrour nitrate. The molecular weight of the enzyme as estimated by gel filtration is approximately 45000 and the subunit size determined by sodium dodecyl sulfate-gel electrophoresis is approximately 23000. The purified enzyme is light brown and has an absorption peak at 410 nm. Reduction of enzyme with sodium dithionite or aldehyde substrate resulted in the appearance of peaks at 523 nm and 552 nm. These results suggest that the enzyme is a hemoprotein. There was no evidence that flavins were present as prosthetic group. The amino acid composition of the enzyme is also presented.Non-Standard Abbreviations PMS phenazine methosulfate - DCPIP 2,6-dichlorophenol indophenol - DEAE diethylaminoethyl     

Adaptation of Pseudomonas sp. GJ1 to 2-bromoethanol caused by overexpression of an NAD-dependent aldehyde dehydrogenase with low affinity for halogenated aldehydes

Pseudomonas sp. GJ1 is able to grow with 2-chloroethanol as the sole carbon and energy source, but not with 2-bromoethanol, which is toxic at low concentrations (1 mM). A muatnt that could grow on 2-bromoethanol with a growth rate of 0.034 h^–1 at concentrations up to 5 mM was isolated and designated strain GJ1M9. Measurement of enzyme activities showed that mutant and wild-type strains contained a PMS-linked alcohol dehydrogenase that was active with halogenated alcohols and that was threefold overexpressed in the mutant when grown on 2-chloroethanol, but only slightly overproduced when grown on 2-bromoethanol. Both strains also contained an NAD-dependent alcohol dehydrogenase that had no activity with halogenated alcohols. Haloacetate dehalogenase levels were similar in the wild-type and the mutant. Activities of NAD-dependent aldehyde dehydrogenase were only slightly higher in extracts of the mutant grown with 2-bromoethanol than in those of the wild-type grown with 2-chloroethanol. SDS-PAGE, however, showed that this enzyme amounted to more than 50% of the total cellular protein in extracts of the mutant from 2-bromoethanol-grown cells, which was fourfold higher than in extracts of the wild-type strain grown on 2-chloroethanol. The enzyme was purified and shown to be a tetrameric protein consisting of subunits of 55 kDa. The enzyme had low K_m values for acetaldehyde and other non-halogenated aldehydes (0.8–4 μM), but much higher K_m values for chloroacetaldehyde (1.7 mM) and bromoacetaldehyde (10.5 mM), while V_max values were similar for halogenated and non-halogenated aldehydes. Cultures that were pregrown on 2-chloroethanol rapidly lost aldehyde dehydrogenase activity after addition of 2-bromoethanol and chloroamphenicol, which indicates that bromoacetaldehyde inactivates the enzyme. To achieve growth with 2-bromoethanol, the high expression of the enzyme thus appears to be necessary in order to compensate for the high K_m for bromoacetaldehyde and for inactivation of the enzyme by bromoacetaldehyde. Received: 31 August 1995 / Accepted: 4 December 1995     

Genetics of ocular NAD+-dependent alcohol dehydrogenase and aldehyde dehydrogenase in the mouse: Evidence for genetic identity with stomach isozymes and localization ofAhd-4 on chromosome 11 near trembler

Electrophoretic and activity variation of the stomach and ocular isozyme of aldehyde dehydrogenase (designated AHD-4) was observed between C57BL/6J and SWR/J inbred strains of mice. The phenotypes were inherited in a normal mendelian fashion, with two alleles at a single locus (Ahd-4) showing codominant expression. The alleles assorted independently of those atAdh-3 [encoding the stomach and ocular isozyme of alcohol dehydrogenase (ADH-C₂)] on chromosome 3. Three chromosome 11 markers, hemoglobin -chain (Hba), trembler (Tr), and rex (Re), were used in backcross analyses which established thatAhd-4 is closely linked to trembler. The distribution patterns for stomach and ocular AHD-4 phenotypes were examined among SWXL recombinant inbred mice, and those for stomach and ocular ADH-C₂ among BXD recombinant inbred strains. The data provided evidence for the genetic identity of stomach and ocular ADH-C₂ and of stomach and ocular AHD-4.This research was supported in part by the U.S. Department of Energy under Contract DE-ACO5-84OR214000 with Martin Marietta Energy Systems, Inc. (to R.A.P.).     

