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.
Highlights
Alcohol dehydrogenases (ADHs; EC 1.1.1.1) are enzymes widely distributed in all living organisms [1] and play an important role in a broad range of physiological processes [2]
To make the assay more amenable to high-throughput formats, we measured the fluorescence of NADPH using quantitative HTS (qHTS)
Designing a high-throughput assay to screen for inhibitors of a hyperthermophilic enzyme like PyAEADHII is challenging for a number of reasons
Summary
Alcohol dehydrogenases (ADHs; EC 1.1.1.1) are enzymes widely distributed in all living organisms (archaea, bacteria, fungi, plants and animals) [1] and play an important role in a broad range of physiological processes (e.g. alcohol and alkane metabolism, cell defense towards exogenous alcohols and aldehydes) [2]. There are on-going efforts to structurally and functionally characterize ADHs from hyperthermophilic bacteria (optimal growth above 100uC). These ADHs are found to display extreme stability at high temperature, high pressure, and high concentrations of chemical denaturants, while demonstrating broad substrate specificity [3]. Many of the functional groups within these structures are found in chemical libraries employed in high-throughput screening (HTS) – large chemical libraries used to identify leads for drug discovery and for studying chemical biology. This suggests that screening ADH enzymes against HTS chemical libraries could yield both inhibitors and substrates of the enzyme
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
