Characterizing UshA Cleavage Activity in Shewanella oneidensis

Abstract

Shewanella oneidensis MR-1 is a facultative anaerobe capable of respiring insoluble, extracellular substrates using flavins, such as FMN, as electron shuttles. MR-1 generates FMN through the cleavage of periplasmic FAD, using multifunctional, 5’-nucleotidase, UshA. A homologous protein, E. coli UshA shares substrates with MR-1 UshA but has little FAD cleavage activity. To understand this difference in substrate specificity, the crystal structure of E. coli UshA was used to model the hypothetical binding pocket of MR-1 UshA. Residues that differed from E. coli UshA were identified, and point mutations were introduced to MR-1 UshA to determine the impact of those residues on FAD cleavage activity. The results highlight the relationship between protein structure and specificity and increase understanding of an enzyme involved in MR-1 extracellular electron transfer.