RESUMEN
(S)-Hydroxynitrile lyase (Hnl) from the tropical rubber tree Hevea brasiliensis catalyzes the formation of (S)-cyanohydrins from hydrocyanic acid and aldehydes or ketones. This enzyme accepts aliphatic, aromatic, and heterocyclic carbonyl compounds as substrates and is therefore considered a potent biocatalyst for the industrial production of optically active chemicals. Limitations in enzyme supply from natural resources were overcome by production of the enzyme in the microbial host systems Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris. Expression of Hnl in the prokaryotic system led to the formation of inclusion bodies whereas in both yeast hosts high levels of soluble protein were obtained. Highest yields were obtained in a high cell density batch fermentation of a P. pastoris transformant that expressed heterologous Hnl to about 50% of the soluble cytosolic protein. At a cell density of 100 g/liter cell dry weight, a volume yield of 22 g/liter of heterologous product was obtained. Attempts to produce the Hnl protein extracellularly with the yeast hosts by applying different leader peptide strategies were not successful. Immunofluorescence microscopy studies indicated that the secretion-directed heterologous Hnl protein accumulated in the plasma membrane forming aggregated clusters of inactive protein.
Asunto(s)
Aldehído-Liasas/genética , Euphorbiaceae/enzimología , Aldehído-Liasas/biosíntesis , Aldehído-Liasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Catálisis , Cromatografía por Intercambio Iónico , Clonación Molecular/métodos , Escherichia coli , Microscopía Fluorescente , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Pichia , Saccharomyces cerevisiaeRESUMEN
(S)-Hydroxynitrile lyase (Hnl) from the tropical rubber tree Hevea brasiliensis is a 29 kDa single chain protein that catalyses the breakdown or formation of a C--C bond by reversible addition of hydrocyanic acid to aldehydes or ketones. The primary sequence of Hnl has no significant homology to known proteins. Detailed homology investigations employing PROFILESEARCH and secondary structure prediction algorithms suggest that Hnl is a member of the alpha/beta hydrolase fold protein family and contains a catalytic triad as functional residues for catalysis. The significance of predicted catalytic residues was tested and confirmed by site-directed mutagenesis and expression of mutant and wild-type proteins in the yeast, Saccharomyces cerevisiae. Based on these data we suggest a mechanistic model for the (S)-cyanohydrin synthesis catalyzed by hydroxynitrile lyase from Hevea brasiliensis.
Asunto(s)
Aldehído-Liasas/química , Aldehído-Liasas/metabolismo , Árboles/enzimología , Aldehído-Liasas/genética , Secuencia de Aminoácidos , Sitios de Unión , Secuencia Conservada , Hidrolasas/química , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Estructura Secundaria de Proteína , Alineación de Secuencia , Homología de Secuencia de AminoácidoAsunto(s)
Aldehído-Liasas/aislamiento & purificación , Aldehído-Liasas/genética , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , ADN Complementario/genética , Escherichia coli , Genes , Datos de Secuencia Molecular , Proteínas Recombinantes , Saccharomyces cerevisiae , Alineación de Secuencia , Relación Estructura-Actividad , ÁrbolesRESUMEN
BACKGROUND: Over three thousand species of plants, including important food crops such as cassava, use cyanogenesis, the liberation of HCN upon tissue damage, as a defense against predation. Detoxification of cyanogenic food crops requires disruption of the cyanogenic pathway. Hydroxynitrile lyase is one of the key enzymes in cyanogenesis, catalyzing the decomposition of an alpha-cyanohydrin to form HCN plus the corresponding aldehyde or ketone. These enzymes are also of potential utility for industrial syntheses of optically pure chiral cyanohydrins, being used to catalyze the reverse reaction. We set out to gain insight into the catalytic mechanism of this important class of enzymes by determining the three-dimensional structure of hydroxynitrile lyase from the rubber tree, Hevea brasiliensis. RESULTS: The crystal structure of the enzyme has been determined to 1.9 A resolution. It belongs to the alpha/beta hydrolase superfamily, with an active site that is deeply buried within the protein and connected to the outside by a narrow tunnel. The catalytic triad is made up of Ser80, His235 and Asp207. By analogy with known mechanisms of other members of this superfamily, catalysis should involve an oxyanion hole formed by the main chain NH of Cys81 and the side chains of Cys81 and Thr11. Density attributed to a histidine molecule or ion is found in the active site. CONCLUSIONS: By analogy with other alpha/beta hydrolases, the reaction catalyzed by hydroxynitrile lyase involves a tetrahedral hemiketal or hemiacetal intermediate formed by nucleophilic attack of Ser80 on the substrate, stabilized by the oxyanion hole. The SH group of Cys81 is probably involved in proton transfer between the HCN and the hydroxynitrile OH. This mechanism is significantly different from the corresponding uncatalyzed solution reaction.
