RESUMO
Glycine betaine is the main osmolyte synthesized and accumulated in mammalian renal cells. Glycine betaine synthesis is catalyzed by the enzyme betaine aldehyde dehydrogenase (BADH) using NAD+ as the coenzyme. Previous studies have shown that porcine kidney betaine aldehyde dehydrogenase (pkBADH) binds NAD+ with different affinities at each active site and that the binding is K+ dependent. The objective of this work was to analyze the changes in the pkBADH secondary and tertiary structure resulting from variable concentrations of NAD+ and the role played by K+ . Intrinsic fluorescence studies were carried out at fixed-variable concentrations of K+ and titrating the enzyme with varying concentrations of NAD+ . Fluorescence analysis showed a shift of the maximum emission towards red as the concentration of K+ was increased. Changes in the exposure of tryptophan located near the NAD+ binding site were found when the enzyme was titrated with NAD+ in the presence of potassium. Fluorescence data analysis showed that the K+ presence promoted static quenching that facilitated the pkBADH-NAD+ complex formation. DC data analysis showed that binding of K+ to the enzyme caused changes in the α-helix content of 4% and 12% in the presence of 25 mM and 100 mM K+ , respectively. The presence of K+ during NAD+ binding to pkBADH increased the thermal stability of the complex. These results indicated that K+ facilitated the pkBADH-NAD+ complex formation and suggested that K+ caused small changes in secondary and tertiary structures that could influence the active site conformation.
Assuntos
Betaína-Aldeído Desidrogenase , Potássio , Animais , Betaína-Aldeído Desidrogenase/metabolismo , Sítios de Ligação , Coenzimas , Cinética , Conformação Molecular , SuínosRESUMO
BACKGROUND: Fragrance is one of the most important quality traits in rice, and the phenotype is attributed to the loss-of-function betaine aldehyde dehydrogenase (BADH2) gene. At least 12 allelic variations of BADH2 have been identified, and some of these have been applied to rice fragrance breeding using traditional molecular markers and Sanger sequencing techniques. However, these traditional methods have several limitations, such as being very expensive, imprecise, inefficient, and having security issues. Thus, a new molecular marker technology must be developed to improve rice fragrance breeding. RESULTS: In this study, more than 95% of the cultivated fragrant rice varieties belonged to a 7-bp deletion in exon 2 (badh2-E2) or an 8-bp deletion and 3-bp variation in exon 7 (badh2-E7). Both allelic variations resulted in the loss of function of the badh2 gene. We developed two novel SNP molecular markers, SNP_badh2-E2 and SNP_badh2- E7, related to the alleles. Their genotype and phenotype were highly cosegregated in the natural variation of rice accessions, with 160 of the 164 fragrant rice varieties detected with the two markers. These markers cosegregated with the fragrance phenotype in the F2 population. CONCLUSIONS: Two functional SNP molecular markers of badh2-E2 and badh2-E7 allelic variations were developed. These functional SNP molecular markers can be used for genotype and genetic improvement of rice fragrance through marker-assisted selection and will significantly improve the efficiency of fragrant rice breeding and promote commercial molecular breeding of rice in the future.
Assuntos
Oryza/enzimologia , Oryza/genética , Betaína-Aldeído Desidrogenase/metabolismo , Marcadores Genéticos , Alelos , Técnicas de Genotipagem/métodos , Genótipo , OdorantesRESUMO
Betaine aldehyde dehydrogenase (BADH) catalyzes the oxidation of betaine aldehyde to glycine betaine using NAD+ as a coenzyme. Studies in porcine kidney BADH (pkBADH) suggested that the enzyme exhibits heterogeneity of active sites and undergoes potassium-induced conformational changes. This study aimed to analyze if potassium concentration plays a role in the heterogeneity of pkBADH active sites through changes in NAD+ affinity constants, in its secondary structure content and stability. The enzyme was titrated with NAD+ 1 mM at fixed-variable KCl concentration, and the interaction measured by Isothermal Titration Calorimetry (ITC) and Circular Dichroism (CD). ITC data showed that K+ increased the first active site affinity in a manner dependent on its concentration; KD values to the first site were 14.4, 13.1, and 10.4 µM, at 25, 50, and 75 mM KCl. ΔG values showed that the coenzyme binding is a spontaneous reaction without changes between active sites or depending on KCl concentration. ΔH and TΔSb values showed that NAD+ binding to the active site is an endothermic process and is carried out at the expense of changes in entropy. α-Helix content increased as KCl increased, enzyme (Tm)app values were 2.6 °C and 3.3 °C higher at 20 mM and 200 mM K+. PkBADH molecular model showed three different interaction K+ sites. Results suggested K+ can interact with pkBADH and cause changes in the secondary structure, it provokes changes in the enzyme affinity by the coenzyme, and in the thermostability.
