MOLECULAR-CLONING OF HYDROXYNITRILE LYASE FROM SORGHUM-BICOLOR (L) - HOMOLOGIES TO SERINE CARBOXYPEPTIDASES

The heterotetrameric enzyme hydroxynitrile lyase (HNL) from sorghum (E C 4.1.2.11)is involved in the catabolism of the cyanogenic glycoside d hurrin. We have isolated a cDNA clone comprising about 90 % of the COO H terminal sequence of a precursor which encodes both subunit of HNL f rom Sorghum bicolor L. (SbHNL). Hence the subunits of SbHNL must be th e result of post-translational processing. The deduced amino acid sequ ence of HNL shares significant sequence homology with members of the s erine carboxypeptidase family. In particular, HNL from sorghum shares the catalytical triad Asp, His, and Ser with these enzymes which evolv ed in 3 groups of enzymes (carboxypeptidase, chymotrypsin, and subtili sin) by convergent evolution. Moreover, like serine carboxypeptidases, HNL from sorghum consists of two pairs of glycosylated cysteine linke d A and B chains forming a heterotetramer of a molecular weight of 105 000 (carboxypeptidases 120000). Thus, HNL from sorghum closely resembl es to serine carboxypeptidases but differs from all other HNLs describ ed so far. Western blotting experiments revealed cross reaction betwee n carboxypeptidase from wheat and anti SbHNL antisera. Therefore, conv ergent evolution of HNLs from various ancestoral enzymes is conceivabl e. Hybridization of SbHNL cDNA to northern blots of total RNAs isolate d from various organs of young sorghum seedlings shows the same expres sion pattern of HNL as found by means of western blotting or enzyme as says. Using PCR and Southern blot analysis, we demonstrated that the g ene of SbHNL is free of introns. Further sequence analysis of cDNA clo nes and genomic DNA revealed a stretch of 23 adenine residues in the 3 '-untranslated part of the gene. Both, intronless organisation of the gene and a genomic stretch of oligo A suggests that SbHNL may have evo lved by a reverse transcription event.