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Activation of Bacterial Thermoalkalophilic Lipases Is Spurred by Dramatic Structural Rearrangements César Carrasco-López, César Godoy, Blanca de las Rivas, Gloria Fernández-Lorente, José M. Palomo, José M. Guisán, Roberto Fernández-Lafuente, Martín Martínez-Ripoll, and Juan A. Hermoso The Journal of Biological Chemistry Vol 284, No. 7, pp. 4365 – 4372

Submitted by Alison Pittman November 5th, 2009

The main point being addressed in this article is discovering the source of mediation of the lid opening of the active site of the lipase Geobacillus thermocatenulatus. Also being determined is the mechanism of activation and structural rearrangements of the enzyme. Several experimental procedures were used to determine the answer to this question. Initially, the gene corresponding to BLT2 (one of two lipases produced from G. thermocatenulatus) was cloned, expressed and purified. Activity of the lipase was analyzed, and several experiments were performed to test BLT2 activation under varying conditions. Native crystals of the lipase were grown, data sets were collected and the images were processed. The crystal structure of the enzyme and the model was then refined. The researchers determined the overall structure of BTL2, as well as resolved its catalytic machinery and binding interactions with its substrate. It was noted that the activation of the lipase involves dramatic conformational rearrangements of two lids that cover the active site and produces new secondary structure elements. The crystal structure of the enzyme showed that the structural rearrangements were required for the activation of this family of lipases. Results also showed that the main driving force of this activation mechanism is the enzyme-substrate interaction. Lipase enzymes, at water/oil interfaces, catalyze the hydrolysis of long-chain triacylglycerides. Lipases often contain a lid domain that controls access to the enzyme active site. Access to the active site involves the displacement of the lid, which is induced by the interaction of lipid aggregates and the enzyme. Substrate molecules can then easily access the active site and the catalytic activity of the enzyme is increased. Bacterial thermoalkalophilic lipases are very resistant to proteases, detergents and chaotropic agents, and are stable at elevated temperatures as well as in organic solvents. These lipases are found in numerous thermophilic aerobic bacteria reclassified recently as genus Geobacillus. Members of this genus demonstrate

optimal activity at pH 8 – 10 and 60 - 75˚C and share approximately 95% amino acid sequence identity. Also characterized by their large molecular sizes, compared to other microbial lipases, the Geobacillus lipases are named the lipase family I.5. The crystal structure of three I.5 lipase species have been previously determined, as well as the presence in each of a long lid helix which buries the active site in the closed conformation. Each lipase contains a zinc-binding site that can account for the I.5 large molecular size. G. Thermocatenulatus is a thermophile that produces two lipases, BTL2 and BTL2. BTL2 is known to be stable at medium temperatures, at alkaline pH (9.0 – 11.0) and in organic solvents. The determination of its crystal structure, activation mechanism and structural rearrangements is significant for developing understanding of how interfacial activation is triggered in this family of lipases. This understanding could potentially be of use for the engineering of lipases with biotechnological functions. Multiple experimental procedures were used to analyze the structure and activation of the BTL2 enzyme. The experimental design appears to be adequate for studying the subject. First, the gene coding for the lipase was cloned into pT1 expression vector, and cells carrying the pT1BTL2 recombinant plasmid were grown and overexpression was induced. The lipase was then purified using a sequential chromatography step procedure in batch. The final washing step of this purification contained Triton X-100, a detergent that results in an open conformation of the enzyme. This method was appropriate, as it allowed the lipase to be in the open conformation for crystallization. The lipase was subsequently immobilized on cyanogen bromide-agarose. This procedure allowed the monomeric form of the lipase to be immobilized without any intermolecular lipase-lipase interactions. The activity of BTL2 was measured by absorbance variation at 348nm of p-nitrophenol in buffer M, using a thermostatted cuvette and magnetic stirring. Adding increasing concentrations of Triton X-100 to the purified, immobilized solution

in an appropriate buffer then tested the effect of activation of BTL2 with the detergent. Lipase inhibition kinetics were also studied using D-pNP in the absence, as well as increasing concentrations, of Triton X-100. Crystals were grown using the suitable hanging drop vapour diffusion method. Good quality crystals were produced, data sets were collected, and images were then processed and scaled using MOSFLM and SCALA programs. The molecular replacement method using the MOLREP program with another Geobacillus lipase as the initial model was used to decipher the BTL2 structure. The methods used seem to be appropriate to answer the questions, and the authors seemed to have considered the best options for their experimental methods. The experiments allowed the authors to determine the overall structure of BTL2. The authors found that the lipase is comprised of 389 residues and is formed of an irregular (α/β) hydrolase fold formed by a central β-sheet of seven strands surrounded by α-helices. No significant differences were found between the zinc-binding domain of BTL2 and those of previously determined I.5 lipases. The structure of the active (open) conformation of BTL2 was solved with two Triton X-100 molecules placed at the active site. The researchers determined that the BTL2 active site contains catalytic triad residues and an oxyanion hole, features also found in other thermoalkalophilic lipases. The conformational changes of the active site, and the residues involved, upon activation, are described in detail. The two molecules of Triton detergent were found to be occupying the hydrophobic active site cleft; the hydrophobic/aromatic side chains allow excellent stabilization of the lipid substrate. The active site was also found to contain three pockets for the branches of the triacylglycerol substrate. Activation of the lipase was unexpectedly found to involve remarkable conformational rearrangements of the two lids that cover the active site when the enzyme is inactive. When activated, new secondary structure

elements are produced in the enzyme, a large hydrophobic cavity is produced and hyperexposure of ten aromatic side chains occurs. The analysis of the open and closed forms allowed the researchers to examine how the large structural rearrangements are produced. The role of the zinc-binding domain was also proven to be necessary in the thermal stabilization of the active form at high temperatures and for stabilizing the structural refolding associated with the activation process. Analysis of the BTL2 at room temperature in the presence of both Triton X100 and the inhibitor D-pNP showed that activity increased with the detergent and was inhibited irreversibly by D-pNP. The authors now believe that the enzyme’s interaction with the substrate is the plausible main cause of the activation mechanism, as it is still active in the absence of high temperatures. The results are believable and all of the data is presented clearly in the article, with the use of helpful, comprehensible figures. All of the author’s statements are backed up by data and explained in detail. The question being addressed is appropriate for scientific inquery, as the structure and activation mechanism for BTL2 provide researchers with the possibility of future research on the engineering of lipases with biotechnological purposes. The results also provide a solid example of determinants that are involved in large structural rearrangements occurring when lipids and proteins interact. The researchers concluded that the enzyme-substrate interaction is the main driving force of the BTL2 activation mechanism. The structural rearrangements necessary for the activation of I.5 lipases was shown and the activation mechanism was determined. The results certainly support the author’s conclusions, but further experiments at varying temperatures with the same concentration of detergent would further solidify the conclusion that it is the enzymesubstrate interaction, and not temperature, which drives the BTL2 activation mechanism.