A. Vanwormhoudt et D. Sellos, CLONING AND SEQUENCING ANALYSIS OF 3 AMYLASE CDNAS IN THE SHRIMP PENAEUS-VANNAMEI (CRUSTACEA-DECAPODA) - EVOLUTIONARY ASPECTS, Journal of molecular evolution, 42(5), 1996, pp. 543-551
In Penaeus vannamei, alpha-amylase is the most important glucosidase a
nd is present as at least two major isoenzymes which have been purifie
d. In order to obtain information on their structure, a hepatopancreas
cDNA library constructed in phage lambda-Zap II (Strategene) was scre
ened using a synthetic oligonucleotide based on the amino acid sequenc
e of a V8 staphylococcal protease peptide of P. vannamei alpha-amylase
. Three clones were selected: AMY SK 37 (EMBL sequence accession numbe
r: X 77318) is the most complete of the analyzed clones and was comple
tely sequenced. It contains the complete cDNA sequence coding for one
of the major isoenzymes of shrimp amylase. The deduced amino acid sequ
ence shows the existence of a 511-residue-long pre-enzyme containing a
highly hydrophobic signal peptide of 16 amino acids. Northern hybridi
zation of total RNA with the amylase cDNA con firms the size of the me
ssenger at around 1,600 bases. AMY SK 28, which contains the complete
mature sequence of amylase, belonged to the same family characterized
by a common 3' terminus and presented four amino acid changes. Some ot
her variants of this family were also partially sequenced. AMY SK 20 w
as found to encode a minor variant of the protein with a different 3'
terminus and 57 amino acid changes. Phylogenetic analysis established
with the conserved amino acid regions of the (beta/alpha) eight-barrel
domain and with the total sequence of P. vannamei showed close evolut
ionary relationships with mammals (59-63% identity) and with insect al
pha-amylase (52-62% identity). The use of conserved sequences increase
d the level of similarity but it did not alter the ordering of the gro
upings. Location of the secondary structure elements confirmed the hig
h level of sequence similarity of shrimp ol-amylase with pig alpha-amy
lase.