Characteristics and aldehyde dehydrogenase activity of four rat hepatoma cell lines produced by diethylnitrosamine-phenobarbital treatment

Summary Recent studies in our laboratory have shown that five established rat hepatoma cell lines provide a wide spectrum of tumor-associated aldehyde dehydrogenase (ALDH) activity representative of the range of activities of this enzyme seen in primary rat hepatocellular carcinomas. Four newly established rat hepatoma cell lines, RLT-2M, RLT-3C, RLT-9F, and RLT-5G, were derived from a primary hepatocellular carcinoma. The primary tumor was induced by a single injection of diethylnitrosamine (15 μM/g body weight) to a 1-d-old female S-D rat followed at weaning by chronic phenobarbital treatment. RLT-2M was established from outgrowths of minced tumor pieces. RLT-3C, RLT-9F, and RLT-5G were cloned from RLT-2M by the serial endpoint dilution. All four lines have been maintained in culture for over 100 passages. The ALDH phenotype in both the primary tumor and the four new cell lines was determined by total activity assay, gel electrophoresis, and histochemistry. By total activity assay, the primary tumor did not possess significant tumor-ALDH activity. In contrast, the four new cell lines expressed tumor-ALDH activity. However, they differed in their basal ALDH activities and in ALDH inducibility by 3-methycholanthrene, benzo(a)pyrene, and phenobarbital. Additionally, significant decreases in tumor-ALDH activity occurred when cells from each line were passaged in vivo. The four lines have been characterized by light and electron microscopic morphology, tumorigenicity, chromosome number, doubling time, and colony formation efficiency, in soft agar. This work was submitted by K.-H. L. in partial fulfillment of the requirements for the Doctor of Philosophy degree in The Graduate School of The University of Alabama. This work was supported by grant CA-21103 from the National Cancer Institute, Bethesda, MD.     

Cloning, expression, and characterization of an aldehyde dehydrogenase from Escherichia coli K-12 that utilizes 3-Hydroxypropionaldehyde as a substrate

For efficient production of isoflavone aglycones from soybean isoflavones, we isolated three novel types of β-glucosidase (BGL1, BGL3, and BGL5) from the filamentous fungi Aspergillus oryzae. Three enzymes were independently displayed on the cell surface of a yeast Saccharomyces cerevisiae as a fusion protein with α-agglutinin. Three β-glucosidase-displaying yeast strains hydrolyzed isoflavone glycosides efficiently but exhibited different substrate specificities. Among these β-glucosidases, BGL1 exhibited the highest activity and also broad substrate specificity to isoflavone glycosides. Although glucose released from isoflavone glycosides are generally known to inhibit β-glucosidase, the residual ratio of isoflavone glycosides in the reaction mixture with BGL1-displaying yeast strain (Sc-BGL1) reached approximately 6.2%, and the glucose concentration in the reaction mixture was maintained at lower level. This result indicated that Sc-BGL1 assimilated the glucose before they inhibited the hydrolysis reaction, and efficient production of isoflavone aglycones was achieved by engineered yeast cells displaying β-glucosidase.     

Patterns of nuclear lamins in diverse animal and plant cells and in germ cells as revealed by immunofluorescence microscopy with polyclonal and monoclonal antibodies

Polyclonal and monoclonal antibodies against rat liver nuclear lamins have been used to evaluate the immunological cross-reactivity of lamins with a given antibody in a variety of animal and plant cells. The results indicated that lamins of all vertebrate cells but not invertebrate cells share at least one antigenic determinant, resulting in immunological cross-reaction with polyclonal antisera to lamina from rat liver. The range of cross-reaction with monoclonal antibody to rat lamins includes all mammalian cells tested but we observed no reaction with other vertebrate and invertebrate cells. Thus, by means of immunological cross-reaction a less stringently conserved pattern is observed for lamins than, for example, cytoskeletal proteins. We have also investigated the fate of the nuclear lamins during meiosis in testes and ovaries of the mouse. Lamins are absent from male meiotic cells and during oogenesis in meiotic prophases.     

Glyceraldehyde dehydrogenases from the thermoacidophilic euryarchaeota Picrophilus torridus and Thermoplasma acidophilum, key enzymes of the non-phosphorylative Entner-Doudoroff pathway, constitute a novel enzyme family within the aldehyde dehydrogenase superfamily

Cells of Picrophilus torridus, grown on glucose, contained all enzyme activities of a non-phosphorylative Entner-Doudoroff pathway, including glucose dehydrogenase, gluconate dehydratase, 2-keto-3-deoxygluconate aldolase, glyceraldehyde dehydrogenase (GADH), glycerate kinase (2-phosphoglycerate forming), enolase and pyruvate kinase. GADH was purified to homogeneity. The 115-kDa homodimeric protein catalyzed the oxidation of glyceraldehyde with NADP+ at highest catalytic efficiency. NAD+ was not used. By MALDI-TOF analysis, open reading frame (ORF) Pto0332 was identified in the genome of P. torridus as the encoding gene, designated gadh, and the recombinant GADH was characterized. In Thermoplasma acidophilum ORF Ta0809 represents a gadh homolog with highest sequence identity; the gene was expressed and the recombinant protein was characterized as functional GADH with properties very similar to the P. torridus enzyme. Sequence comparison and phylogenetic analysis define both GADHs as members of novel enzyme family within the aldehyde dehydrogenase superfamily.     

