The sequence, crystal structure determination and refinement of two crystal forms of lipase B from Candida antarctica

Uppenberg, J.; Hansen, Mogens Trier; Patkar, Shamkant; Jones, T. Alwyn

doi:10.1016/s0969-2126(00)00031-9

Cited by 837 publications

(680 citation statements)

References 45 publications

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“…T40-O γ H participates only sparsely. The available crystal structures of CAL-B [51,39] reveal that T40 accepts H-bonds from the amide side chains of Q106 and Q157 (Fig. 1), and at least one is maintained in the simulations, accompanied by a slight distortion of the backbone, also observed in the uncomplexed variants (ESM Fig.…”

Section: Simulations Of Substratesmentioning

confidence: 83%

“…[39] The entry was chosen due to the presence of a large lipid analog covalently bound to the catalytic S105, which putatively induces a fully active conformation of the enzyme. The all-atom RMSD to two other common structures in modeling is small; 1TCA [51] = 0.29, 1LBS [39] = 0.44. The 1LBT structure was stripped of all water molecules and protonated (default protonation state) in the viewer Hermes, a part of the GOLD Suite.…”

Section: Enzyme Variantsmentioning

confidence: 92%

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Computational design of a lipase for catalysis of the Diels-Alder reaction

et al. 2010

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Combined molecular docking, molecular dynamics (MD) and density functional theory (DFT) studies have been employed to study catalysis of the Diels-Alder reaction by a modified lipase. Six variants of the versatile enzyme {\em Candida Antarctica} lipase B (CALB) have been rationally engineered {\em in silico} based on the specific characteristics of the pericyclic addition. A kinetic analysis reveals that hydrogen bond stabilization of the transition state and substrate binding are key components of the catalytic process. In the case of substrate binding, which has the greater potential for optimization, both binding strength and positioning of the substrates are important for catalytic efficiency. The binding strength is determined by hydrophobic interactions and can be tuned by careful selection of solvent and substrates. The MD simulations show that substrate positioning is sensitive to cavity shape and size, and can be controlled by a few rational mutations. The well-documented S105A mutation is essential to enable sufficient space in the vicinity of the oxyanion hole. Moreover, bulky residues on the edge of the active site hinders the formation of a sandwich-like near-attack conformer (NAC), and the I189A mutation is needed to obtain enough space above the face of the $\alpha$,$\beta$-double bond on the dienophile. The double mutant S105A/I189A performs quite well for two of three dienophiles. Based on binding constants and NAC energies obtained from MD simulations combined with activation energies from DFT computations, relative catalytic rates ($v_{cat}/v_{uncat}$) of up to $10^3$ are predicted.Response to Reviewers: We are happy with the favorable comments by the reviewers. We have have corrected the manuscript as suggested by reviewer 1 1. A sentence explaining catalytic promiscuity has been added. 2. A paragraph discussing the relationship between the NAC concept and the Reaction Force has been added.We have shortened the manuscript as suggested by reviewer 2. In particular, we have moved information regarding the structural relaxation of protein structure in MD simulations to the supplementary material. Abstract Combined molecular docking, molecular dynamics (MD) and density functional theory (DFT) studies have been employed to study catalysis of the DielsAlder reaction by a modified lipase. Six variants of the versatile enzyme Candida Antarctica lipase B (CALB) have been rationally engineered in silico based on the specific characteristics of the pericyclic addition. A kinetic analysis reveals that hydrogen bond stabilization of the transition state and substrate binding are key components of the catalytic process. In the case of substrate binding, which has the greater potential for optimization, both binding strength and positioning of the substrates are important for catalytic efficiency. The binding strength is determined by hydrophobic interactions and can be tuned by careful selection of solvent and substrates. The MD simulations show that substrate positioning is sensitive to cavity s...

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Section: Simulations Of Substratesmentioning

confidence: 83%

Section: Enzyme Variantsmentioning

confidence: 92%

Computational design of a lipase for catalysis of the Diels-Alder reaction

et al. 2010

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“…Motivated by these findings, we examined the enzymatic activity of individual molecules of the 33-kDa lipase B from Candida antarctica molecules (15,16) by using confocal fluorescence microscopy. This lipase catalyzes the hydrolysis of esters in aqueous solution following the same reaction mechanism as that of a serine protease (17).…”

mentioning

confidence: 99%

Stretched exponential decay and correlations in the catalytic activity of fluctuating single lipase molecules

