title: S-selective hydroxynitrile lyase from a plant Baliospermum montanum: molecular characterization of recombinant enzyme.
abstract: A novel S-hydroxynitrile lyase (HNL) was purified from leaves of a plant, Baliospermum montanum, by ammonium sulfate fractionation and column chromatographies. Full-length cDNA and genomic DNA were cloned and sequenced. The latter contained two introns and one ORF encoding a 263-residue protein (subunit: 29.5 kDa). The hnl gene was expressed in Escherichia coli and the enzyme was characterized including detailed kinetic studies of 20 substrates for (S)-cyanohydrin synthesis. The enzyme exhibited the highest specific activity (178 U/mg), k(cat) (98/s) and k(cat)/K(m) ratio for piperonal. k(cat)/K(m) ratio for aromatic aldehydes was much larger than those of aliphatic aldehydes and ketones. It was strongly inhibited by AgNO₃, PMSF, phenol and methyl ethyl ketone, showed an optimum at pH 5, while having activity at range of 4-6.5. It exhibited stability at wide pH range 2.4-11, the highest activity at 20 °C, being active at 0-65 °C. The enzyme showed variations in residues involved in substrate pocket and substrate entrance channel compared to other S-selective HNLs, based on a model was built. C-terminal short truncations provided more enzyme production. Gel filtration revealed a 60-65 kDa molecular mass for this non-FAD enzyme and its C-terminally truncated forms using three buffer compositions, indicating dimeric structures.
authors.affiliation: Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Dadashipour
authors.forename: Mizue
authors.initials: M
authors.lastname: Yamazaki
authors.forename: Kazumi
authors.initials: K
authors.lastname: Momonoi
authors.forename: Ken'ichirou
authors.initials: K
authors.lastname: Tamura
authors.forename: Ken-Ichi
authors.initials: K
authors.lastname: Fuhshuku
authors.forename: Yurina
authors.initials: Y
authors.lastname: Kanase
authors.forename: Etsuzoh
authors.initials: E
authors.lastname: Uchimura
authors.forename: Guan
authors.initials: G
authors.lastname: Kaiyun
authors.forename: Yasuhisa
authors.initials: Y
authors.lastname: Asano
mesh_terms: D000446:Aldehyde-Lyases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001547:Benzaldehydes; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D019665:Euphorbiaceae / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009570:Nitriles / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D001547:Benzaldehydes; D009570:Nitriles; D010940:Plant Proteins; D011994:Recombinant Proteins; C430593:cyanohydrin; D000446:Aldehyde-Lyases