Molecular cloning of the full-length cDNA of (S)-hydroxynitrile lyase from Hevea brasiliensis. Functional expression in Escherichia coli and Saccharomyces cerevisiae and identification of an active site residue

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 aliph...

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Bibliographic Details
Published in:The Journal of biological chemistry 1996-03, Vol.271 (10), p.5884-5891
Main Authors: Hasslacher, M, Schall, M, Hayn, M, Griengl, H, Kohlwein, S D, Schwab, H
Format: Article
Language:English
Subjects:
Aldehyde-Lyases - biosynthesis
Aldehyde-Lyases - chemistry
Aldehyde-Lyases - metabolism
Amino Acid Sequence
Animals
Base Sequence
Binding Sites
Blotting, Northern
Blotting, Southern
Blotting, Western
Cloning, Molecular
DNA, Complementary
DNA, Plant - isolation & purification
Epoxide Hydrolases - chemistry
Escherichia coli
Hevea brasiliensis
Humans
Kinetics
Mammals
Molecular Sequence Data
Molecular Weight
Plant Leaves
Recombinant Proteins - biosynthesis
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
Rubber
Saccharomyces cerevisiae
Sequence Homology, Amino Acid
Substrate Specificity
Transcription, Genetic
Trees - enzymology
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Summary: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.
ISSN:0021-9258
DOI:10.1074/jbc.271.10.5884