58th Annual Report on Research 2013 Under Sponsorship of the ACS Petroleum Research Fund

The goal of this project is to reveal the mechanism behind interfacial enzymes such as certain lipases that are torrent or even thrive in organic solvents. Most biological catalysts are not active in oil rich media as they are evolved to perform functions in predominantly aqueous environments. Still, interfacial proteins and a group of extremophilic enzymes provide interesting examples to explore the design of biomolecules that can manipulate potential petroleum products in organic solvents.

Low polarity solvent induced peptide conformational switch: During the first funding year, we were able to follow our plan to examine a particular bacterial lipase's gating motion. The gating motion of the enzyme is critical for the admission of the substrate, triglyceride (TAG), an important ester. In aqueous solutions, the enzyme has a closed gate, so that no substrate can access the active site to be catalyzed. When the lipase encounters a water-oil interface, the gate is opened to allow the entrance of the substrate. Previously, we characterized the gating motion at the phenomenological level (Johnson et al. Biochemistry, 2012). With the help of the ACS PRF, we have been able to look at the triggering mechanism of lipase gating in atomistic detail this year. Indeed, an amphiphilic peptide, α5, is the critical part of the access channel. We have studied the conformations of the α5 peptide in various solvent conditions. We found that α5 adopts α-helix conformations in a high polarity environment but changes to 3₁₀helix conformations in low polarity solvents such as hexane and other aliphatic solvents. To our knowledge, this is the first report of organic-solvent-induced secondary structural changes of a natural protein. We further tie how this conformational change is related to the gating motion. With the support of ACS PRF, this work has been published recently (Nellas et al. Biochemistry, 2013).

DMSO activation: Besides understanding the lipase in aliphatic solvents (such as hexane), we also set up and performed molecular dynamics simulations of the lipase in DMSO (dimethyl sulfoxide), which has an intriguing effect on lipase activity. With increasing DMSO concentration (in water), the activity of many lipases first shows a strong increase at low concentrations (<50 %  w/w) and then a decrease at high concentrations. How the conformation of the enzyme contributes to the DMSO activation phenomenon is not clear. We thus set up lipase simulations in various concentrations of DMSO this year Indeed, for the initial 50 ns simulation we performed we observed that the level of lipase gating is strongly affected by DMSO. The gate opening increases with DMSO concentration till the high DMSO concentration. Our next step is to observe the mechanism of DMSO's effect on lipase gating and to provide a molecular explanation.

Aliphatic vs. Aromatic solvent: There is evidence of preference in the pairing of specific lipases and specific organic solvent types. We hypothesize that the preference comes from specific interactions of the solvents with side-chains. For example, lipase Pseudomonas aeruginosa PAL-LST03 functions better (110%) in decane-containing solution than in aqueous solution, while its activity is reduced to 19%, if it is in a benzene-containing solution. Further, in contrast to our focus lipase PAL-LST03, a lipase from Acinetobacter baylyi seems to be very tolerant to benzene but does not work well with decane. This phenomenon cannot be explained using the argument of octanol/water partitioning (log P_O/W values, measuring the polarity) of organic solvents, since both solvents are hydrophobic and the correlation is very poor between the activity and log P_O/W for a series of organic solvents. This year,we set up lipase PAL-LST03 in aromatic solvent (benzene), and have preformed a 100 ns simulation. Indeed, we found that the gating behavior in benzene is very different from that of previously reported lipase gating in aliphatic solvent (hexane). Our next step is to study the solvent-protein interaction at molecular detail to reveal the origin of the difference. We will apply the recently developed solvent density mapping techniques to compare the solvent hot spots surrounding the lipase for these two types of solvents in the coming year.