We have recently reported that Shewanella oneidensis, a Gram-negative ␥-proteobacterium with a rich arsenal of redox proteins, possesses four old yellow enzyme (OYE) homologues. Here, we report a series of high resolution crystal structures for one of these OYEs, Shewanella yellow enzyme 1 (SYE1), in its oxidized form at 1.4 Å resolution, which binds a molecule of PEG 400 in the active site, and in its NADH-reduced and p-hydroxybenzaldehyde-and p-hydroxyacetophenone-bound forms at 1.7 Å

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... on. Although the overall structure of SYE1 reveals a monomeric enzyme based on the ␣ 8 ␤ 8 barrel scaffold observed for other OYEs, the active site exhibits a unique combination of features: a strongly butterfly-bent FMN cofactor both in the oxidized and NADH-reduced forms, a collapsed and narrow active site tunnel, and a novel combination of conserved residues involved in the binding of phenolic ligands. Furthermore, we identify a second p-hydroxybenzaldehyde-binding site in a hydrophobic cleft next to the entry of the active site tunnel in the capping subdomain, formed by a restructuring of Loop 3 to an "open" conformation. This constitutes the first evidence to date for the entire family of OYEs that Loop 3 may indeed play a dynamic role in ligand binding and thus provides insights into the elusive NADH complex and into substrate binding in general. Structure-based sequence alignments indicate that the novelties we observe in SYE1 are supported by conserved residues in a number of structurally uncharacterized OYEs from the ␤and ␥-proteobacteria, suggesting that SYE1 represents a new subfamily of bacterial OYEs.