Flomenbom

Velonia²,

Loos³

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

287

261

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Single-molecule techniques offer a unique tool for studying the dynamical behavior of individual molecules and provide the possibility to construct distributions from individual events rather than from a signal stemming from an ensemble of molecules. In biological systems, known for their complexity, these techniques make it possible to gain insights into the detailed spectrum of molecular conformational changes and activities. Here, we report on the direct observation of a single lipase-catalyzed reaction for extended periods of time (hours), by using confocal fluorescence microscopy. When adding a profluorescent substrate, the monitored enzymatic activity appears as a trajectory of ''on-state'' and ''off-state'' events. The waiting time probability density function of the off state and the state-correlation function fit stretched exponentials, independent of the substrate concentration in a certain range. The data analysis unravels oscillations in the logarithmic derivative of the off-state waiting time probability density function and correlations between off-state events. These findings imply that the fluctuating enzyme model, which involves a spectrum of enzymatic conformations that interconvert on the time scale of the catalytic activity, best describes the observed enzymatic activity. Based on this model, values for the coupling and reaction rates are extracted.single enzyme activity ͉ two-state trajectories D ynamics of chemical reactions are conventionally investigated by ensemble measurements. Recent advances in single-molecule spectroscopy have enabled the real-time study of biophysical processes (1-10) and conformational changes (11, 12) of single biomolecules. These studies have demonstrated that new information about such processes can be extracted from single-molecule measurements. In particular, deviations from the standard Michaelis-Menten behavior (13,14), which is expected for bulk enzymatic activity, have been observed (6 -8, 12).Motivated by these findings, we examined the enzymatic activity of individual molecules of the 33-kDa lipase B from Candida antarctica molecules (15, 16) by using confocal fluorescence microscopy. This lipase catalyzes the hydrolysis of esters in aqueous solution following the same reaction mechanism as that of a serine protease (17). To study the catalysis by single lipase, we used a fluorogenic substrate, namely the nonf luorescent ester 2Ј,7Ј-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester, which upon hydrolysis forms a highly fluorescent carboxylic acid product (18,19). This method enabled us to probe the enzymatic activity by monitoring the fluorescence emission from single enzymes. The fluorescence emission displayed blinking of ''on'' and ''off'' events depending on the presence (or absence) of the fluorescent product in the confocal focus (20). By using this approach, we have been able to obtain long trajectories (for time periods of hours) suitable for reliable statistical analysis while varying the concentration of the substrate, thus ...

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“…Model visualization and analysis was conducted with GROMACS package and VMD 1.9 (Humphrey et al, 1996). CALB crystal structure was PDB 1TCA (Uppenberg et al, 1994) with every water molecule and N-acetyl-D-glucosamine in the crystal removed prior to simulation. AMBER99 (Wang et al, 2000) force field was used to define potential energy of the model.…”

Section: Molecular Dynamic Simulationmentioning

confidence: 99%

DESIGN OF <i>CANDIDA ANTARCTICA</i> LIPASE B THERMOSTABILITY IMPROVEMENT BY INTRODUCING EXTRA DISULFIDE BOND INTO THE ENZYME

Tambunan¹

2014

OnLine Journal of Biological Sciences

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Candida Antarctica Lipase B (CALB) is extensively studied in enzymatic production of biodiesel, pharmaceutical products, detergents and other chemicals. One drawback of using CALB is its relatively low optimum temperature at 313 K (40°C). The objective of this research is to design CALB mutant with improved thermostability by introducing extra disulfide bond. Molecular dynamic simulation was conducted to get better insight into the process of thermal denaturation or unfolding in CALB. Thermal denaturation of CALB was accelerated by conducting simulation at high temperature. Molecular dynamic simulation of CALB was performed with GROMACS software package at 300-700 K. Prediction of possible mutation was done using "Disulfide by Design TM " software. Selection of mutated residues was based on flexibility analysis of CALB. From those analyses, three mutants were designed, which are Mutant-1 (73LeuCys/151AlaCys), Mutant-2 (155TrpCys/294GluCys) and Mutant-3 (43ThrCys/67SerCys). Parameters that were used to compare the thermostability of mutant with wild type enzyme were Root Mean Square Deviations (RMSD), Solvent Accessible Surface Area (SASA), Radius of gyration (Rg) and secondary structure. Molecular dynamic simulation conducted on those three mutants showed that Mutant-1 has better thermostability compared to wild type CALB. We proposed the order of mutant thermostability improvement as follows: Mutant-1, Mutant-2 and Mutant-3, with Mutant-1 having better potential thermostability improvement and Mutant-3, the least stable.

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The sequence, crystal structure determination and refinement of two crystal forms of lipase B from Candida antarctica

Cited by 837 publications

References 45 publications

Computational design of a lipase for catalysis of the Diels-Alder reaction

Computational design of a lipase for catalysis of the Diels-Alder reaction

Stretched exponential decay and correlations in the catalytic activity of fluctuating single lipase molecules

DESIGN OF <i>CANDIDA ANTARCTICA</i> LIPASE B THERMOSTABILITY IMPROVEMENT BY INTRODUCING EXTRA DISULFIDE BOND INTO THE ENZYME

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