Assuntos
Betaína-Aldeído Desidrogenase/metabolismo , NAD/metabolismo , Potássio/metabolismo , Sítios de Ligação , Modelos MolecularesRESUMO
In marine animals, glycine betaine is one of the main osmolytes accumulated under osmotic stress conditions; nevertheless, in penaeids, shrimps little is known about the pathways involved in glycine betaine biosynthesis. In animal cells, glycine betaine is synthesized by the enzyme betaine aldehyde dehydrogenase (BADH). We herein investigated the salinity effect on the synthesis and concentration of glycine betaine on white shrimp Litopenaeus vannamei. Shrimps were subjected to 10, 20, 35, 40, 50, and 60â¯ppt salinity conditions for seven days. BADH activity increased in hepatopancreas and gills of shrimps subjected to salinities above 35â¯ppt salinity. In muscle, the BADH activity decreased at 35â¯ppt salinity. In hepatopancreas from shrimps subjected to 50 and 60â¯ppt salinities, BADH activity increased 1.1 and 1.7-fold. At 60â¯ppt salinity, BADH activity increased 1.5-fold respect to 35â¯ppt in gills. Glycine betaine concentration increased in hepatopancreas, gills, muscle, and hemolymph in shrimps subjected to salinities above 35â¯ppt. Glycine betaine concentration also increased at 20â¯ppt salinity, while at 10â¯ppt, not detected significant differences. The catch of glycine betaine from hemolymph by the cell likely is carried out to avoid protein denaturalization. Ammonia concentration in the aquarium's water only increased at salinities of 20â¯ppt and 10â¯ppt (1.1-fold relative to 35â¯ppt). Our data demonstrated that in L. vannamei, salinity regulates BADH activity and glycine betaine content in a tissue-specific manner.
Assuntos
Betaína-Aldeído Desidrogenase/metabolismo , Betaína/metabolismo , Osmorregulação , Pressão Osmótica , Penaeidae/metabolismo , Salinidade , Animais , Hemolinfa/metabolismo , Hepatopâncreas/metabolismo , Penaeidae/efeitos dos fármacosRESUMO
The enzyme betaine aldehyde dehydrogenase (BADH) catalyzes the irreversible oxidation of betaine aldehyde to glycine betaine (GB), a very efficient osmolyte accumulated during osmotic stress. In this study, we determined the nucleotide sequence of the cDNA for the BADH from the white shrimp Litopenaeus vannamei (LvBADH). The cDNA was 1882 bp long, with a complete open reading frame of 1524 bp, encoding 507 amino acids with a predicted molecular mass of 54.15 kDa and a pI of 5.4. The predicted LvBADH amino acid sequence shares a high degree of identity with marine invertebrate BADHs. Catalytic residues (C-298, E-264 and N-167) and the decapeptide VTLELGGKSP involved in nucleotide binding and highly conserved in BADHs were identified in the amino acid sequence. Phylogenetic analyses classified LvBADH in a clade that includes ALDH9 sequences from marine invertebrates. Molecular modeling of LvBADH revealed that the protein has amino acid residues and sequence motifs essential for the function of the ALDH9 family of enzymes. LvBADH modeling showed three potential monovalent cation binding sites, one site is located in an intra-subunit cavity; other in an inter-subunit cavity and a third in a central-cavity of the protein. The results show that LvBADH shares a high degree of identity with BADH sequences from marine invertebrates and enzymes that belong to the ALDH9 family. Our findings suggest that the LvBADH has molecular mechanisms of regulation similar to those of other BADHs belonging to the ALDH9 family, and that BADH might be playing a role in the osmoregulation capacity of L. vannamei.
Assuntos
Betaína-Aldeído Desidrogenase/metabolismo , Betaína/metabolismo , Modelos Moleculares , Penaeidae/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Betaína-Aldeído Desidrogenase/classificação , Betaína-Aldeído Desidrogenase/genética , Sítios de Ligação , Biocatálise , Clonagem Molecular , Humanos , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de SequênciaRESUMO
The fragrance gene, betaine aldehyde dehydrogenase 2 (Badh2), has been well studied in many plant species. The objectives of this study were to clone Badh2 and compare the sequences between aromatic and non-aromatic coconuts. The complete coding region was cloned from cDNA of both aromatic and non-aromatic coconuts. The nucleotide sequences were highly homologous to Badh2 genes of other plants. Badh2 consisted of a 1512-bp open reading frame encoding 503 amino acids. A single nucleotide difference between aromatic and non-aromatic coconuts resulted in the conversion of alanine (non-aromatic) to proline (aromatic) at position 442, which was the substrate binding site of BADH2. The ring side chain of proline could destabilize the structure leading to a non-functional enzyme. Badh2 genomic DNA was cloned from exon 1 to 4, and from exon 5 to 15 from the two coconut types, except for intron 4 that was very long. The intron sequences of the two coconut groups were highly homologous. No differences in Badh2 expression were found among the tissues of aromatic coconut or between aromatic and non-aromatic coconuts. The amino acid sequences of BADH2 from coconut and other plants were compared and the genetic relationship was analyzed using MEGA 7.0. The phylogenetic tree reconstructed by the Bayesian information criterion consisted of two distinct groups of monocots and dicots. Among the monocots, coconut (Cocos nucifera) and oil palm (Elaeis guineensis) were the most closely related species. A marker for coconut differentiation was developed from one-base substitution site and could be successfully used.
