Purification, characterization, and cloning of alpha-hydroxynitrile lyase from cassava (Manihot esculenta Crantz).
alpha-Hydroxynitrile lyase (HNL, acetone cyanohydrin lyase, EC 4.1.2.37) was purified to homogeneity from young leaves of the cyanogenic tropical crop plant cassava (Manihot esculenta Crantz). The purified protein is a homo-trimer with a subunit relative molecular mass of 28,500 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The active protein is not glycosylated and does not contain a flavin group. HNL exhibits complex kinetics which vary according to substrate concentration and may be related to aggregation of the enzyme. HNL activity against two natural substrates, acetone cyanohydrin and 2-butanone cyanohydrin, and one nonphysiological substrate, 2-pentanone cyanohydrin, was demonstrated. N-terminal and internal peptide sequences, obtained from HNL digested with the endoproteinase Glu-C, were used to design degenerate oligonucleotide primers for polymerase chain reaction with single-strand cDNA, using purified mRNA from cotyledons as template. The resulting DNA fragment was used to probe a cassava cotyledon cDNA library. Four cDNA clones were isolated, sequenced, and shown to contain derived amino acid sequences identical to those obtained from the purified protein.
Hughes
J
J
Department of Biochemistry and Genetics, Medical School, University of Newcastle upon Tyne, United Kingdom.
Carvalho
F J
FJ
Hughes
M A
MA
D000446:Aldehyde-Lyases / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D002365:Manihot / Q000201:enzymology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity
D017931:DNA Primers; D018076:DNA, Complementary; D010446:Peptide Fragments; D000446:Aldehyde-Lyases; C087495:acetone-cyanohydrin lyase