RESUMO
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
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.