Assuntos
Betaína-Aldeído Desidrogenase/genética , Cocos/genética , Sequência de Aminoácidos , Cocos/enzimologia , Éxons , Genes de Plantas , Odorantes , Fenótipo , Filogenia , Proteínas de Plantas/genética , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNARESUMO
Glycine betaine is an important quaternary ammonium compound that is produced in response to several abiotic stresses in many organisms. The synthesis of glycine betaine requires the catalysis of betaine aldehyde dehydrogenase (BADH), which can convert betaine aldehyde into glycine betaine in plants, especially in halotolerant plants. In this study, we isolated the full-length cDNA of BADH from Suaeda corniculata (ScBADH) using reverse transcriptase-polymerase chain reaction and rapid amplification of cDNA ends. Next, we analyzed the expression profile of ScBADH using real-time PCR. The results showed that ScBADH expression was induced in the roots, stems, and leaves of S. corniculata seedlings under salt and drought stress. Next, ScBADH was overexpressed in Arabidopsis, resulting in the transgenic plants exhibiting enhanced tolerance over wild-type plants under salt and drought stress. We then analyzed the levels of glycine betaine and proline, as well as superoxide dismutase (SOD) activity, during salt stress in WT and transgenic Arabidopsis. The results indicated that overexpression of ScBADH produced more glycine betaine and proline, and increased SOD activity under NaCl treatment. Our results suggest that ScBADH might be a positive regulator in plants during the response to NaCl.
Assuntos
Betaína-Aldeído Desidrogenase/genética , Chenopodiaceae/genética , Proteínas de Plantas/genética , Betaína/metabolismo , Betaína-Aldeído Desidrogenase/metabolismo , Chenopodiaceae/enzimologia , Clonagem Molecular , Secas , Regulação da Expressão Gênica de Plantas , Glicina/metabolismo , Proteínas de Plantas/metabolismo , Prolina/metabolismo , Salinidade , Estresse Fisiológico , Superóxido Dismutase/metabolismoRESUMO
In plants, the last step in the biosynthesis of the osmoprotectant glycine betaine (GB) is the NAD(+)-dependent oxidation of betaine aldehyde (BAL) catalysed by some aldehyde dehydrogenase (ALDH) 10 enzymes that exhibit betaine aldehyde dehydrogenase (BADH) activity. Given the irreversibility of the reaction, the short-term regulation of these enzymes is of great physiological relevance to avoid adverse decreases in the NAD(+):NADH ratio. In the present study, we report that the Spinacia oleracea BADH (SoBADH) is reversibly and partially inactivated by BAL in the absence of NAD(+)in a time- and concentration-dependent mode. Crystallographic evidence indicates that the non-essential Cys(450)(SoBADH numbering) forms a thiohemiacetal with BAL, totally blocking the productive binding of the aldehyde. It is of interest that, in contrast to Cys(450), the catalytic cysteine (Cys(291)) did not react with BAL in the absence of NAD(+) The trimethylammonium group of BAL binds in the same position in the inactivating or productive modes. Accordingly, BAL does not inactivate the C(450)SSoBADH mutant and the degree of inactivation of the A(441)I and A(441)C mutants corresponds to their very different abilities to bind the trimethylammonium group. Cys(450)and the neighbouring residues that participate in stabilizing the thiohemiacetal are strictly conserved in plant ALDH10 enzymes with proven or predicted BADH activity, suggesting that inactivation by BAL is their common feature. Under osmotic stress conditions, this novel partial and reversible covalent regulatory mechanism may contribute to preventing NAD(+)exhaustion, while still permitting the synthesis of high amounts of GB and avoiding the accumulation of the toxic BAL.
