The lid is a structural and functional determinant of lipase activity and selectivity

Secundo, Francesco; Carrea, Giacomo; Tarabiono, Chiara; Gatti‐Lafranconi, Pietro; Brocca, Stefania; Lotti, Marina; Jaeger, Karl‐Erich; Puls, Michael; Eggert, Thorsten

doi:10.1016/j.molcatb.2006.01.018

Cited by 119 publications

(67 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…A similar strategy of "lid swapping" has previously been explored by Secundo, Jaeger and co-workers, who mainly worked with Candida rugosa lipase isoenzymes, but they also investigated lid mutations in the Pseudomonas fragi lipase, as well as insertion of lid fragments from homologues on the lid-less Bacillus subtilis li-A C H T U N G T R E N N U N G pase A. [39] In our work with CALB, in particular the transfer of lids from the N. crassa and G. zeae homologues resulted in chimeric lipases with interesting properties. These enzymes were "CALB-N. crassa" (with a lid from Q7RYD2): Y135F, K136H, V139M, G142Y, P143G, D145C, L147G, A148N, V149F, S150G, KVAKAGAPC, A151P, W155L, and "CALB-G. zeae" (with a lid from Q4HUY1): V139I, G142N, P143I, L144G, D145G, L147T, A148G, V149L, S150IN, A151T, S153A, W155V.…”

Section: Resultsmentioning

confidence: 99%

Understanding the Plasticity of the α/β Hydrolase Fold: Lid Swapping on the Candida antarctica Lipase B Results in Chimeras with Interesting Biocatalytic Properties

et al. 2009

View full text Add to dashboard Cite

The best of both worlds. Long molecular dynamics (MD) simulations of Candida antarctica lipase B (CALB) confirmed the function of helix alpha5 as a lid structure. Replacement of the helix with corresponding lid regions from CALB homologues from Neurospora crassa and Gibberella zeae resulted in new CALB chimeras with novel biocatalytic properties. The figure shows a snapshot from the MD simulation. The Candida antarctica lipase B (CALB) has found very extensive use in biocatalysis reactions. Long molecular dynamics simulations of CALB in explicit aqueous solvent confirmed the high mobility of the regions lining the channel that leads into the active site, in particular, of helices alpha5 and alpha10. The simulation also confirmed the function of helix alpha5 as a lid of the lipase. Replacing it with corresponding lid regions from the CALB homologues from Neurospora crassa and Gibberella zeae resulted in two new CALB mutants. Characterization of these revealed several interesting properties, including increased hydrolytic activity on simple esters, specifically substrates with C(alpha) branching on the carboxylic side, and much increased enantioselectivity in hydrolysis of racemic ethyl 2-phenylpropanoate (E>50), which is a common structure of the profen drug family.

show abstract

Section: Resultsmentioning

confidence: 99%

Understanding the Plasticity of the α/β Hydrolase Fold: Lid Swapping on the Candida antarctica Lipase B Results in Chimeras with Interesting Biocatalytic Properties

et al. 2009

View full text Add to dashboard Cite

show abstract

“…[32] Researchers replaced the lid region (residues 66-93) in lip1 with the corresponding sequence in lip3 to create lip1lid3. This replacement introduced six changes in the amino acids sequence at positions 69, 74, 76, 88, 91, and 93.…”

Section: Orientation Of the Slow-reacting Enantiomermentioning

confidence: 99%

Molecular Basis of Chiral Acid Recognition by Candida rugosa Lipase: X‐Ray Structure of Transition State Analog and Modeling of the Hydrolysis of Methyl 2‐Methoxy‐2‐phenylacetate

