TY - BOOK
T1 - Structure and Function of Lipase
T2 - Altering the Activation Mechanism in Thermomyces lanuginosus Lipase by Rational Design
AU - Skjold-Jørgensen, Jakob
PY - 2015
Y1 - 2015
N2 - Lipases are triacylglycerol hydrolases (EC 3.1.1.3) which are able to act on water-insoluble esters, butdisplay very low activity towards water-soluble, monomeric substrates. This is ascribed to theircharacteristic activation mechanism occurring at the boundary between water and lipid, i.e. the waterlipidinterface. For Thermomyces lanuginosus lipase (TlL) and related lipases, activation of the enzymeinvolves a rearrangement of a structural domain, called the “lid”, which covers the active site inhomogenous aqueous solution. At the water-lipid interface, the lid is displaced from the active site andmoves towards an open conformation enabling the substrate to gain access, thus initiating catalysis.Lipases have been studied for decades and their functional features have drawn much attention withinindustrial applications since their first discovery. However, given that their molecular action takes placeat the water-lipid interface, structural movements occurring during activation have been difficult to probeexperimentally. In this work, novel variants of TlL were constructed based on rational design with amutated lid-region in order to elucidate the impact of the lid-residue composition and characteristics onthe activation mechanism. From characterization studies of these variants we have shown (Paper I) thatthe lid-region plays a crucial role in governing interfacial activation and enzymatic activity. Specifically,using a combination of spectroscopic and enzymatic activity-based methods we have characterized thelipase variants to show that the lid-mutations enable enzymatic activity on water-soluble substrates, fasteractivation at the water-lipid interface and a higher degree of calcium independence compared to wild-typeTlL. To investigate whether the observed activation differences between TlL and the designed lid-variantsinvolved differences in the conformation of the lid-region, a site-directed-fluorescence-labeling (SDFL)method called Tryptophan Induced Quenching (TrIQ) (Paper II) was applied. Using this method, it waspossible to probe the delicate movement (below 2 nm) of the lid in TlL as a function of solvent polarity,which showed pronounced differences in open vs. closed states of the lid between TlL and the lidvariants.To elucidate whether the observed differences in activation could be ascribed to a lowering ofthe energy barrier of lid-opening, molecular dynamic simulations were carried out to calculate the energydifference between the open and closed lid conformation for TlL and a selection of lid-variants (PaperIII). Here, a correlation between experimental and theoretical data was discovered supporting the notionlid plays a key role in governing activation at the interface, and that mutagenesis of the lid enables TlL tobecome activated at higher solvent polarities.Using the findings and acquired understanding of the lid’s impact on interfacial activation, a lipasemutant was designed containing an intrinsic switch enabling control of both activity and interfacialbinding (Paper IV). The crystal structure of this mutant was obtained and revealed the presence of asuccessfully formed disulfide bond spanning the active site pocket locking the lid in a closed, slightlystrained conformation. Using a conventional reducing agent, this bond was readily and specificallyreduced resulting in establishment of the lipase activity and interfacial binding.Combined, this work shows that the lid-region in TlL governs binding, interfacial activation, andenzymatic activity in TlL and related lipases. The protein design of novel TlL lid-variants facilitated achange in activation mechanism and enabled investigations structural movements associated with lipaseactivation at medium to high solvent polarities. The findings strengthen and expand on the understandingof the lid’s role in the activation of lipases, and have created interesting perspectives in designing futurelipases with faster activation, controlled regulation of binding, and catalysis at the water-lipid interface.
AB - Lipases are triacylglycerol hydrolases (EC 3.1.1.3) which are able to act on water-insoluble esters, butdisplay very low activity towards water-soluble, monomeric substrates. This is ascribed to theircharacteristic activation mechanism occurring at the boundary between water and lipid, i.e. the waterlipidinterface. For Thermomyces lanuginosus lipase (TlL) and related lipases, activation of the enzymeinvolves a rearrangement of a structural domain, called the “lid”, which covers the active site inhomogenous aqueous solution. At the water-lipid interface, the lid is displaced from the active site andmoves towards an open conformation enabling the substrate to gain access, thus initiating catalysis.Lipases have been studied for decades and their functional features have drawn much attention withinindustrial applications since their first discovery. However, given that their molecular action takes placeat the water-lipid interface, structural movements occurring during activation have been difficult to probeexperimentally. In this work, novel variants of TlL were constructed based on rational design with amutated lid-region in order to elucidate the impact of the lid-residue composition and characteristics onthe activation mechanism. From characterization studies of these variants we have shown (Paper I) thatthe lid-region plays a crucial role in governing interfacial activation and enzymatic activity. Specifically,using a combination of spectroscopic and enzymatic activity-based methods we have characterized thelipase variants to show that the lid-mutations enable enzymatic activity on water-soluble substrates, fasteractivation at the water-lipid interface and a higher degree of calcium independence compared to wild-typeTlL. To investigate whether the observed activation differences between TlL and the designed lid-variantsinvolved differences in the conformation of the lid-region, a site-directed-fluorescence-labeling (SDFL)method called Tryptophan Induced Quenching (TrIQ) (Paper II) was applied. Using this method, it waspossible to probe the delicate movement (below 2 nm) of the lid in TlL as a function of solvent polarity,which showed pronounced differences in open vs. closed states of the lid between TlL and the lidvariants.To elucidate whether the observed differences in activation could be ascribed to a lowering ofthe energy barrier of lid-opening, molecular dynamic simulations were carried out to calculate the energydifference between the open and closed lid conformation for TlL and a selection of lid-variants (PaperIII). Here, a correlation between experimental and theoretical data was discovered supporting the notionlid plays a key role in governing activation at the interface, and that mutagenesis of the lid enables TlL tobecome activated at higher solvent polarities.Using the findings and acquired understanding of the lid’s impact on interfacial activation, a lipasemutant was designed containing an intrinsic switch enabling control of both activity and interfacialbinding (Paper IV). The crystal structure of this mutant was obtained and revealed the presence of asuccessfully formed disulfide bond spanning the active site pocket locking the lid in a closed, slightlystrained conformation. Using a conventional reducing agent, this bond was readily and specificallyreduced resulting in establishment of the lipase activity and interfacial binding.Combined, this work shows that the lid-region in TlL governs binding, interfacial activation, andenzymatic activity in TlL and related lipases. The protein design of novel TlL lid-variants facilitated achange in activation mechanism and enabled investigations structural movements associated with lipaseactivation at medium to high solvent polarities. The findings strengthen and expand on the understandingof the lid’s role in the activation of lipases, and have created interesting perspectives in designing futurelipases with faster activation, controlled regulation of binding, and catalysis at the water-lipid interface.
UR - https://rex.kb.dk/primo-explore/fulldisplay?docid=KGL01010158232&context=L&vid=NUI&search_scope=KGL&tab=default_tab&lang=da_DK
M3 - Ph.D. thesis
BT - Structure and Function of Lipase
PB - Department of Chemistry, Faculty of Science, University of Copenhagen
ER -