Upgrading ethanol to higher order alcohols is desired but difficult using current biotechnological methods.

The ovarian tumor domain (OTU) deubiquitinylating cysteine proteases OTU ubiquitin aldehyde binding 1 (OTUB1) and OTUB2 are representative members of the OTU subfamily of deubiquitinylases. Deubiquitinylation critically regulates a multitude of important cellular processes, such as apoptosis, cell signaling, and growth. Moreover, elevated OTUB expression has been observed in various cancers, including glioma, endometrial cancer, ovarian cancer, and breast cancer. Here, using molecular dynamics simulation approaches, we found that both OTUB1 and OTUB2 display a catalytic triad characteristic of proteases, but differ in their configuration and protonation states. The OTUB1 protein had a pre-arranged catalytic site, with strong electrostatic interactions between the active-site residues His-265 and Asp-267. In OTUB2, however, the arrangement of the catalytic triad was different. In the absence of ubiquitin, the neutral states of the catalytic-site residues in OTUB2 were more stable, resulting in larger distances between these residues. Only upon ubiquitin binding, did the catalytic triad in OTUB2 re-arrange and bring the active site into a catalytically feasible state. An analysis of water access channels revealed only a few diffusion trajectories for the catalytically active form of OTUB1, whereas in OTUB2 the catalytic site was solvent accessible, and a larger number of water molecules reached and left the binding pocket. Interestingly, in OTUB2, the catalytic residues His-224 and Asn-226 formed a stable hydrogen bond. We propose that the observed differences in activation kinetics, protonation states, water channels, and active-site accessibility between OTUB1 and OTUB2 may be relevant for the selective design of OTU inhibitors.