Molecular analysis of (R)-(+)-mandelonitrile lyase microheterogeneity in black cherry

The flavoprotein (R)-(+)-mandelonitrile lyase (MDL; EC 4.1.2.10), which pla ys a key role in cyanogenesis in rosaceous stone fruits, occurs in black ch erry (Prunus serotina Ehrh.) homogenates as several closely related isoform s. Biochemical and molecular biological methods were used to investigate MD L microheterogeneity and function in this species. Three novel MDL cDNAs of high sequence identity (designated MDL2, MDL4, and MDL5) were isolated. Li ke MDL1 and MDL3 cDNAs (Z. Hu, J.E. Poulton [1997] Plant Physiol 115: 1359- 1369), they had open reading frames that predicted a flavin adenine dinucle otide-binding site, multiple N-glycosylation sites, and an N-terminal signa l sequence. The N terminus of an MDL isoform purified from seedlings matche d the derived amino acid sequence of the MDL4 cDNA. Genomic sequences corre sponding to the MDL1, MDL2, and MDL4 cDNAs were obtained by polymerase chai n reaction amplification of genomic DNA. Like the previously reported mdl3 gene, these genes are interrupted at identical positions by three short, co nserved introns. Given their overall similarity, we conclude that the genes mdl1, mdl2, mdl3, mdl4, and mdl5 are derived from a common ancestral gene and constitute members of a gene family. Genomic Southern-blot analysis sho wed that this family has approximately eight members. Northern-blot analysi s using gene-specific probes revealed differential expression of the genes mdl1, mdl2, mdl3, mdl4, and mdl5.