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 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.
Hasslacher
M
M
Institut für Biochemie, Technische Universität Graz, A8010 Graz, Austria.
Schall
M
M
Hayn
M
M
Griengl
H
H
Kohlwein
S D
SD
Schwab
H
H
D000446:Aldehyde-Lyases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D003001:Cloning, Molecular; D018076:DNA, Complementary; D018744:DNA, Plant / Q000302:isolation & purification; D004851:Epoxide Hydrolases / Q000737:chemistry; D004926:Escherichia coli; D006801:Humans; D007700:Kinetics; D008322:Mammals; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018515:Plant Leaves; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012408:Rubber; D012441:Saccharomyces cerevisiae; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014158:Transcription, Genetic; D014197:Trees / Q000201:enzymology*
D018076:DNA, Complementary; D018744:DNA, Plant; D011994:Recombinant Proteins; D012408:Rubber; D004851:Epoxide Hydrolases; D000446:Aldehyde-Lyases; C079359:hydroxymandelonitrile lyase