RESUMO
Fragrant rice (Oryza sativa L.) betaine aldehyde dehydrogenase 2 (OsBADH2) is a key enzyme in the synthesis of fragrance aroma compounds. The extremely low activity of OsBADH2 in catalyzing the oxidation of acetaldehyde is believed to be crucial for the accumulation of the volatile compound 2-acetyl-1-pyrroline (2AP) in many scented plants, including fragrant rice. Recombinant fragrant rice OsBADH2 was expressed in Escherichia coli as an N-terminal hexahistidine fusion protein, purified using Ni Sepharose affinity chromatography and crystallized using the microbatch method. Initial crystals were obtained within 24 h using 0.1 M Tris pH 8.5 with 30%(w/v) PEG 4000 and 0.2 M magnesium chloride as the precipitating agent at 291 K. Crystal quality was improved when the enzyme was cocrystallized with NAD(+). Improved crystals were grown in 0.1 M HEPES pH 7.4, 24%(w/v) PEG 4000 and 0.2 M ammonium chloride and diffracted to beyond 2.95 Å resolution after being cooled in a stream of N(2) immediately prior to X-ray diffraction experiments. The crystals belonged to space group C222(1), with unit-cell parameters a = 66.03, b = 183.94, c = 172.28 Å. An initial molecular-replacement solution has been obtained and refinement is in progress.
Assuntos
Betaína-Aldeído Desidrogenase/química , Oryza/enzimologia , Betaína-Aldeído Desidrogenase/isolamento & purificação , Cristalografia por Raios X , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Agriculture productivity is severely hampered by soil salinity, drought and other environmental stresses. Studies on stress-resistant plants (halophytes, xerophytes, accumulating plants for specific toxic ions) have illuminated some mechanisms of stress tolerance in plants at metabolic or molecular levels, which gave some clues on how to genetically engineer stress-tolerant crops. With the isolation of more stress-responsive genes, genetic engineering with modified expression of stress responsive genes may be an effective way to produce stress-tolerant crops. In the present report, two genes (PEAMT and BADH) encoding the corresponding key enzymes for choline and glycine betaine (an important osmoprotectant) biosynthesis in plants were isolated in oilseed rape, an important oil crop in the world. Effects of salt stress on their expression were studied with quantitative PCR and their potential use in the genetic engineering of oilseed rape was discussed.
Assuntos
Betaína-Aldeído Desidrogenase/genética , Brassica napus/genética , Metiltransferases/genética , Sequência de Aminoácidos , Sequência de Bases , Betaína/metabolismo , Betaína-Aldeído Desidrogenase/biossíntese , Betaína-Aldeído Desidrogenase/isolamento & purificação , Brassica napus/efeitos dos fármacos , Brassica napus/metabolismo , Colina/metabolismo , Clonagem Molecular , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Metiltransferases/biossíntese , Metiltransferases/isolamento & purificação , Dados de Sequência Molecular , Filogenia , RNA de Plantas/genética , Homologia de Sequência de Aminoácidos , Cloreto de Sódio/farmacologiaRESUMO
Betaine aldehyde dehydrogenase (BADH) catalyses the irreversible oxidation of betaine aldehyde to glycine betaine with the concomitant reduction of NAD(P)(+) to NAD(P)H. In the opportunistic pathogen Pseudomonas aeruginosa, this enzyme (PaBADH) could be an antimicrobial target. Several aldehyde dehydrogenases (ALDHs) are inactivated by arsenite in the presence of a low molecular thiol, a finding that was interpreted as a demonstration of the existence of vicinal thiols in these enzymes. As part of our studies on the susceptibility to chemical modification of the catalytic cysteine (C286) of PaBADH, we treated the enzyme with two arsenical reagents widely used to inhibit enzymes that have vicinal thiols: sodium m-arsenite plus 2,3-dimercaptopropanol (arsenite-BAL) and phenylarsine oxide (PAO). Here we report that they readily and reversibly inactivate PaBADH, even though the four cysteine residues of this enzyme (C286, C353, C377, and C439) are far from each other in the three-dimensional structure. Modification of PaBADH by both reagents was reversible by an excess of a dithiol (dithiothreitol), but only the PAO-modified enzyme could be reactivated by a monothiol (2-mercaptoethanol). C286 is the reactive residue as indicated by the following findings: (i) betaine aldehyde and NADP(+) afforded full protection against enzyme inactivation; (ii) the mutant proteins C353A, C377A, and C439A showed similar inactivation kinetics that the wild-type enzyme, and (iii) pretreatment of PaBADH with arsenite-BAL prevented irreversible inactivation by N-ethylmaleimide. Our results confirm previous findings on other ALDHs, and indicate that these vicinal thiol-specific reagents readily react with certain monothiols, such as the one of the catalytic cysteinyl residue of ALDHs. As arsenicals are being recently used to treat certain cancers, human ALDHs, even those not having conformationally vicinal thiols, may be unsuspected targets in these treatments.
Assuntos
Arsenicais/metabolismo , Arsenitos/metabolismo , Betaína-Aldeído Desidrogenase/metabolismo , Cisteína/metabolismo , Dimercaprol/metabolismo , Pseudomonas aeruginosa/enzimologia , Betaína-Aldeído Desidrogenase/antagonistas & inibidores , Betaína-Aldeído Desidrogenase/química , Betaína-Aldeído Desidrogenase/isolamento & purificação , Biocatálise , Cinética , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Rice (Oryza sativa) has two betaine aldehyde dehydrogenase homologs, BAD1 and BAD2, encoded on chromosome four and chromosome eight respectively. BAD2 is responsible for the characteristic aroma of fragrant rice. Complementary DNA clones of both BAD1 and BAD2 were isolated and expressed in E. coli. BAD2 had optimum activity at pH 10, little to no affinity towards N-acetyl-gamma-aminobutyraldehyde (NAGABald) with a Km of approximately 10 mM and moderate affinity towards gamma-guanidinobutyraldehyde (GGBald) and betaine aldehyde (bet-ald) with Km values of approximately 260 microM and 63 microM respectively. A lower Km of approximately 9 microM was observed with gamma-aminobutyraldehyde (GABald), suggesting BAD2 has a higher affinity towards this substate in vivo. The enzyme encoded on chromosome four, BAD1, had optimum activity at pH 9.5, showed little to no affinity towards bet-ald with a Km of 3 mM and had moderate affinity towards GGBald, NAGABald and GABald with Km values of approximately 545, 420 and 497 microM respectively. BAD1 had a half life roughly double that of BAD2. We discuss the implications of these findings on the pathway of fragrance generation in Basmati and Jasmine rice and the potential of rice to accumulate the osmoprotectant glycine betaine.