Assuntos
Betaína-Aldeído Desidrogenase/química , Betaína/análogos & derivados , Mutação de Sentido Incorreto , Proteínas de Plantas/química , Spinacia oleracea/enzimologia , Substituição de Aminoácidos , Betaína/química , Betaína-Aldeído Desidrogenase/genética , Domínio Catalítico , Cristalografia por Raios X , Ativação Enzimática , Proteínas de Plantas/genética , Spinacia oleracea/genéticaRESUMO
Crustaceans overcome osmotic disturbances by regulating their intracellular concentration of ions and osmolytes. Glycine betaine (GB), an osmolyte accumulated in response to hyperosmotic stress, is synthesized by betaine aldehyde dehydrogenase (BADH EC 1.2.1.8) through the oxidation of betaine aldehyde. A partial BADH cDNA sequence from the white shrimp Litopenaeus vannamei was obtained and its organ-specific expression during osmotic stress (low and high salinity) was evaluated. The partial BADH cDNA sequence (LvBADH) is 1103bp long and encodes an open reading frame for 217 protein residues. The amino acid sequence of LvBADH is related to that of other BADHs, TMABA-DH and ALDH9 from invertebrate and vertebrate homologues, and includes the essential domains of their function and regulation. LvBADH activity and mRNA expression were detected in the gills, hepatopancreas and muscle with the highest levels in the hepatopancreas. LvBADH mRNA expression increased 2-3-fold in the hepatopancreas and gills after 7days of osmotic variation (25 and 40ppt). In contrast, LvBADH mRNA expression in muscle decreased 4-fold and 15-fold after 7days at low and high salinity, respectively. The results indicate that LvBADH is ubiquitously expressed, but its levels are organ-specific and regulated by osmotic stress, and that LvBADH is involved in the cellular response of crustaceans to variations in environmental salinity.
Assuntos
Betaína-Aldeído Desidrogenase/genética , Betaína-Aldeído Desidrogenase/metabolismo , Decápodes/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Betaína-Aldeído Desidrogenase/química , DNA Complementar/química , DNA Complementar/genética , Decápodes/enzimologia , Decápodes/metabolismo , Dados de Sequência Molecular , Especificidade de Órgãos , Pressão Osmótica , RNA Mensageiro/metabolismoRESUMO
In response to salinity or drought stress, many plants accumulate glycine betaine, which is a regulator of osmosis. In plants, the last step in betaine synthesis is catalyzed by betaine aldehyde dehydrogenase (BADH), a nuclear-encoded chloroplastic enzyme. Based on the conserved oligo amino acid residues of the published BADH genes from other higher plant species, a cDNA sequence, designated CtBADH, was isolated from safflower (Carthamus tinctorius L.) using a polymerase chain reaction approach. The clones were 1.7 kb on average, and contained an open reading frame predicting a polypeptide of 503 amino acids with 84.5% identity to that of Helianthus annuus. The deduced amino acid sequence showed a decapeptide, Val-Thr-Leu-Geu-Leu-Gly-Gly-Lys-Ser-Pro and Cys, which is essential for proper functioning of BADH. Phylogenetic analysis indicated that CtBADH grouped with other dicotyledonous plant BADH genes, and subgrouped in the composite family. Prediction of secondary structure and subcellular localization suggested that the protein encoded by CtBADH contains 33 coils, 15 alpha helixes, and 21 beta strands, and most likely targets the chloroplast or mitochondria.
Assuntos
Betaína-Aldeído Desidrogenase/genética , Carthamus tinctorius/enzimologia , Genes de Plantas , Proteínas de Plantas/genética , Sequência de Aminoácidos , Sequência de Bases , Betaína-Aldeído Desidrogenase/química , Betaína-Aldeído Desidrogenase/metabolismo , Carthamus tinctorius/química , Carthamus tinctorius/genética , Clonagem Molecular , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Estrutura Terciária de Proteína , Transporte ProteicoRESUMO
Plant Aldehyde Dehydrogenase10 (ALDH10) enzymes catalyze the oxidation of ω-primary or ω-quaternary aminoaldehydes, but, intriguingly, only some of them, such as the spinach (Spinacia oleracea) betaine aldehyde dehydrogenase (SoBADH), efficiently oxidize betaine aldehyde (BAL) forming the osmoprotectant glycine betaine (GB), which confers tolerance to osmotic stress. The crystal structure of SoBADH reported here shows tyrosine (Tyr)-160, tryptophan (Trp)-167, Trp-285, and Trp-456 in an arrangement suitable for cation-π interactions with the trimethylammonium group of BAL. Mutation of these residues to alanine (Ala) resulted in significant K(m)(BAL) increases and V(max)/K(m)(BAL) decreases, particularly in the Y160A mutant. Tyr-160 and Trp-456, strictly conserved in plant ALDH10s, form a pocket where the bulky trimethylammonium group binds. This space is reduced in ALDH10s with low BADH activity, because an isoleucine (Ile) pushes the Trp against the Tyr. Those with high BADH activity instead have Ala (Ala-441 in SoBADH) or cysteine, which allow enough room for binding of BAL. Accordingly, the mutation A441I decreased the V(max)/K(m)(BAL) of SoBADH approximately 200 times, while the mutation A441C had no effect. The kinetics with other ω-aminoaldehydes were not affected in the A441I or A441C mutant, demonstrating that the existence of an Ile in the second sphere of interaction of the aldehyde is critical for discriminating against BAL in some plant ALDH10s. A survey of the known sequences indicates that plants have two ALDH10 isoenzymes: those known to be GB accumulators have a high-BAL-affinity isoenzyme with Ala or cysteine in this critical position, while non GB accumulators have low-BAL-affinity isoenzymes containing Ile. Therefore, BADH activity appears to restrict GB synthesis in non-GB-accumulator plants.
