Design and realization of a tailor-made enzyme to modify the molecular recognition of 2-arylpropionic esters by Candida rugosa lipase

Manetti, Fabrizio; Mileto, Daniela; Corelli, Federico; Soro, Simonetta; Palocci, Cleofe; Cernia, E.; D’Acquarica, Ilaria; Lotti, Marina; Alberghina, Lilia; Botta, Maurizio

doi:10.1016/s0167-4838(00)00185-0

Cited by 28 publications

(28 citation statements)

References 33 publications

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“…These tetrahedral intermediates were built using the crystal structure of the phosphonate analog for hydrolysis of (S)-1 as the model. The modeling used the OPLS 2005 force field [24] for the lipase and tetrahedral intermediate and a TIP3P model [25] for the water solvent. A molecular dynamics simulation generated multiple conformations of each possible orientation to mimic the multiple conformations possible during the reaction.…”

Section: Modeling To Find Possible Orientations Of the Slowreacting Ementioning

confidence: 99%

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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: Modeling To Find Possible Orientations Of the Slowreacting Ementioning

confidence: 99%

“…[24,25,26] Modeling naproxen (aryl = 6-methoxy-2-naphthyl) in the active site of CRL placed the aryl group in the tunnel. For the fast-reacting enantiomer, the methyl group at the stereocenter pointed toward Phe296, as does the methoxy group in the phosphonate structure in this paper.…”

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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“…These known 3-D structures will definitely help scientists improve on the catalytic activity, substrate specificity, enantioselectivity, lid structures, chain length specificity, and enzyme stability of CRL isoforms for possible industrial applications, by applying gene shuffling (directed evolution) or rational design protein engineering. A tailor-made enzyme to modify the molecular recognition of 2-arylpropionic esters by recombinant LIP1 using site-directed mutagenesis of Phe 344 and Phe 345 was reported (50). Enzyme activity and selectivity were significantly decreased by Phe345Val (substituting Phe with Val at position 345) mutation and Phe344,345Val double mutation.…”

Section: Protein Engineering Of Crl Isoforms-tailoring Enzyme Functionmentioning

confidence: 99%

Protein engineering and applications of Candida rugosa lipase isoforms

Akoh

Lee

Shaw

2004

Lipids

102

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Commercial preparations of Candida rugosa lipase (CRL) are mixtures of lipase isoforms used for the hydrolysis and synthesis of various esters. The presence of variable isoforms and the amount of lipolytic protein in the crude lipase preparations lead to a lack of reproducibility of biocatalytic reactions. Purification of crude CRL improve their substrate specificity, enantioselectivity, stability, and specific activities. The expression of the isoforms is governed by culture or fermentation conditions. Unfortunately, the nonsporogenic yeast C. rugosa does not utilize the universal codon CTG for leucine; therefore, most of the CTG codons were converted to universal serine triplets by site-directed mutagenesis to gain expression of functional lipase in heterologous hosts. Recombinant expressions by multiple-site mutagenesis or complete synthesis of the lipase gene are other possible ways of obtaining pure and different CRL isoforms, in addition to culture engineering. Protein engineering of purified CRL isoforms allows the tailoring of enzyme function. This involves computer modeling based on available 3-D structures of lipase isoforms. Lid swapping and DNA shuffling techniques can be used to improve the enantioselectivity, thermostability, and substrate specificity of CRL isoforms and increase their biotechnological applications. Lid swapping can result in chimera proteins with new functions. The sequence of the lid can affect the activity and specificity of recombinant CRL isoforms. Candida rugosa lipase is toxicologically safe for food applications. Protein engineering through lid swapping and rationally designed site-directed mutagenesis will continue to lead to the production of CRL isoforms with improved catalytic power, thermostability, enantioselectivity, and substrate specificity, while providing evidence for the mechanisms of actions of the various isoforms.

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“…Several attempts to vary enantioselectivity of esterases and lipases by directed evolution, [11][12][13] and by rational protein engineering were successful. [14,15] The latter method also yields insight into the molecular mechanism of enantiorecognition. X-ray structures of several lipases and esterases complexed with chiral substrate analogous inhibitors are available.…”

Section: Introductionmentioning

confidence: 99%

A Molecular Mechanism of Enantiorecognition of Tertiary Alcohols by Carboxylesterases

et al. 2003

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Design and realization of a tailor-made enzyme to modify the molecular recognition of 2-arylpropionic esters by Candida rugosa lipase

Cited by 28 publications

References 33 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

Protein engineering and applications of Candida rugosa lipase isoforms

A Molecular Mechanism of Enantiorecognition of Tertiary Alcohols by Carboxylesterases

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