What’s My Substrate? Computational Function Assignment of <i>Candida parapsilosis</i> ADH5 by Genome Database Search, Virtual Screening, and QM/MM Calculations

Dhoke, Gaurao V.; Ensari, Yunus; Davari, Mehdi D.; Ruff, Anna Joëlle; Schwaneberg, Ulrich; Bocola, Marco

doi:10.1021/acs.jcim.6b00076

Cited by 9 publications

(20 citation statements)

References 62 publications

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“…As previously reported, cpADH5 mainly prefers carbonyl substrates instead of alcoholic ones ( V max values toward phenylacetaldehyde and corresponding alcohol 2‐phenylethanol are 51.74 and 0.25 U mg −1 , respectively). In terms of reduction reactions, the L119M variant has an improved reduction activity toward bulky substrates (e.g.…”

Section: Resultssupporting

confidence: 77%

“…The distance D1 between the hydroxyl oxygen and the catalytic zinc ion was determined to be below 2 Å for Me‐( S )‐3‐OHC 4 ( 1 a ), which indicates that catalytic activity should be initiated by the catalytic zinc ion. This is due to fact that substrates with a single methyl substituent at their alkyl branch are sterically similar to the natural substrates (butyraldehydes) of cpADH5 . All other variants showed a decrease of up to 95 % in activity for the oxidation of Me‐( S )‐3‐OHC 4 ( 1 a ) with the exception of L119M.…”

Section: Resultsmentioning

confidence: 98%

“…Interestingly, L119M/W286S also has an cooperative effect toward Me‐( R )‐3‐OHC 4 ( 1 b ) and showed a 23‐fold activity increase toward Me‐( R )‐3‐OHC 4 ( 1 b ) compared to cpADH5 WT. cpADH5 WT shows Prelog specificity and thus preferentially converts Me‐( S )‐3‐OHC 4 ( 1 a ) and has no activity toward Me‐( R )‐3‐OHC 4 ( 1 b ) (Figure ).…”

Section: Resultsmentioning

confidence: 99%

“…Candida parapsilosis alcohol dehydrogenase 5 (cpADH5), formerly also named as Candida parapsilosis carbonyl reductase 2 (CPCR2), belongs to the zinc‐dependent medium chain alcohol dehydrogenases (MDRs) family . cpADH5 has a broad substrate range toward aliphatic ketones, keto esters, cyclic ketones, and aryl or aromatic substituted secondary alcohols . Yamamoto and co‐workers reported that cpADH5 is capable of oxidizing secondary alcohols such as ( S )‐2‐butanol, ( S )‐1‐phenylethanol, and achiral 2‐propanol .…”

Section: Introductionmentioning

confidence: 99%

“…Yamamoto and co‐workers reported that cpADH5 is capable of oxidizing secondary alcohols such as ( S )‐2‐butanol, ( S )‐1‐phenylethanol, and achiral 2‐propanol . cpADH5 has mainly been used for asymmetric reduction of prochiral aldehydes or ketones to the enantiomerically pure ( S )‐enantiomeric alcohols as the reduction rate of cpADH5 is more than 200 times higher than its oxidation rate . Recently, cpADH5 was engineered to extend its substrate scope toward methylated cyclic in order to generate new synthesis routes through enantiomeric alcohols with a high added value, such as 2‐methyl cyclohexanone and 2‐chloroacetophenone .…”

Section: Introductionmentioning

confidence: 99%

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Inversion of cpADH5 Enantiopreference and Altered Chain Length Specificity for Methyl 3‐Hydroxyalkanoates

Ensari

Dhoke

Davari

et al. 2017

Chemistry A European J

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Expanding the substrate scope of enzymes opens up new routes for synthesis of valuable chemicals. Ketone-functionalized fatty acid derivatives and corresponding chiral alcohols are valuable building blocks for the synthesis of a variety of chemicals including pharmaceuticals. The alcohol dehydrogenase from Candida parapsilosis (cpADH5) catalyzes the reversible oxidations of chiral alcohols and has a broad substrate range; a challenge for cpADH5 is to convert alcohols with small substituents (methyl or ethyl) next to the oxidized alcohol moiety. Molecular docking studies revealed that W286 is located in the small binding pocket and limits the access to substrates that contain aliphatic chains longer than ethyl substituent. In the current manuscript, we report that positions L119 and W286 are key residues to boost oxidation of medium chain methyl 3-hydroxy fatty acids; interestingly the enantiopreference toward methyl 3-hydroxybutyrate was inverted. Kinetic characterization of W286A showed a 5.5 fold increase of V and a 9.6 fold decrease of K values toward methyl 3-hydroxyhexanoate (V : 2.48 U mg and K : 4.76 mm). Simultaneous saturation at positions 119 and 286 library yielded a double mutant (L119M/W286S) with more than 30-fold improved activity toward methyl 3-hydroxyoctanoate (WT: no conversion; L119M/W286S: 30 %) and inverted enantiopreference (S-enantiomer ≥99 % activity decrease and R-enantiomer >20-fold activity improvement) toward methyl 3-hydroxybutyrate.

