Linda Fransson

Enantioselectivity in Candida antarctica lipase B: A molecular dynamics study

A major problem in predicting the enantioselectivity of an enzyme toward substrate molecules is that even high selectivity toward one substrate enantiomer over the other corresponds to a very small difference in free energy. However, total free energies in enzyme‐substrate systems are very large and fluctuate significantly because of general protein motion. Candida antarctica lipase B (CALB), a serine hydrolase, displays enantioselectivity toward secondary alcohols. Here, we present a modeling study where the aim has been to develop a molecular dynamics‐based methodology for the prediction of enantioselectivity in CALB. The substrates modeled (seven in total) were 3‐methyl‐2‐butanol with various aliphatic carboxylic acids and also 2‐butanol, as well as 3,3‐dimethyl‐2‐butanol with octanoic acid. The tetrahedral reaction intermediate was used as a model of the transition state. Investigative analyses were performed on ensembles of nonminimized structures and focused on the potential energies of a number of subsets within the modeled systems to determine which specific regions are important for the prediction of enantioselectivity. One category of subset was based on atoms that make up the core structural elements of the transition state. We considered that a more favorable energetic conformation of such a subset should relate to a greater likelihood for catalysis to occur, thus reflecting higher selectivity. The results of this study conveyed that the use of this type of subset was viable for the analysis of structural ensembles and yielded good predictions of enantioselectivity.

Size as a parameter for solvent effects on Candida antarctica lipase B enantioselectivity

Changes in solvent type were shown to yield significant improvement of enzyme enantioselectivity. The resolution of 3-methyl-2-butanol catalyzed by Candida antarctica lipase B, CALB, was studied in eight liquid organic solvents and supercritical carbon dioxide, SCCO(2). Studies of the temperature dependence of the enantiomeric ratio allowed determination of the enthalpic (Delta(R-S)Delta H(++)) as well as the entropic (Delta(R-S)Delta S(++)) contribution to the overall enantioselectivity (Delta(R-S)Delta G(++)= -RTlnE). A correlation of the enantiomeric ratio, E, to the van der Waals volume of the solvent molecules was observed and suggested as one of the parameters that govern solvent effects on enzyme catalysis. An enthalpy-entropy compensation relationship was indicated between the studied liquid solvents. The enzymatic mechanism must be of a somewhat different nature in SCCO(2), as this reaction in this medium did not follow the enthalpy-entropy compensation relation.

Substrate entropy in enzyme enantioselectivity: An experimental and molecular modeling study of a lipase

The temperature dependence of the enantioselectivity of Candida antarctica lipase B for 3‐hexanol, 2‐butanol, 3‐methyl‐2‐butanol, 3,3‐dimethyl‐2‐butanol, and 1‐bromo‐2‐butanol revealed that the differential activation entropy, ΔR−SΔS‡, was as significant as the differential activation enthalpy, ΔR−SΔH‡, to the enantiomeric ratio, E. 1‐Bromo‐2‐butanol, with isosteric substituents, displayed the largest ΔR−SΔS‡. 3‐Hexanol displayed, contrary to other sec‐alcohols, a positive ΔR−SΔS‡. In other words, for 3‐hexanol the preferred R‐enantiomer is not only favored by enthalpy but also by entropy. Molecular dynamics (MD) simulations and systematic search calculations of the substrate accessible volume within the active site revealed that the (R)‐3‐hexanol transition state (TS) accessed a larger volume within the active site than the (S)‐3‐hexanol TS. This correlates well with the higher TS entropy of (R)‐3‐hexanol. In addition, this enantiomer did also yield a higher number of allowed conformations, N, from the systematic search routines, than did the S‐enantiomer. The substrate accessible volume was greater for the enantiomer preferred by entropy also for 2‐butanol. For 3,3‐dimethyl‐2‐butanol, however, neither MD‐simulations nor systematic search calculations yielded substrate accessible volumes that correlate to TS entropy. Ambiguous results were achieved for 3‐methyl‐2‐butanol.

Understanding promiscuous amidase activity of an esterase from Bacillus subtilis

Water works. Bacillus subtilis esterase BS2 is a promiscuous esterase that shows amidase activity. This amidase activity was shown to depend on a hydrogen-bond network with the substrate amide hydr ...

