Genomic Organization of the Zinc-Endopeptidase Astacin

The crayfish digestive protease astacin is the first described member of the astacin family of zinc-endopeptidases, for which it is regarded as a prototype. We have isolated and characterized the genomic sequence of astacin which spans a region of 2616 bp. The coding sequence is distributed over five exons and is interrupted by four introns. It was observed that structurally and functionally essential units of the protein, like the three α-helices, the five β-strands, the Zn-binding motif, and the Met turn are never disrupted by introns. The start site of transcription was determined by primer extension analysis, confirming the existence of a pre-pro-protein of 49 amino acids which so far had not been detectable at the protein level. In addition, when compared to the amino acid sequence of mature astacin, a carboxy-terminal extension of two additional amino acids was also found. The exon–intron pattern of the astacin gene was compared to those of three other astacin family members with known genomic sequences, i.e., tolloid ofDrosophila,the fish hatching enzyme LCE, and the human BMP1 gene. In each of the four proteins one intron was found to be inserted in the codon for a similar Gly residue which is highly conserved in this position within the astacin family.