Assuntos
Aminoácidos/metabolismo , Betaína-Aldeído Desidrogenase/metabolismo , Betaína/análogos & derivados , Spinacia oleracea/enzimologia , Aminoácidos Aromáticos/metabolismo , Betaína/química , Betaína/metabolismo , Betaína-Aldeído Desidrogenase/química , Sítios de Ligação , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação Proteica , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Concentrated urine formation in the kidney is accompanied by conditions that favor the accumulation of reactive oxygen species (ROS). Under hyperosmotic conditions, medulla cells accumulate glycine betaine, which is an osmolyte synthesized by betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8). All BADHs identified to date have a highly reactive cysteine residue at the active site, and this cysteine is susceptible to oxidation by hydrogen peroxide. Porcine kidney BADH incubated with H(2)O(2) (0-500 µM) lost 25% of its activity. However, pkBADH inactivation by hydrogen peroxide was limited, even after 120 min of incubation. The presence of coenzyme NAD(+) (10-50 µM) increased the extent of inactivation (60%) at 120 min of reaction, but the ligands betaine aldehyde (50 and 500 µM) and glycine betaine (100 mM) did not change the rate or extent of inactivation as compared to the reaction without ligand. 2-Mercaptoethanol and dithiothreitol, but not reduced glutathione, were able to restore enzyme activity. Mass spectrometry analysis of hydrogen peroxide inactivated BADH revealed oxidation of M278, M243, M241 and H335 in the absence and oxidation of M94, M327 and M278 in the presence of NAD(+). Molecular modeling of BADH revealed that the oxidized methionine and histidine residues are near the NAD(+) binding site. In the presence of the coenzyme, these oxidized residues are proximal to the betaine aldehyde binding site. None of the oxidized amino acid residues participates directly in catalysis. We suggest that pkBADH inactivation by hydrogen peroxide occurs via disulfide bond formation between vicinal catalytic cysteines (C288 and C289).
Assuntos
Betaína-Aldeído Desidrogenase/metabolismo , Peróxido de Hidrogênio/farmacologia , Rim/enzimologia , Animais , Betaína-Aldeído Desidrogenase/química , Ativação Enzimática/efeitos dos fármacos , Reativadores Enzimáticos/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Cinética , Ligantes , Modelos Moleculares , Conformação Proteica , SuínosRESUMO
In the human pathogen Pseudomonas aeruginosa, the NAD(P)(+)-dependent betaine aldehyde dehydrogenase (PaBADH) may play the dual role of assimilating carbon and nitrogen from choline or choline precursors--abundant at infection sites--and producing glycine betaine and NADPH, potentially protective against the high-osmolarity and oxidative stresses prevalent in the infected tissues. Disruption of the PaBADH gene negatively affects the growth of bacteria, suggesting that this enzyme could be a target for antibiotic design. PaBADH is one of the few ALDHs that efficiently use NADP(+) and one of the even fewer that require K(+) ions for stability. Crystals of PaBADH were obtained under aerobic conditions in the presence of 2-mercaptoethanol, glycerol, NADP(+) and K(+) ions. The three-dimensional structure was determined at 2.1-A resolution. The catalytic cysteine (C286, corresponding to C302 of ALDH2) is oxidized to sulfenic acid or forms a mixed disulfide with 2-mercaptoethanol. The glutamyl residue involved in the deacylation step (E252, corresponding to E268 of ALDH2) is in two conformations, suggesting a proton relay system formed by two well-conserved residues (E464 and K162, corresponding to E476 and K178, respectively, of ALDH2) that connects E252 with the bulk water. In some active sites, a bound glycerol molecule mimics the thiohemiacetal intermediate; its hydroxyl oxygen is hydrogen bonded to the nitrogen of the amide groups of the side chain of the conserved N153 (N169 of ALDH2) and those of the main chain of C286, which form the "oxyanion hole." The nicotinamide moiety of the nucleotide is not observed in the crystal, and the adenine moiety binds in the usual way. A salt bridge between E179 (E195 of ALDH2) and R40 (E53 of ALDH2) moves the carboxylate group of the former away from the 2'-phosphate of the NADP(+), thus avoiding steric clashes and/or electrostatic repulsion between the two groups. Finally, the crystal shows two K(+) binding sites per subunit. One is in an intrasubunit cavity that we found to be present in all known ALDH structures. The other--not described before for any ALDH but most likely present in most of them--is located in between the dimeric unit, helping structure a region involved in coenzyme binding and catalysis. This may explain the effects of K(+) ions on the activity and stability of PaBADH.
