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Alison Pittman Article Critique Final
Alison Pittman Article Critique Final
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
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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. 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
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
temperature, 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
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
assumed that high temperatures could activate BTL2. The purpose of this paper was to analyze
temperature, high kinetic energy will be the main cause of activation, and if it is mediated by
enzyme-substrate interaction, the enzyme will active at lower temperatures in the presence of
lipid substrate. The determination of the crystal structure, activation mechanism and structural
is triggered in this family of lipases. This understanding could potentially be of use for the
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. Crystals were grown using the 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
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model was used to decipher the BTL2 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 may
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 findings. 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.
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Two Triton X-100 detergent molecules were found in the active site of BTL2, imitating chains of
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
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 backed
up 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
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