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Section: Resultssupporting

confidence: 77%

Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Inversion of cpADH5 Enantiopreference and Altered Chain Length Specificity for Methyl 3‐Hydroxyalkanoates

Ensari

Dhoke

Davari

et al. 2017

Chemistry A European J

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A Comparative Reengineering Study of cpADH5 through Iterative and Simultaneous Multisite Saturation Mutagenesis

et al. 2018

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Positions identified in directed evolution campaigns or by (semi)rational design can be recombined iteratively or simultaneously. Iterative recombination has yielded many success stories and is beneficially used if screening capabilities are limited (four iterative SSMs generate 20×4=80 different enzyme variants). Simultaneous site saturation mutagenesis offers significantly higher diversity (20 =160 000 variants) and enables greater improvements to be found, especially if the selected positions are in close proximity to each other (cooperative effects). Here we report a first comprehensive comparison of iterative and simultaneous saturation of four residues in Candida parapsilosis alcohol dehydrogenase 5 (cpADH5) with methyl 3-hydroxyhexanoate as substrate. Screening of 7200 clones from 33 site saturation mutagenesis libraries (exploring 17 recombination paths) yielded the cpADH5 W286A variant, with a 82-fold improved initial activity toward methyl 3-hydroxyhexanoate. Screening 3500 clones from a single OmniChange library with four positions (C57, W116, L119, and W286; 1.8 % of the generated sequence space) yielded the cpADH5 C57V/W286S variant, with a 108-fold improvement in initial activity toward methyl 3-hydroxyhexanoate. A 1.8 % coverage of the sequence space of the simultaneous multisite saturation library was, in comparison to the investigated 17 recombination paths, sufficient to identify a cpADH5 variant with improved activity.

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Reversal of Regioselectivity in Zinc‐Dependent Medium‐Chain Alcohol Dehydrogenase from Rhodococcus erythropolis toward Octanone Derivatives

et al. 2020

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The zinc-dependent medium-chain alcohol dehydrogenase from Rhodococcus erythropolis (ReADH) is one of the most versatile biocatalysts for the stereoselective reduction of ketones to chiral alcohols. Despite its known broad substrate scope, ReADH only accepts carbonyl substrates with either a methyl or an ethyl group adjacent to the carbonyl moiety; this limits its use in the synthesis of the chiral alcohols that serve as a building blocks for pharmaceuticals. Protein engineering to expand the substrate scope of ReADH toward bulky substitutions next to carbonyl group (ethyl 2-oxo-4-phenylbutyrate) opens up new routes in the synthesis of ethyl-2-hydroxy-4phenylbutanoate, an important intermediate for anti-hypertension drugs like enalaprilat and lisinopril. We have performed computer-aided engineering of ReADH toward ethyl 2-oxo-4phenylbutyrate and octanone derivatives. W296, which is located in the small binding pocket of ReADH, sterically restricts the access of ethyl 2-oxo-4-phenylbutyrate, octan-3-one or octan-4-one toward the catalytic zinc ion and thereby limits ReADH activity. Computational analysis was used to identify position W296 and site-saturation mutagenesis (SSM) yielded an improved variant W296A with a 3.6-fold improved activity toward ethyl 2-oxo-4-phenylbutyrate when compared to WT ReADH (ReADH W296A: 17.10 U/mg and ReADH WT: 4.7 U/mg). In addition, the regioselectivity of ReADH W296A is shifted toward octanone substrates. ReADH W296A has a more than 16-fold increased activity toward octan-4-one (ReADH W296A: 0.97 U/mg and ReADH WT: 0.06 U/mg) and a more than 30-fold decreased activity toward octan-2-one (ReADH W296A: 0.23 U/ mg and ReADH WT: 7.69 U/mg). Computational and experimental results revealed the role of position W296 in controlling the substrate scope and regiopreference of ReADH for a variety of carbonyl substrates.

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What’s My Substrate? Computational Function Assignment of Candida parapsilosis ADH5 by Genome Database Search, Virtual Screening, and QM/MM Calculations

Cited by 9 publications

References 62 publications

Inversion of cpADH5 Enantiopreference and Altered Chain Length Specificity for Methyl 3‐Hydroxyalkanoates

Inversion of cpADH5 Enantiopreference and Altered Chain Length Specificity for Methyl 3‐Hydroxyalkanoates

A Comparative Reengineering Study of cpADH5 through Iterative and Simultaneous Multisite Saturation Mutagenesis

Reversal of Regioselectivity in Zinc‐Dependent Medium‐Chain Alcohol Dehydrogenase from Rhodococcus erythropolis toward Octanone Derivatives

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