CHARACTERIZATION OF THE C-TERMINAL PROPEPTIDE INVOLVED IN BACTERIAL WALL SPANNING OF ALPHA-AMYLASE FROM THE PSYCHROPHILE ALTEROMONAS-HALOPLANCTIS

The antarctic psychrophile Alteromonas haloplanctis secretes a Ca2+- a nd Cl--dependent alpha-amylase. The nucleotide sequence of the amy gen e and the amino acid sequences of the gene products indicate that the alpha-amylase precursor is a preproenzyme composed by the signal pepti de (24 residues), the mature alpha-amylase (453 residues, 49 kDa), and a long C-terminal propeptide or secretion helper (192 residues, 21 kD a). In cultures of the wild-type strain, the 70-kDa precursor is secre ted at the mid-exponential phase and is cleaved by a nonspecific prote ase into the mature enzyme and the propeptide. The purified C-terminal propeptide displays several features common to beta-pleated transmemb rane proteins. It has no intramolecular chaperone function because act ive alpha-amylase is expressed by Escherichia coli in the absence of t he propeptide coding region, In E. coli, the 70-kDa precursor is direc ted toward the supernatant, When the alpha-amylase coding region is ex cised from the gene, the secretion helper can still promote its own me mbrane spanning. It can also accept a foreign passenger, as shown by t he extracellular routing of a beta-lactamase-propeptide fusion protein .