GENOMIC ORGANIZATION AND STRUCTURE OF ALPHA-HYDROXYNITRILE LYASE IN CASSAVA (MANIHOT-ESCULENTA CRANTZ)

Two clones with homology to the alpha-hydroxynitrile lyase (HNL) cDNA clone, MeHNL10, were isolated from a lambda EMBL3 cassava (Manihot esc ulenta Crantz) genomic library. Analysis of the sequences showed that both genomic clones contain HNL genes (Me-HNL4, MeHNL24) which are int errupted by two introns. RT-PCR analysis of MeHNL4 shows that it is ex pressed at high levels in seedling roots and at lower levels in cotyle dons and young leaves. The deduced amino acid sequences of MeHNL4, MeH NL10, and MeHNL24 show high sequence identity and homology to the HNL from Hevea brasiliensis whose tertiary structure has been solved at 1. 9-Angstrom resolution by X-ray crystallography. This high homology all owed the construction of model structures for all of the cassava prote ins using the MODELLER program. Homology modeIing indicates that the s hort variable exon 2 encodes the ''cap'' region which is thought to in fluence the substrate specificity of the protein. Two hybrid proteins were modeled using the core alpha/beta domain of MeHNL10 and the cap r egion of either the Hevea HNL or a structurally related Zea protein of unknown function. This analysis suggests that changes in the active s ite can be engineered by swapping exons. (C) 1998 Academic Press.