PMID:15327949
| Citation |
Sulzenbacher, G, Alvarez, K, Van Den Heuvel, RH, Versluis, C, Spinelli, S, Campanacci, V, Valencia, C, Cambillau, C, Eklund, H and Tegoni, M (2004) Crystal structure of E.coli alcohol dehydrogenase YqhD: evidence of a covalently modified NADP coenzyme. J. Mol. Biol. 342:489-502 |
|---|---|
| Abstract |
In the course of a structural genomics program aiming at solving the structures of Escherichia coli open reading frame (ORF) products of unknown function, we have determined the structure of YqhD at 2.0A resolution using the single wavelength anomalous diffraction method at the Pt edge. The crystal structure of YqhD reveals that it is an NADP-dependent dehydrogenase, a result confirmed by activity measurements with several alcohols. The current interpretation of our findings is that YqhD is an alcohol dehydrogenase (ADH) with preference for alcohols longer than C(3). YqhD is a dimer of 2x387 residues, each monomer being composed of two domains, a Rossmann-type fold and an alpha-helical domain. The crystals contain two dimers in the asymmetric unit. While one of the dimers contains a cofactor in both subunits, only one of the subunits in the second dimer contains it, making it possible to compare bound and unbound active sites. The active site contains a Zn atom, as verified by EXAFS on the crystals. The electron density maps of NADP revealed modifications of the nicotinamide ring by oxygen atoms at positions 5 and 6. Further analysis by electrospray mass spectrometry and comparison with the mass spectra of NADP and NADPH revealed the nature of the modification and the incorporation of two hydroxyl moieties at the 5 and 6 position in the nicotinamide ring, yielding NADPH(OH)(2). These modifications might be due to oxygen stress on an enzyme, which would functionally work under anaerobic conditions. |
| Links |
PubMed Online version:10.1016/j.jmb.2004.07.034 |
| Keywords |
Alcohol Dehydrogenase/chemistry; Alcohol Dehydrogenase/metabolism; Catalytic Domain; Coenzymes/chemistry; Coenzymes/metabolism; Crystallography, X-Ray; Escherichia coli/chemistry; Escherichia coli/enzymology; Escherichia coli/metabolism; Kinetics; Mass Spectrometry; Models, Molecular; NADP/metabolism; Niacinamide/chemistry; Niacinamide/metabolism; Protein Structure, Quaternary; Protein Structure, Tertiary |
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