A Water Molecule in the Stereospecificity Pocket of Candida Antarctica Lipase B Enhances Enantioselectivity towards Pentan‐2‐ol

The effect of water activity on enzyme‐catalyzed enantioselective transesterification was studied by using a solid/gas reactor. The experimental results were compared with predictions from molecular modelling. The system studied was the esterification of pentan‐2‐ol with methylpropanoate as acyl donor and lipase B from Candida antarctica as catalyst. The data showed a pronounced water‐activity effect on both reaction rate and enantioselectivity. The enantioselectivity increased from 100, at water activity close to zero, to a maximum of 320, at a water activity of 0.2. Molecular modelling revealed how a water molecule could bind in the active site and obstruct the binding of the slowly reacting enantiomer. Measurements of enantioselectivity at different water‐activity values and temperatures showed that the water molecule had a high affinity for the stereospecificity pocket of the active site with a binding energy of 9 kJ mol−1, and that it lost all its degrees of rotation, corresponding to an entropic energy of 37 J mol−1 K−1.

Suppressed Native Hydrolytic Activity of a Lipase to Reveal Promiscuous Michael Addition Activity in Water

Suppression of the native hydrolytic activity of Pseudozyma antarctica lipase B (PalB) (formerly Candida antarctica lipase B) in water is demonstrated. By replacing the catalytic Ser 105 residue with an alanine unit, promiscuous Michael addition activity is favored. A Michael addition reaction between methyl acrylate and acetylacetone was explored as a model system. For the PalB Ser 105 Ala mutant, the hydrolytic activity was suppressed more than 1000 times and, at the same time, the Michael addition activity was increased by a factor of 100. Docking studies and molecular dynamics simulations revealed an increased ability of the PalB Ser 105 Ala mutant to harbor the substrates close to a catalytically competent conformation.

Lactone size dependent reactivity in Candida antarctica lipase B: A molecular dynamics and docking study

Size matters: Lactones have extensively been studied as monomers in enzymatic polymerization reactions. Large lactones showed an unexpectedly high reactivity in these reactions. A combination of docking and molecular dynamics studies have been used to explain this high reactivity in terms of productive binding due to the transoid nature of the ester bond in these substrates.

Minor Enantiomer Recycling: Metal Catalyst, Organocatalyst and Biocatalyst Working in Concert

A minor enantiomer recycling one-pot procedure employing two reinforcing chiral catalysts has been developed. Continuous regeneration of the achiral starting material is effected via selective enzyme-catalyzed hydrolysis of the minor product enantiomer from Lewis acid-Lewis base catalyzed addition of acyl cyanides to prochiral aldehydes in a two-phase solvent system. The process provides O-acylated cyanohydrins in close to perfect enantioselectivities, higher than those obtained in the direct process, and in high yields. A combination of a (S,S)-salen Ti Lewis acid and Candida antarctica lipase B provides the products with R absolute configuration, whereas the opposite enantiomer is obtained from the (R,R)-salen Ti complex and Candida rugosa lipase.

Switched enantiopreference of Humicola lipase for 2-phenoxyalkanoic acid ester homologs can be rationalized by different substrate binding modes

Humicola lanuginosa lipase was used for enantioselective hydrolyses of a series of homologous 2-phenoxyalkanoic acid ethyl esters. The enantioselectivity (E-value) of the enzyme changed from an (R) ...

Gaining Selectivity by Combining Catalysts: Sequential versus Recycling Processes

Highly enantioenriched chiral products may be obtained by using a combination of two moderately selective catalysts. Sequential enantioselective transformations comprising an asymmetric reaction followed by a kinetic resolution of the scalemic product mixture obtained in the first step are well known. In such processes, the minor, undesired enantiomer is transformed to a compound that can be more easily separated from the major enantiomer. Although chiral compounds may be obtained with high enantiopurity by such coupled processes, the yield of the desired product necessarily suffers. Recycling processes, whereby the minor enantiomer is transformed to prochiral starting material, avoid this limitation. In this Minireview, different types of sequential catalytic processes using two reinforcing catalysts are surveyed and their advantages and limitations discussed in relation to recycling processes.