et al. 2011

View full text Add to dashboard Cite

Lipase from Candida rugosa shows high enantioselectivity toward a-substituted chiral acids such as 2-arylpropionic acids. To understand how Candida rugosa lipase (CRL) distinguishes between enantiomers of chiral acids, we determined the X-ray crystal structure of a transition-state analog covalently linked to CRL. CRL shows moderate enantioselectivity (E = 23) toward methyl 2-methoxy-2-phenylacetate, 1-methyl ester, favoring the (S)-enantiomer. We synthesized phosphonate (R C ,R P S P )-3, which, upon reaction with CRL, mimics the transition state for hydrolysis of (S)-1-methyl ester, the fast-reacting enantiomer. An X-ray crystal structure of this complex shows a catalytically productive orientation with the phenyl ring in the hydrophobic tunnel of the lipase. Phe345 crowds the region near the substrate stereocenter. Computer modeling of the slow-reacting enantiomer examined four possible conformations for the corresponding slow-reacting enantiomer: three conformations where two substituents at the stereocenter have been exchanged relative to the fast-reacting enantiomer and one conformation with an umbrella-like inversion orientation. Each of these orientations disrupts the orientation of the catalytic histidine, but the molecular basis for disruption differs in each case showing that multiple mechanisms are required for high enantioselectivity.

show abstract

“…It is important to note that, for both lipase BC and CALB, the comparison of activity between the various enzyme forms and that with the enzyme in water was performed referring to the same amount of lipase. A 6.4 -fold increase in the transesterifi cation activity was also observed in the case of wild type Bacillus subtilis lipase A ( wtBSLA ) when it was previously lyophilized with PEG (Table 5.3 ) [8] .…”

Section: Lipase Formulations and Their Activity And Enantioselectivitmentioning

confidence: 71%

Importance of Enzyme Formulation for the Activity and Enantioselectivity of Lipases in Organic Solvents

Secundo

2008

Modern Biocatalysis

Self Cite

View full text Add to dashboard Cite

5.1 IntroductionLipases are of remarkable practical interest since they have been used in numerous biocatalytic applications, such as kinetic resolution of alcohols and carboxyl esters (both in water and in non -aqueous media) [1] , regioselective acylations of polyhydroxylated compounds, and the preparation of enantiopure amino acids and amides [2,3] . Moreover, lipases are stable in organic solvents, do not require cofactors, possess broad substrate specifi city, and exhibit, in general, a high enantioselectivity. All these features have contributed to make lipases the class of enzyme with the highest number of biocatalytic applications carried out in neat organic solvents.The use of organic solvents is especially advantageous in transforming substrates that are unstable or poorly soluble in water. Furthermore, in the absence of water, the synthesis of ester and amide bonds by hydrolases (mainly lipases and proteases) can be favored over hydrolysis and many side reactions that are water dependent can be prevented. By varying the organic solvent it is also possible to control the substrate specifi city and the regio -and enantioselectivity of a given enzyme. However, although enzymes in organic media show numerous advantages, their catalytic effi ciency is, in most cases, orders of magnitude lower than in aqueous systems. This behavior can be ascribed to different causes such as high saturating substrate concentrations, diffusional limitations, low stabilization of the transition state of the enzyme -substrate intermediate, restricted protein fl exibility, and aggregation and distortions of the enzyme molecules caused by dehydration, which become irreversible in anhydrous solvents [4] .The present chapter briefl y examines and discusses the effects of some additives, namely lyoprotectants (sugars, crown ethers, and methoxypoly(ethylene glycol)), on the activity and enantioselectivity of several lipases. A possible rationale for the increment usually observed when using lyoprotectants, on the basis of conformational data obtained by means of Fourier -transform infrared (FT -IR) spectroscopy is also suggested.

show abstract

The lid is a structural and functional determinant of lipase activity and selectivity

Cited by 119 publications

References 24 publications

Understanding the Plasticity of the α/β Hydrolase Fold: Lid Swapping on the Candida antarctica Lipase B Results in Chimeras with Interesting Biocatalytic Properties

Understanding the Plasticity of the α/β Hydrolase Fold: Lid Swapping on the Candida antarctica Lipase B Results in Chimeras with Interesting Biocatalytic Properties

Molecular Basis of Chiral Acid Recognition by Candida rugosa Lipase: X‐Ray Structure of Transition State Analog and Modeling of the Hydrolysis of Methyl 2‐Methoxy‐2‐phenylacetate

Importance of Enzyme Formulation for the Activity and Enantioselectivity of Lipases in Organic Solvents

Contact Info

Product

Resources

About