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The view has been widely held that pepsin-like aspartic proteinases are found only in eukaryotes, and not in bacteria. However, a recent bioinfor-matics search [Rawlings ND & Bateman A (2009)BMC Genomics10, 437] revealed that, in seven of 1000 completely sequenced bacterial genomes, genes were present encoding polypeptides that displayed the requisite hall-mark sequence motifs of pepsin-like aspartic proteinases. | IFEBS Journal Shewasin A an active pepsin homolog from the bacterium Shewanella amazonensis Isaura Simoes1 2 Rosario Faro1 Daniel Bur3 John Kay4 and Carlos Faro1 2 1 CNC-Center for Neuroscience and CellBiology University of Coimbra Portugal 2 Biocant Biotechnology Innovation Center Cantanhede Portugal 3 Actelion Pharmaceuticals Ltd Allschwil Switzerland 4 Schoolof Biosciences Cardiff University UK Keywords aspartic proteinase bacteria pepsin-like Correspondence I. Simcoes Biocant Parque Tecnologico de Cantanhede Nucleo 4 Lote 3 3060-197 Cantanhede Portugal Fax 351 231 419O49 Tel 351 231 419040 E-mail isimoes@biocant.pt Received 19 April 2011 revised 4 July 2011 accepted 8 July 2011 doi 10.1111 j.1742-4658.2011.08243.x 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 PT positions of the substrate. It was completely inhibited by the general inhibitor of aspartic proteinases pepstatin and mutation of
