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Molecular cloning of hydroxynitrile lyase from Sorghum bicolor (L.). Homologies to serine carboxypeptidases

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Abstract

The heterotetrameric enzyme hydroxynitrile lyase (HNL) from sorghum (EC 4.1.2.11) is involved in the catabolism of the cyanogenic glycoside dhurrin. We have isolated a cDNA clone comprising about 90% of the COOH terminal sequence of a precursor which encodes both subunit of HNL from Sorghum bicolor L. (SbHNL). Hence the subunits of SbHNL must be the result of post-translational processing. The deduced amino acid sequence of HNL shares significant sequence homology with members of the serine carboxypeptidase family. In particular, HNL from sorghum shares the catalytical triad Asp, His, and Ser with these enzymes which evolved in 3 groups of enzymes (carboxypeptidase, chymotrypsin, and subtilisin) by convergent evolution. Moreover, like serine carboxypeptidases, HNL from sorghum consists of two pairs of glycosylated cysteine linked A and B chains forming a heterotetramer of a molecular weight of 105000 (carboxypeptidases 120000). Thus, HNL from sorghum closely resembles to serine carboxypeptidases but differs from all other HNLs described so far. Western blotting experiments revealed cross reaction between carboxypeptidase from wheat and anti SbHNL antisera. Therefore, convergent evolution of HNLs from various ancestoral enzymes is conceivable. Hybridization of SbHNL cDNA to northern blots of total RNAs isolated from various organs of young sorghum seedlings shows the same expression pattern of HNL as found by means of western blotting or enzyme assays. Using PCR and Southern blot analysis, we demonstrated that the gene of SbHNL is free of introns. Further sequence analysis of cDNA clones 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 evolved by a reverse transcription event.

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Abbreviations

CPDB:

barley serine carboxypeptidase

CPDW:

wheat serine carboxypeptidase

CPDY:

yeast serine carboxypeptidase

DFP:

diisopropylfluorophosphate

HNL:

hydroxynitrile lyase

mAb:

monoclonal antibody

PCR:

polymerase chain reaction

SbHNL:

hydroxynitrile lyase from Sorghum bicolor L.

SDS-PAGE:

sodium dodecyl sulfate polyacrylamide gel electrophoresis

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Authors and Affiliations

  1. Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany

    Harald Wajant & Klaus Pfizenmaier

  2. Biological Institute, Department of Botany, University of Stuttgart, Pfaffenwaldring 57, 70569, Stuttgart, Germany

    Karl-Wolfgang Mundry

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  1. Harald Wajant
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  2. Karl-Wolfgang Mundry
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Wajant, H., Mundry, KW. & Pfizenmaier, K. Molecular cloning of hydroxynitrile lyase from Sorghum bicolor (L.). Homologies to serine carboxypeptidases. Plant Mol Biol 26, 735–746 (1994). https://doi.org/10.1007/BF00013758

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  • DOI: https://doi.org/10.1007/BF00013758

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