Assuntos
Betaína-Aldeído Desidrogenase/química , Cátions/metabolismo , NADP/metabolismo , Potássio/metabolismo , Pseudomonas aeruginosa/enzimologia , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
The NAD+-dependent animal betaine aldehyde dehydrogenases participate in the biosynthesis of glycine betaine and carnitine, as well as in polyamines catabolism. We studied the kinetics of inactivation of the porcine kidney enzyme (pkBADH) by the drug disulfiram, a thiol-reagent, with the double aim of exploring the enzyme dynamics and investigating whether it could be an in vivo target of disulfiram. Both inactivation by disulfiram and reactivation by reductants were biphasic processes with equal limiting amplitudes. Under certain conditions half of the enzyme activity became resistant to disulfiram inactivation. NAD+ protected almost 100% at 10 microM but only 50% at 5mM, and vice versa if the enzyme was pre-incubated with NAD+ before the chemical modification. NADH, betaine aldehyde, and glycine betaine also afforded greater protection after pre-incubation with the enzyme than without pre-incubation. Together, these findings suggest two kinds of active sites in this seemingly homotetrameric enzyme, and complex, unusual ligand-induced conformational changes. In addition, they indicate that, in vivo, pkBADH is most likely protected against disulfiram inactivation.
Assuntos
Aldeído Desidrogenase/química , Aldeído Desidrogenase/ultraestrutura , Betaína-Aldeído Desidrogenase/química , Betaína-Aldeído Desidrogenase/ultraestrutura , Dissulfiram/química , Rim/enzimologia , Modelos Químicos , Animais , Simulação por Computador , Estabilidade Enzimática , Modelos Moleculares , Conformação Proteica , SuínosRESUMO
In the human pathogen Pseudomonas aeruginosa, betaine aldehyde dehydrogenase (PaBADH) may play the dual role of assimilating carbon and nitrogen from choline or choline precursors--abundant at infection sites--and producing glycine betaine, which protects the bacterium against the high-osmolality stress prevalent in the infected tissues. This tetrameric enzyme contains four cysteine residues per subunit and is a potential drug target. In our search for specific inhibitors, we mutated the catalytic Cys286 to alanine and chemically modified the recombinant wild-type and the four Cys-->Ala single mutants with thiol reagents. The small methyl-methanethiosulfonate inactivated the enzymes without affecting their stability while the bulkier dithionitrobenzoic acid (DTNB) and bis[diethylthiocarbamyl] disulfide (disulfiram) induced enzyme dissociation--at 23 degrees C--and irreversible aggregation--at 37 degrees C. Of the four Cys-->Ala mutants only C286A retained its tetrameric structure after DTNB or disulfiram treatments, suggesting that steric constraints arising upon the covalent attachment of a bulky group to C286 resulted in distortion of the backbone configuration in the active site region followed by a severe decrease in enzyme stability. Since neither NAD(P)H nor betaine aldehyde prevented disulfiram-induced PaBADH inactivation or aggregation, and reduced glutathione was unable to restore the activity of the modified enzyme, we propose that disulfiram could be a useful drug to combat infection by P. aeruginosa.