Molecular cloning and differential expression of an aldehyde dehydrogenase gene in rice leaves in response to infection by blast fungus

Aldehyde dehydrogenase (ALDH) superfamily is a group of enzymes metabolizing endogenous and exogenous aldehydes. Using differential display RT-PCR and cDNA library screening, a full-length aldehyde dehydrogenase cDNA (ALDH7B7) was isolated from rice leaves infected by incompatible race of blast fungus Magnaporthe grisea. The deduced amino acid sequence consists of 509 amino acid residues and shares 74∼81% identity with those of ALDH7Bs from other plants. ALDH7B7 expression was induced by blast fungus infection, ultraviolet, mechanical wound in rice leaves and was not detected in untreated rice organs. This gene has also been found to be inducible after exogenous phytohormones application, such as salicylic acid, methyl ester of jasmonic acid and abscisic acid. The function of ALDH7B7 in the interaction process between blast fungus and rice is discussed.     

Structure of aldehyde reductase in ternary complex with coenzyme and the potent 20alpha-hydroxysteroid dehydrogenase inhibitor 3,5-dichlorosalicylic acid: implications for inhibitor binding and selectivity

The structure of aldehyde reductase (ALR1) in ternary complex with the coenzyme NADPH and 3,5-dichlorosalicylic acid (DCL), a potent inhibitor of human 20α-hydroxysteroid dehydrogenase (AKR1C1), was determined at a resolution of 2.41 Å. The inhibitor formed a network of hydrogen bonds with the active site residues Trp22, Tyr50, His113, Trp114 and Arg312. Molecular modelling calculations together with inhibitory activity measurements indicated that DCL was a less potent inhibitor of ALR1 (256-fold) when compared to AKR1C1. In AKR1C1, the inhibitor formed a 10-fold stronger binding interaction with the catalytic residue (Tyr55), non-conserved hydrogen bonding interaction with His222, and additional van der Waals contacts with the non-conserved C-terminal residues Leu306, Leu308 and Phe311 that contribute to the inhibitor’s selectivity advantage for AKR1C1 over ALR1.     

Binding and incorporation of 4-trans-(N,N-dimethylamino) cinnamaldehyde by aldehyde dehydrogenase

4-trans-(N,N-Dimethylamino)cinnamaldehyde (DACA) is a chromophoric substrate of aldehyde dehydrogenase (EC 1.2.1.3) whose fate can be followed during catalysis. During this investigation we found that DACA also fluoresces and that this fluorescence is enhanced and blue-shifted upon binding to aldehyde dehydrogenase. Binding of DACA to aldehyde dehydrogenase also occurs in the absence of coenzyme. Benzaldehyde (a substrate), acetophenone (a substrate-competitive inhibitor), and the substrate-competitive affinity reagent bromoaceto-phenone interfere with DACA binding. Thus, DACA binds to the active site and can be employed for titration of active aldehyde dehydrogenase. Both E1 and E2 isozymes, which are homotetramers, bind DACA with dissociation constants of 1–4 M with a stoichiometry of 2 mol DACA/mol enzyme. The stoichiometry of enzyme–acyl intermediate was also found to be 2 mol DACA/mol enzyme for both E1 and E2 isozymes. Thus, both enzymes appear to have only two substrate-binding sites which participate in catalysis. The level of enzyme–acyl intermediate remained constant at different pH values, showing that enhancement of velocity with pH was not due to altered DACA–enzyme levels. When the reaction velocity was increased even further by using 150 M Mg²⁺ the intermediate level was decreased, suggesting that both increased pH and Mg²⁺ promote decomposition of the DACA–enzyme intermediate. Titration with DACA permits study of aldehyde substrate catalysis before central complex interconversion.     

Feasibility of prophylaxis and therapy against Gram-negative infections by human monoclonal antibodies

Abstract In a murine model of Gram-negative sepsis, we have shown that the prophylactic application of human monoclonal antibodies (HmAbs) with specificity for lipopolysaccharides (LPS) of Pseudomonas aeruginosa protected against bacterial infection. In this paper we show that the therapeutical application of 5 μg of these HmAbs up to 6 h after challenge with a lethal dose of live P. aeruginosa results in a protection rate of 70–90%. Administration 18 h after bacterial challenge, diminished the protection to 43% survival rate. Furthermore, using a mixture of HmAbs recognizing a total of six different P. aeruginosa serotypes, no interference in their protective capacities was found. Finally, these HmAbs also protected galactosamine-sensitized mice against lethal challenge with LPS. Our data show that the described HmAbs confer bactericidal activity as well as anti-endotoxic activity in vivo.