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+- and Cl--dependent alpha-amylase. The nucleotide sequence of the amy gene and the amino acid sequences of the gene products indicate that the alpha-amylase precursor is a preproenzyme composed by the signal peptide (24 residues), the mature alpha-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 beta-pleated transmembrane proteins. It has no intramolecular chaperone function because active alpha-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 alpha-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 beta-lactamase-propeptide fusion protein.
Feller
G
G
Laboratory of Biochemistry, Institute of Chemistry B6, University of Liege, B-4000 Liege, Belgium. gfeller@ulg.ac.be
D'Amico
S
S
Benotmane
A M
AM
Joly
F
F
Van Beeumen
J
J
Gerday
C
C
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D002473:Cell Wall / Q000201:enzymology*; D002942:Circular Dichroism; D004269:DNA, Bacterial; D004792:Enzyme Precursors / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006090:Gram-Negative Bacteria / Q000201:enzymology*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001618:beta-Lactamases / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D000516:alpha-Amylases; C491797:alpha-amylase carboxy terminal propeptide, Pseudoalteromonas haloplanktis; D001618:beta-Lactamases