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J. Biol. Chem., Vol. 281, Issue 38, 28152-28161, September 22, 2006

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Ligand-induced Conformational Changes in the Capping Subdomain of a Bacterial Old Yellow Enzyme Homologue and Conserved Sequence Fingerprints Provide New Insights into Substrate Binding^*

From the Department of Biochemistry, Physiology and Microbiology, Laboratory for Protein Biochemistry and Protein Engineering, K.L. Ledeganckstraat 35, Ghent University, 9000 Ghent, Belgium

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 1.4Å 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 1.7Å resolution. Although the overall structure of SYE1 reveals a monomeric enzyme based on the ₈₈ 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.

Received for publication, April 25, 2006 , and in revised form, July 5, 2006.

^* This work was supported in part by the Research Infrastructure Action under EU-FP6 "Structuring the European Research Area Specific Programme" Contract RII3-CT-2004-506008. This work was also supported by Grant GOA 120154 from the Concerted Research Actions Program of Ghent University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The atomic coordinates and structure factors (codes 2GOU, 2GQ8, 2GQ9, and 2GQA) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

¹ To whom correspondence may be addressed. Tel.: 32-9-264-5124; Fax: 32-9-264-5338; E-mail: savvas.savvides{at}ugent.be. ² To whom correspondence may be addressed. Tel.: 32-9-264-5109; Fax: 32-9-264-5338; E-mail: jozef.vanbeeumen{at}ugent.be.

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