S-selective hydroxynitrile lyase from a plant Baliospermum montanum: molecular characterization of recombinant enzyme.

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.