Asunto(s)
Aldehído-Liasas/química , Cianuro de Hidrógeno/metabolismo , Árboles/metabolismo , Aldehído-Liasas/metabolismo , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Histidina/metabolismo , Modelos Moleculares , Árboles/enzimologíaRESUMEN
Crystals of the hydroxynitrile lyase from Hevea brasiliensis overexpressed in Pichia pastoris have been obtained by the hanging-drop technique at 294 K with ammonium sulfate and PEG 400 as precipitants. The crystals belong to the orthorhombic space group C222(1) with cell dimensions of a = 47.6, b = 106.8 and c = 128.2 A. The crystals diffract to about 2.5 A resolution on a rotating-anode X-ray source.
RESUMEN
The full-length cDNA of (S)-hydroxynitrile lyase (Hnl) from leaves of Hevea brasiliensis (tropical rubber tree) was cloned by an immunoscreening and sequenced. Hnl from H. brasiliensis is involved in the biodegradation of cyanogenic glycosides and also catalyzes the stereospecific synthesis of aliphatic, aromatic, and heterocyclic cyanohydrins, which are important as precursors for pharmaceutical compounds. The open reading frame identified in a 1. 1-kilobase cDNA fragment codes for a protein of 257 amino acids with a predicted molecular mass of 29.2 kDa. The derived protein sequence is closely related to the (S)-hydroxynitrile lyase from Manihot esculenta (Cassava) and also shows significant homology to two proteins of Oryza sativa with as yet unknown enzymatic function. The H. brasiliensis protein was expressed in Escherichia coli and Saccharomyces cerevisiae and isolated in an active form from the respective soluble fractions. Replacement of cysteine 81 by serine drastically reduced activity of the heterologous enzyme, suggesting a role for this amino acid residue in the catalytic action of Hnl.
Asunto(s)
Aldehído-Liasas/química , Aldehído-Liasas/metabolismo , Árboles/enzimología , Aldehído-Liasas/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Northern Blotting , Southern Blotting , Western Blotting , Clonación Molecular , ADN Complementario , ADN de Plantas/aislamiento & purificación , Epóxido Hidrolasas/química , Escherichia coli , Humanos , Cinética , Mamíferos , Datos de Secuencia Molecular , Peso Molecular , Hojas de la Planta , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Goma , Saccharomyces cerevisiae , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Transcripción GenéticaRESUMEN
When Aspergillus panamensis, CBS 120.45, was grown on a medium with high carbon and low nitrogen content, it was found to produce six antibiotically active metabolites. All compounds were identified as tetronic acid derivatives; three of them were found to be identical with gregatins A, B, and D, previously reported as metabolites of Cephalosporium gregatum. The other three compounds are new. All six compounds show antibacterial activity towards Gram-negative and Gram-positive bacteria. Five of the antibiotics exhibit inhibitory effects on the macromolecular syntheses in cells of the ascitic form of EHRLICH carcinoma (ECA) of mice.