Article Critique

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

December 3rd, 2009

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 The main point of the article was to discover what mediates the opening of the active site

of the lipase BTL2 from Geobacillus thermocatenulatus, temperature or interaction with the lipid

substrate. Structural and biochemical studies were used to answer this question. Activity of

BTL2 was analyzed at low temperatures with the addition of lipid substrate. X-ray

crystallography was used to determine the crystal structure of the lipase in the active state.

Native crystals of BTL2 were grown using the hanging drop vapour diffusion method and the

crystal structure of the enzyme was then solved. It was noted that the activation of the lipase

involves dramatic conformational rearrangements of two lids that cover the active site. The

crystal structure of the enzyme showed that these structural rearrangements are required for the

activation of BTL2. Results also showed that the main driving force of this activation

mechanism is the enzyme-substrate interaction, not elevated temperatures.

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. Substrate access to the active site involves the displacement of the lid, which is induced by

the interaction of lipid aggregates and the enzyme. Thermophiles are organisms that thrive at

relatively high temperatures. These organisms contain enzymes that are able to function at high

temperatures, which is essential to their survival. Bacterial thermoalkalophilic lipases are known

to be 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, as well as share

approximately 95% amino acid sequence identity. Geobacillus lipases are named the lipase

family I.5. The crystal structures of three I.5 lipase species have been previously solved. A long

lid helix, which buries the active site in the closed conformation, has been determined in each

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species. Each lipase also contains a zinc-binding site that can account for their large molecular

size. G. Thermocatenulatus is a thermophile that produces two lipases, BTL1 and BTL2. BTL2

is known to be stable at medium temperatures (50°C). Because it is a thermophilic enzyme, it is

assumed that high temperatures could activate BTL2. The purpose of this paper was to analyze

the mechanism of BTL2 activation and determine whether it is mediated by temperature or

enzyme-substrate interaction, a usual mechanism for lipases. If activation is mediated by

temperature, high kinetic energy will be the main cause of activation. If it is mediated by

enzyme-substrate interaction however, the enzyme will activate at lower temperatures in the

presence of lipid substrate. The determination of the crystal structure, activation mechanism and

structural rearrangements of BTL2 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.

The main methods used, as described in the article, appear to be appropriate to determine

the structure and activation of the BTL2 enzyme. First, the gene coding for the lipase was

cloned into pT1 expression vector 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. The monomeric form of the lipase was then immobilized to inhibit intermolecular

lipase-lipase interactions. The activation of BTL2 with varying concentrations of Triton X-100

at low temperatures was also tested. Good quality crystals were grown using the hanging drop

vapour diffusion method. Data sets were collected and images were 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

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structure. The X-ray crystallography method produced excellent density maps for the bulk of the

BTL2 structure. Fragments of Triton X-100, simulating substrate molecules, were found in the

catalytic groove of BTL2. The authors should have considered running NMR analysis of the

enzyme because it is comprised of 389 residues, which is still within an acceptable range for

NMR. Another method of determining the enzyme structure would have definitely

supplemented their findings, confirming their results. As well, because the authors are trying to

determine if temperature is the main mediator of activation for BTL2, perhaps an analysis of

enzyme activity at different temperatures would have been informative.

The methods used allowed the crystal structure of BTL2 to be solved in an open

configuration. It was found that BTL2 activation involves dramatic structural rearrangements of

approximately 70 amino acids. Activation of the lipase was also found to involve remarkable

conformational rearrangements of the two lids that cover the active site when the enzyme is

inactive. When the two lids, the α6 and α7-helices, are displaced, the active site becomes

unmasked. The transfer of bulky hydrophobic residues out of the N-terminal end of the α6-helix

is essential to the restructuring process of the lids. The incorporation of short side chain residues

to the α6 C-terminal end is also central to the displacement of the lids. The asymmetric amino

acid composition of the α6-helix and the adjustable loop appear to be important in the activation

mechanism. The structure of BTL2 shows that the zinc-binding domain also plays a significant

role in lid displacement. Residues Asp-239 and Gln-211 comprise the molecular hinge of the α7

lid, with Asp-239 being directly involved in the zinc cation coordination. In previous

experiments, the zinc domain has proven to be critical in stabilizing the structural rearrangements

during activation and in the thermal stabilization of the open conformation at high temperatures.

Two Triton X-100 detergent molecules were found in the active site of BTL2, imitating chains of

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the triglyceride substrate. This showed the position of the catalytic triad, three pockets that

accommodate the sn-1, sn-2 and sn-3 fatty acid chains. The crystal structure was solved in an

open conformation in the absence of high temperatures, indicating that the activation mechanism

is not temperature-mediated. The structural and biochemical studies of detergent-mediated

activation at low temperatures also showed that the BTL2 enzyme could be active at low

temperatures in the presence of substrate. All of the statements made by the authors are

supported by experimental data and the results are convincing. However, the authors did not

consider whether both temperature and enzyme-substrate interaction could together activate the

enzyme. Potentially, the greatest activation could occur under the influence of these two

variables at once. The authors did not test enzyme activity in lipid substrate at high

temperatures, which could have either confirmed or negated that possibility.

The question being addressed is appropriate for scientific inquiry, 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 that occur when lipids and

proteins interact. There are several pieces of evidence presented that support the conclusion that

the enzyme-substrate interaction is the driving force of the activation mechanism. The

researchers succeeded very well in showing that interaction with the lipid substrate can activate

the enzyme at low temperatures, but they did not show that high temperatures had negligible

influence on the activation mechanism. More experiments testing the effect of high temperatures

on BTL2 activation would certainly strengthen their conclusion. Overall, the paper was well

written, comprehensible and informative.