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By contrast, pepsin homologs from bacteria have only recently started to be characterized. The work with recombinant shewasin A from<jats:italic>Shewanella amazonensis<\/jats:italic>provided the first documentation of this activity in prokaryotes. Here we extend our studies to shewasin D, the pepsin homolog from<jats:italic>Shewanella denitrificans<\/jats:italic>, to gain further insight into this group of bacterial peptidases that likely represent ancestral versions of modern eukaryotic pepsin-like enzymes. We demonstrate that the enzymatic properties of recombinant shewasin D are strongly reminiscent of eukaryotic pepsin homologues. We determined the specificity preferences of both shewasin D and shewasin A using proteome-derived peptide libraries and observed remarkable similarities between both shewasins and eukaryotic pepsins, in particular with BACE-1, thereby confirming their phylogenetic proximity. Moreover, we provide first evidence of expression of active shewasin D in<jats:italic>S. denitrificans<\/jats:italic>cells, confirming its activity at acidic pH and inhibition by pepstatin. Finally, our results revealed an unprecedented localization for a family A1 member by demonstrating that native shewasin D accumulates preferentially in the cytoplasm.<\/jats:p>","DOI":"10.1038\/srep23869","type":"journal-article","created":{"date-parts":[[2016,3,31]],"date-time":"2016-03-31T09:39:47Z","timestamp":1459417187000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Enzymatic properties, evidence for in vivo expression, and intracellular localization of shewasin D, the pepsin homolog from Shewanella denitrificans"],"prefix":"10.1038","volume":"6","author":[{"given":"Ana Rita","family":"Leal","sequence":"first","affiliation":[]},{"given":"Rui","family":"Cruz","sequence":"additional","affiliation":[]},{"given":"Daniel","family":"Bur","sequence":"additional","affiliation":[]},{"given":"Pitter F.","family":"Huesgen","sequence":"additional","affiliation":[]},{"given":"Ros\u00e1rio","family":"Faro","sequence":"additional","affiliation":[]},{"given":"Bruno","family":"Manadas","sequence":"additional","affiliation":[]},{"given":"Alexander","family":"Wlodawer","sequence":"additional","affiliation":[]},{"given":"Carlos","family":"Faro","sequence":"additional","affiliation":[]},{"given":"Isaura","family":"Sim\u00f5es","sequence":"additional","affiliation":[]}],"member":"297","published-online":{"date-parts":[[2016,3,31]]},"reference":[{"key":"BFsrep23869_CR1","first-page":"437","volume":"10","author":"ND Rawlings","year":"2009","unstructured":"N. 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