Shewasin A, an active pepsin homolog from the bacterium Shewanella amazonensis

The view has been widely held that pepsin‐like aspartic proteinases are found only in eukaryotes, and not in bacteria. However, a recent bioinformatics search [Rawlings ND & Bateman A (2009) BMC Genomics 10 , 437] revealed that, in seven of ∼ 1000 completely sequenced bacterial genomes, genes were present encoding polypeptides that displayed the requisite hallmark sequence motifs of pepsin‐like aspartic proteinases. The implications of this theoretical observation prompted us to generate biochemical data to validate this finding experimentally. The aspartic proteinase gene from one of the seven identified bacterial species, Shewanella amazonensis , was expressed in Escherichia coli . The recombinant protein, termed shewasin A, was produced in soluble form, purified to homogeneity, and shown to display properties remarkably similar to those of pepsin‐like aspartic proteinases. Shewasin A was maximally active at acidic pH values, cleaving a substrate that has been widely used for assessment of the proteolytic activity of other aspartic proteinases, and displayed a clear preference for cleaving peptide bonds between hydrophobic residues in the P1*P1′ positions of the substrate. It was completely inhibited by the general inhibitor of aspartic proteinases, pepstatin, and mutation of one of the catalytic Asp residues (in the Asp‐Thr‐Gly motif of the N‐terminal domain) resulted in complete loss of enzymatic activity. It can thus be concluded unequivocally that this Shewanella gene encodes an active pepsin‐like aspartic proteinase. It is now beyond doubt that pepsin‐like aspartic proteinases are not confined to eukaryotes, but are encoded within some species of bacteria. The distinctions between the bacterial and eukaryotic polypeptides are discussed and their evolutionary relationships are outlined. Structured digital abstract•   Shewasin A   cleaves   Oxidized Insulin B chain  by  protease assay  (View Interaction  1 ,  2 )