Physicochemical Characterization of a Thermostable Alcohol Dehydrogenase from Pyrobaculum aerophilum
dc.contributor.author | Vitale, Annalisa | |
dc.contributor.author | Thorne, Natasha | |
dc.contributor.author | Lovell, Scott | |
dc.contributor.author | Battaile, Kevin P. | |
dc.contributor.author | Hu, Xin | |
dc.contributor.author | Shen, Min | |
dc.contributor.author | D'Auria, Sabato | |
dc.contributor.author | Auld, Douglas S. | |
dc.date.accessioned | 2014-03-20T16:17:47Z | |
dc.date.available | 2014-03-20T16:17:47Z | |
dc.date.issued | 2013-06-05 | |
dc.identifier.citation | Vitale, A., Thorne, N., Lovell, S., Battaile, K. P., Hu, X., Shen, M., … Auld, D. S. (2013). Physicochemical Characterization of a Thermostable Alcohol Dehydrogenase from Pyrobaculum aerophilum. PLoS ONE, 8(6). http://dx.doi.org/10.1371/journal.pone.0063828 | |
dc.identifier.uri | http://hdl.handle.net/1808/13297 | |
dc.description.abstract | In this work we characterize an alcohol dehydrogenase (ADH) from the hyperthermophilic archaeon Pyrobaculum aerophilum (PyAeADHII). We have previously found that PyAeADHII has no activity when standard ADH substrates are used but is active when α-tetralone is used as substrate. Here, to gain insights into enzyme function, we screened several chemical libraries for enzymatic modulators using an assay employing α-tetralone. The results indicate that PyAeADHII activity in the presence of α-tetralone was inhibited by compounds such as flunarizine. We also examined metal coordination of the enzyme in solution by performing metal substitution of the enzyme-bound zinc (Zn2+) with cobalt. The solution-based absorption spectra for cobalt substituted PyAeADHII supports substitution at the structural Zn2+ site. To gain structural insight, we obtained the crystal structure of both wild-type and cobalt-substituted PyAeADHII at 1.75 Å and 2.20 Å resolution, respectively. The X-ray data confirmed one metal ion per monomer present only at the structural site with otherwise close conservation to other ADH enzymes. We next determined the co-crystal structure of the NADPH-bound form of the enzyme at 2.35 Å resolution to help define the active site region of the enzyme and this data shows close structural conservation with horse ADH, despite the lack of a catalytic Zn2+ ion in PyAeADHII. Modeling of α-tetralone into the NADPH bound structure suggests an arginine as a possible catalytic residue. The data presented here can yield a better understanding of alcohol dehydrogenases lacking the catalytic zinc as well as the structural features inherent to thermostable enzymes. | |
dc.description.sponsorship | This work was supported by the Molecular Libraries Initiative of the National Institutes of Health Roadmap for Medical Research and the Intramural Research Program of the National Human Genome Research Institute, National Institutes of Health. Use of the IMCA-CAT beamline 17-ID at the Advanced Photon Source was supported by the companies of the Industrial Macromolecular Crystallography Association through a contract with Hauptman-Woodward Medical Research Institute. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. Use of the KU COBRE-PSF Protein Structure Laboratory was supported by grants from the National Center for Research Resources (5P20RR017708-10) and the National Institute of General Medical Sciences (8 P20 GM103420-10) from the National Institutes of Health. | |
dc.publisher | Public Library of Science | |
dc.rights | ©2013 Vitale et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. | |
dc.rights.uri | http://creativecommons.org/licenses/by/4.0/ | |
dc.subject | Alcohols | |
dc.subject | Cobalt | |
dc.subject | Crystal structure | |
dc.subject | Enzyme inhibitors | |
dc.subject | Enzyme structure | |
dc.subject | Enzymes | |
dc.subject | Library screening | |
dc.subject | Zinc | |
dc.title | Physicochemical Characterization of a Thermostable Alcohol Dehydrogenase from Pyrobaculum aerophilum | |
dc.type | Article | |
kusw.kuauthor | Lovell, Scott | |
kusw.kudepartment | Molecular Biosciences | |
kusw.kudepartment | Protein Structure Laboratory | |
kusw.oastatus | fullparticipation | |
dc.identifier.doi | 10.1371/journal.pone.0063828 | |
kusw.oaversion | Scholarly/refereed, publisher version | |
kusw.oapolicy | This item meets KU Open Access policy criteria. | |
dc.rights.accessrights | openAccess |
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Except where otherwise noted, this item's license is described as: ©2013 Vitale et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.