A structural basis for enantioselective inhibition of <i>Candida rugosa</i> lipase by long‐chain aliphatic alcohols

Holmquist, Mats; Hæffner, Fredrik; Norin, Torbjörn; Hult, Karl

doi:10.1002/pro.5560050110

Cited by 65 publications

(21 citation statements)

References 29 publications

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“…[29] The experimental evidence is that adding long-chain alcohols decreases the enantioselectivity of the CRL-catalyzed esterification of 2-methyldecanoic acid from E = 83 (90 mM n-heptanol) to E = 37 (900 mM n-heptanol). [30] The decreased enantioselectivity stems from slower reaction of the fast enantiomer, but no change in the reaction rate of the slow enantiomer.…”

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

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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.

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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

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“…Molecular modeling studies (Kazlauskas 2000) on the stereoselectivity of lipases mostly perform conformational analysis (Uppenberg et al 1995; Holmquist et al 1996; Botta et al 1997; Yagnik et al 1997), the rational design of the active site (Scheib et al 1998, 1999; Manetti et al 2000) or substrates (Stadler et al 1995; Tafi et al 2000) or energy based evaluation of enantioselectivity (Haeffner et al 1998).…”

mentioning

confidence: 99%

Directed Evolution of N‐Carbamyl‐d‐amino Acid Amidohydrolase for Simultaneous Improvement of Oxidative and Thermal Stability

Nam

Kim

2002

Biotechnology Progress

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Directed evolution of N-carbamyl-D-amino acid amidohydrolase from Agrobacterium tumefaciens NRRL B11291 was attempted in order to simultaneously improve oxidative and thermal stability. A mutant library was generated by DNA shuffling, and positive clones with improved oxidative and thermal stability were screened on the basis of the activity staining method on a solid agar plate containing pH indicator (phenol red) and substrate (N-carbamyl-D-p-hydroxyphenylglycine). Two rounds of directed evolution resulted in the best mutant 2S3 with a significantly improved stability. Oxidative stability of the evolved enzyme 2S3 was about 18-fold higher than that of the wild type, and it also showed an 8-fold increased thermostability. The K(m) value of 2S3 was comparable to that of wild-type enzyme, but k(cat) was slightly decreased. DNA sequence analysis revealed that six amino acid residues (Q23L, V40A, H58Y, G75S, M184L, and T262A) were substituted in 2S3. From the mutational analysis, four mutations (Q23L, H58Y, M184L, and T262A) were found to lead to an improvement of both oxidative and thermal stability. Of them, T262A had the most significant effect, and V40A and G75S only increased the oxidative stability.

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“…Although lipases have no general similarity, a consensus sequence common to all lipases is the amino acid sequence of the nucleophilic elbow; G-X-S-X-G-Sm, where Sm is a small residue. Molecular modeling studies (Kazlauskas 2000) on the stereoselectivity of lipases mostly perform conformational analysis (Uppenberg et al 1995;Holmquist et al 1996;Botta et al 1997;Yagnik et al 1997), the rational design of the active site (Scheib et al 1998(Scheib et al , 1999Manetti et al 2000) or substrates (Stadler et al 1995;Tafi et al 2000) or energy based evaluation of enantioselectivity (Haeffner et al 1998).…”

mentioning

confidence: 99%

A model of the pressure dependence of the enantioselectivity of Candida rugosalipase towards (±)‐menthol

2001

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Transesterification of (±)-menthol using propionic acid anhydride and Candida rugosa lipase was performed in chloroform and water at different pressures (1, 10, 50, and 100 bar) to study the pressure dependence of enantioselectivity E. As a result, E significantly decreased with increasing pressure from E ‫ס‬ 55 (1 bar) to E ‫ס‬ 47 (10 bar), E ‫ס‬ 37 (50 bar), and E ‫ס‬ 9 (100 bar). To rationalize the experimental findings, molecular dynamics simulations of Candida rugosa lipase were carried out. Analyzing the lipase geometry at 1, 10, 50, and 100 bar revealed a cavity in the Candida rugosa lipase. The cavity leads from a position on the surface distinct from the substrate binding site to the core towards the active site, and is limited by F415 and the catalytic H449. In the crystal structure of the Candida rugosa lipase, this cavity is filled with six water molecules. The number of water molecules in this cavity gradually increased with increasing pressure: six molecules in the simulation at 1 bar, 10 molecules at 10 bar, 12 molecules at 50 bar, and 13 molecules at 100 bar. Likewise, the volume of the cavity progressively increased from about 1864 Å 3 in the simulation at 1 bar to 2529 Å 3 at 10 bar, 2526 Å 3 at 50 bar, and 2617 Å 3 at 100 bar. At 100 bar, one water molecule slipped between F415 and H449, displacing the catalytic histidine side chain and thus opening the cavity to form a continuous water channel. The rotation of the side chain leads to a decreased distance between the H449-N and the (+)-menthyl-oxygen (nonpreferred enantiomer) in the acyl enzyme intermediate, a factor determining the enantioselectivity of the lipase. Although the geometry of the preferred enantiomer is similar in all simulations, the geometry of the nonpreferred enantiomer gets gradually more reactive. This observation correlates with the gradually decreasing enantioselectivity E.

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A structural basis for enantioselective inhibition of Candida rugosa lipase by long‐chain aliphatic alcohols

Cited by 65 publications

References 29 publications

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

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

Directed Evolution of N‐Carbamyl‐d‐amino Acid Amidohydrolase for Simultaneous Improvement of Oxidative and Thermal Stability

A model of the pressure dependence of the enantioselectivity of Candida rugosalipase towards (±)‐menthol

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