Assuntos
Anti-Infecciosos/farmacologia , Betaína-Aldeído Desidrogenase/metabolismo , Dissulfiram/farmacologia , Inibidores Enzimáticos/farmacologia , Pseudomonas aeruginosa/metabolismo , Alanina/química , Sítios de Ligação , Catálise , Cromatografia , Cisteína/química , Ácido Ditionitrobenzoico/farmacologia , Glutationa/química , Cinética , Metanossulfonato de Metila/análogos & derivados , Metanossulfonato de Metila/farmacologia , Modelos Químicos , Mutagênese Sítio-Dirigida , Mutação , NADP/química , Conformação Proteica , Estrutura Quaternária de Proteína , Proteínas/química , Compostos de Sulfidrila , Temperatura , Fatores de TempoRESUMO
In the human pathogen Pseudomonas aeruginosa, betaine aldehyde dehydrogenase (BADH) may play a dual role assimilating carbon and nitrogen from choline or choline precursors--abundant at infection sites--and producing glycine betaine, which protects the bacteria against the high-osmolarity stress prevalent in the infected tissues. We cloned the P. aeruginosa BADH gene and expressed the BADH protein in Escherichia coli. The recombinant protein appears identical to its native counterpart, as judged by Western blot, N-terminal amino acid sequence, tryptophan-fluorescence emission spectra, circular-dichroism spectroscopy, size-exclusion chromatography, and kinetic properties. Computational analysis indicated that the promoter sequence of the putative operon that includes the BADH gene has a consensus-binding site for the choline-sensing transcription repressor BetI, and putative boxes for ArcA and Lrp transcription factors but no known elements of response to osmotic stress. This is consistent with the strong induction of BADH expression by choline and with the lack of effect of NaCl. As there were significant amounts of BADH protein and activity in P. aeruginosa cells grown on glucose plus choline, as well as the BADH activity exhibiting tolerance to salt, it is likely that glycine betaine is synthesized in vivo and could play an important osmoprotectant role under conditions of infection.
Assuntos
Betaína-Aldeído Desidrogenase/genética , Colina/metabolismo , Pseudomonas aeruginosa/enzimologia , Cloreto de Sódio/metabolismo , Sequência de Bases , Betaína-Aldeído Desidrogenase/biossíntese , Betaína-Aldeído Desidrogenase/metabolismo , Colina/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Glucose/genética , Glucose/metabolismo , Biologia Molecular , Dados de Sequência Molecular , Pseudomonas aeruginosa/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
Betaine aldehyde dehydrogenase (BADH) from the human pathogen Pseudomonas aeruginosa is a tetrameric enzyme that contains a catalytic Cys286 and three additional cysteine residues, Cys353, 377, and 439, per subunit. In the present study, we have investigated the role of the three non-essentials in enzyme activity and stability by homology modeling and site-directed mutagenesis. Cys353 and Cys377 are located at the protein surface with their sulfur atoms buried, while Cys439 is at the subunit interface between the monomers forming a dimeric pair. All three residues were individually mutated to alanine and Cys439 also to serine and valine. The five mutant proteins were expressed in Escherichia coli and purified to homogeneity. Their steady-state kinetics was not significantly affected, neither was their structure as indicated by circular dicroism spectropolarimetry, protein intrinsic fluorescence, and size-exclusion chromatography. However, stability was severely reduced in the Cys439 mutants particularly in C439S and C439V, which were inactive when expressed at 37 degrees C. They also exhibited higher sensitivity to thermal and chemical inactivation, and higher propensity to dissociation by dilution or exposure to low ionic strength than the wild-type enzyme. Size-exclusion chromatography indicates that substitution of Cys439 lead to unstable dimers or to stable dimeric conformations not compatible with a stable tetrameric structure. To the best of our knowledge, this is the first study of an aldehyde dehydrogenase revealing a residue at the dimer interface involved in holding the dimer, and consequently the tetramer, together.
Assuntos
Betaína-Aldeído Desidrogenase/genética , Betaína-Aldeído Desidrogenase/metabolismo , Citosina , Pseudomonas aeruginosa/enzimologia , Substituição de Aminoácidos , Animais , Betaína-Aldeído Desidrogenase/química , Dicroísmo Circular , Peixes , Cinética , Fígado/enzimologia , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Desnaturação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TermodinâmicaRESUMO
The reaction catalyzed by betaine aldehyde dehydrogenase (BADH) involves the nucleophilic attack of a catalytic cysteinyl residue on the aldehyde substrate. As a possible mechanism of regulation, we have studied the modulation by ligands of the reactivity and/or accessibility of the essential thiol of the enzyme from the human pathogen Pseudomonas aeruginosa and the leaves of the plant Amaranthus hypochondriacus (amaranth). In the absence of ligands, the kinetics of inactivation by thiol modifying reagents of both enzymes were biphasic, suggesting the existence of two enzyme conformers differing in the reactivity of their catalytic thiolate. Preincubation of P. aeruginosa BADH with the coenzymes or the aldehyde prior to the chemical modification brought about active site rearrangements that resulted in an important decrease in the inactivation rate. Amaranth BADH responded similarly to the preincubation with NADH or betaine aldehyde but NAD(+) elicited opposite changes, increasing the rate of inactivation after prolonged preincubation. In amaranth BADH, the different behavior of both coenzymes, and the observed biphasic inactivation kinetics are consistent with the previously proposed iso kinetic mechanism, characterized by the existence of two interconvertible apoenzyme forms, one able to bind NAD(+) and the other NADH. Taken together, our results suggest that ligand-induced conformational changes in BADH from the two sources studied might be important for both proper enzyme function and protection against oxidation.
