Studies on hydrolysis of chiral, achiral and racemic alcohol esters with Pseudomonas cepacia lipase: mechanism of stereospecificity of the enzyme

Nishizawa, Kanji; Ohgami, Yasutaka; Matsuo, Nobutake; Kisida, Hiroshi; Hirohara, Hideo

doi:10.1039/a608618g

Cited by 33 publications

(17 citation statements)

References 34 publications

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“…Kinetic studies of another lipase showed that acylation was the rate-determining step. [17] A tetrahedral intermediate exists for only a short time, but a stable phosphonate mimics this intermediate. We synthesized phosphonate inactivators that could react with the active site serine in CRL and make a stable phosphonate.…”

Section: Introductionmentioning

confidence: 98%

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: Introductionmentioning

confidence: 98%

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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“…Esterase activities were determined by measuring the initial rates of hydrolysis of the substrates. Initial rate measurements were carried out using a pH-stat autotitration system (718 STAT Titrino Autotitrator, Metrohm, Herisau, Switzerland; the capillary nozzle replaced with that of a Radiometer Analytical System, Radiometer, Lyon, France), as described previously, 34) with some modifications. The substrate solution consisted of a suitable amount of each substrate, dimethyl sulfoxide (DMSO) in which the substrate was first dissolved at 30 C or a higher temperature, and then mixed into 25 mM Tris-HCl buffer (pH 8.0).…”

Section: )mentioning

confidence: 99%

A Novel Alkaline Esterase fromSporosarcinasp. nov. Strain eSP04 Catalyzing the Hydrolysis of a Wide Variety of Aryl-carboxylic Acid Esters

Takehara

Kinoshita

Miyamoto

et al. 2012

Bioscience, Biotechnology, and Biochemistry

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A novel esterase showing activity specific for esters of aryl-carboxylic acids was discovered in Sporosarcina sp. nov., which was identified by the 16S rDNA sequencing method in addition to morphological and physiological analyses. The aryl-carboxylesterase (named EstAC) was purified 780-fold from crude cell extracts by a 5-step procedure. EstAC was characterized as a monomeric protein with a molecular weight of 43,000, an optimum pH of around 9.0, and an optimum temperature of 40 C. The pH optimum and the effects of inhibitors together with an internal amino acid sequence suggested that EstAC is a member of family VIII esterases. EstAC was found to be highly active on a wide variety of substrates such as alkyl benzoates, alkyl phenylacetates, ethyl -or -substituted phenylpropionates, dialkyl terephthalates, dimethyl isophthalate, and ethylene glycol dibenzoate. However, monomethyl terephthalate was not hydrolyzed. It was suggested that EstAC had 4-hydroxybenzoyl and cinnamoyl esterase activities as well.

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“…Since differences of lipase activity [20,21] toward two enantiomers and diastereomers results in experimentally observable enantioselectivity (…”

Section: å)mentioning

confidence: 99%

Structural basis of stereoselectivity in Candida rugosa lipase-catalyzed hydrolysis of secondary alcohols

Schulz¹,

Schmid²,

Pleiss³

2001

J Mol Model

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Lipases are widely applied catalysts for highly enantioselective resolution of chiral secondary alcohols. While stereopreference is determined predominantly by the substrate structure, stereoselectivity (enantioselectivity and diastereoselectivity) depends on atomic details of interactions between substrate and lipase. Experimentally (≤ 1.8 Å) for RR stereo isomers, which were also experimentally found to be hydrolyzed most rapidly; distances d(H Nε -O alc ) were about 2 Å for SS and SR stereo isomers, which were converted at similar rates but at lower rate than RR stereo isomers; finally, distances d(H Nε -O alc ) for SR stereo isomers were greater than 4 Å, in accordance with very slow conversion of SR stereo isomers.

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Studies on hydrolysis of chiral, achiral and racemic alcohol esters with Pseudomonas cepacia lipase: mechanism of stereospecificity of the enzyme

Cited by 33 publications

References 34 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

A Novel Alkaline Esterase fromSporosarcinasp. nov. Strain eSP04 Catalyzing the Hydrolysis of a Wide Variety of Aryl-carboxylic Acid Esters

Structural basis of stereoselectivity in Candida rugosa lipase-catalyzed hydrolysis of secondary alcohols

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