DROSOPHILA-MELANOGASTER ANGIOTENSIN-I-CONVERTING ENZYME EXPRESSED IN PICHIA-PASTORIS RESEMBLES THE C-DOMAIN OF THE MAMMALIAN HOMOLOG AND DOES NOT REQUIRE GLYCOSYLATION FOR SECRETION AND ENZYMATIC-ACTIVITY

Drosophila melanogaster angiotensin I-converting enzyme (AnCE) is a se creted single-domain homologue of mammalian angiotensin I-converting e nzyme (ACE) which comprises two domains (N and C domains). In order to characterize in detail the enzymic properties of AnCE and to study th e influence of glycosylation on the secretion and enzymic activity of this enzyme, we overexpressed AnCE (expression level, 160 mg/l) and an unglycosylated mutant (expression level, 43 mg/l) in the yeast Pichia pastoris. The recombinant enzyme was apparently homogeneous on SDS/PA GE without purification and partial deglycosylation demonstrated that all three potential sites for N-linked glycosylation were occupied by oligosaccharide chains. Each N-glycosylation sequence (Asn-Xaa-Ser/Thr ) was disrupted by substituting a glutamine for the asparagine residue at amino acid positions 53, 196 and 311 by site-directed mutagenesis to produce a single mutant, Expression of the unglycosylated mutant in Pichia produced a secreted catalytically active enzyme (AnCE(Delta CH O)). This mutant displayed unaltered kinetics for the hydrolyses of hi ppuryl-His-leu, angiotensin I and N-acetyl-Ser-Asp-Lys-Pro (AcSDKP) an d was equally sensitive to ACE inhibitors compared with wild-type AnCE , However, AnCE(Delta CHO) was less stable, displaying a half-life of 4.94 h at 37 degrees C, compared with AnCE which retained full activit y under the same conditions, Two catalytic criteria demonstrate the fu nctional resemblance of AnCE with the human ACE C domain: first, the k (eat)/K-m of AcSDKP hydrolysis and secondly, the k(eat)/K-m and optima l chloride concentration for hippuryl-His-Leu hydrolysis. A range of A CE inhibitors were far less potent towards AnCE compared with the huma n ACE domains, except for captopril which suggests an alternative stru cture in AnCE corresponding to the region of the S-1 subsite in the hu man ACE active sites.