Genomic Organization and Structure of α-Hydroxynitrile Lyase in Cassava (Manihot esculentaCrantz)

Two clones with homology to the α-hydroxynitrile lyase (HNL) cDNA clone,MeHNL10, were isolatedfrom a λEMBL3 cassava (Manihot esculentaCrantz) genomic library. Analysis of the sequences showed that both genomic clones contain HNL genes (MeHNL4, MeHNL24) which are interrupted by two introns. RT–PCR analysis of MeHNL4 shows that it is expressed at high levels in seedling roots and at lower levels in cotyledons and young leaves. The deduced amino acid sequences ofMeHNL4, MeHNL10, andMeHNL24show high sequence identity and homology to the HNL fromHevea brasiliensiswhose tertiary structure has been solved at 1.9-Å resolution by X-ray crystallography. This high homology allowed the construction of model structures for all of the cassava proteins using the MODELLER program. Homology modeling indicates that the short variable exon 2 encodes the “cap” region which is thought to influence the substrate specificity of the protein. Two hybrid proteins were modeled using the core α/β domain ofMeHNL10and the cap region of either theHeveaHNL or a structurally relatedZeaprotein of unknown function. This analysis suggests that changes in the active site can be engineered by swapping exons.