Characterization of the C-terminal propeptide involved in bacterial wall spanning of α-amylase from the psychrophile Alteromonas haloplanctis

Abstract

The antarctic psychrophile Alteromonas haloplanctis secretes a Ca2+- and Cl--dependent α-amylase. The nucleotide sequence of the amy gene and the amine acid sequences of the gene products indicate that the α-amylase precursor is a preproenzyme composed by the signal peptide (24 residues), the mature α-amylase (453 residues, 49 kDa), and a long C-terminal propeptide or secretion helper (192 residues, 21 kDa). In cultures of the wild-type strain, the 70-kDa precursor is secreted at the mid-exponential phase and is cleaved by a nonspecific protease into the mature enzyme and the propeptide. The purified C-terminal propeptide displays several features common to β- pleated transmembrane proteins. It has no intramolecular chaperone function because active α-amylase is expressed by Escherichia coli in the absence of the propeptide coding region. In E. coli, the 70-kDa precursor is directed toward the supernatant. When the α-amylase coding region is excised from the gene, the secretion helper can still promote its own membrane spanning. It can also accept a foreign passenger, as shown by the extracellular routing of a β-lactamase-propeptide fusion protein.