Assuntos
Aldeído Oxirredutases/metabolismo , Amaranthus/enzimologia , Pseudomonas aeruginosa/enzimologia , Compostos de Sulfidrila/metabolismo , Aldeído Oxirredutases/química , Betaína-Aldeído Desidrogenase , Catálise , Cinética , Ligantes , Folhas de Planta/enzimologia , Conformação ProteicaRESUMO
Betaine aldehyde dehydrogenase from the human pathogen Pseudomonas aeruginosa requires K(+) ions for maintenance of its active conformation. In order to explore if this property is shared by other BADHs of different origins and to further understand the mechanism underlying the effects of these ions, we carried out a comparative study on the stability and quaternary structure of P. aeruginosa, porcine kidney and amaranth leaves BADHs in the absence of K(+) ions. At low enzyme concentrations, the bacterial and porcine enzymes were totally inactivated upon removal of K(+) following biphasic and monophasic kinetics, respectively, whereas the amaranth enzyme retained its activity. Inactivation of P. aeruginosa BADH was much faster than that of the porcine enzyme. The oxidized coenzyme protected both enzymes against inactivation by the absence of K(+), whereas betaine aldehyde afforded partial protection to the bacterial BADH and increased the inactivation rate of the porcine. Reactivation of the inactive enzymes, by adding back to the incubation medium K(+) ions, was dependent on enzyme concentration, suggesting that enzyme dissociation takes place in the absence of K(+). In the bacterial enzyme, NH(4)(+) but not Na(+) ions could mimic the effects of K(+), whereas the three cations tested reactivated porcine BADH, indicating a requirement of this enzyme for high ionic strength rather than for a specific monovalent cation. Size exclusion chromatography of the inactivated enzymes confirmed that K(+) ions or other monovalent cations are required for the maintenance of the quaternary structure of these two BADHs. At pH 7.0, in the absence of K(+) in a buffer of low ionic strength, the active tetrameric form of P. aeruginosa BADH dissociated into inactive monomers and that of porcine kidney BADH into inactive dimers. Once reactivated, both enzymes reassociated into active tetramers.
Assuntos
Aldeído Oxirredutases/metabolismo , Amaranthus/enzimologia , Rim/enzimologia , Pseudomonas aeruginosa/enzimologia , Aldeído Oxirredutases/antagonistas & inibidores , Animais , Betaína-Aldeído Desidrogenase , Cátions Monovalentes , Estabilidade Enzimática , Folhas de Planta/enzimologia , Especificidade por Substrato , SuínosRESUMO
Betaine aldehyde dehydrogenase (BADH) activity might be crucial for the growth of the human pathogen Pseudomonas aeruginosa under conditions of infection and therefore appears to be a suitable target for antimicrobial agents. As a first step in the search for BADH inhibitors, we have tested the effects of the known aldehyde dehydrogenase inhibitor disulfiram (DSF) on the activity of P. aeruginosa and Amaranthus hypochondriacus (amaranth) leaf BADHs. DSF totally inactivated both enzymes in a time- and dose-dependent manner. In the case of the Pseudomonas enzyme, inactivation kinetics were monophasic with a second-order inactivation rate constant at pH 6.9 of 4.9+/-0.4 M(-1) s(-1), whereas the plant enzyme was inactivated in a biphasic process with second-order inactivation rate constants at pH 7.5 of 6.8+/-0.6 and 0.33+/-0.04 M(-1) s(-1). At pH 8.8, the second-order rate constants for inactivation of the bacterial enzyme was 1 x 10(3) M(-1) s(-1), which compare well with that reported for human liver mitochondrial aldehyde dehydrogenase (ALDH2), the target of DSF inhibition in the aversion therapy of alcoholism. Both BADHs were inactivated faster in the presence of NAD(P)(+) than in its absence, whereas NAD(P)H and betaine aldehyde protected the bacterial, but increased the inactivation rate of the plant enzyme. The inactivated enzymes were reactivated by dithiothreitol, but not by a high concentration of the physiological reductant glutathione. The high in vitro sensitivity of the Pseudomonas BADH to DSF, particularly in the presence of NAD(P)(+), together with the lack of reversibility of DSF modification by glutathione, makes this inhibitor a potential antimicrobial agent and suggests that it might be worth testing its effects and those of its metabolites in vivo, under culture conditions in which the activity of BADH is required for growth of the bacteria.