Changing the substrate specificity of P450cam towards diphenylmethane by semi-rational enzyme engineering

Hoffmann, Gregor; Bönsch, Kathrin; Greiner-Stöffele, Thomas; Ballschmiter, Meike

doi:10.1093/protein/gzq119

Cited by 20 publications

(11 citation statements)

References 33 publications

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“…The 96 in‐house P450cam–RhFRed variants were pooled according to the mutated residues generating five pooled libraries (A, B, C, D, EFG, see Experimental Section) of roughly equal size. In general, clustering strategies allow one to reduce the screening effort, avoiding the need for screen on a single‐clone level . In the following step, positive clusters may be broken down to the single‐clone levelto identify mutations improving the desired activity.…”

Section: Resultsmentioning

confidence: 99%

One‐Pot Biocatalytic Double Oxidation of α‐Isophorone for the Synthesis of Ketoisophorone

et al. 2017

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The chemical synthesis of ketoisophorone, a valuable building block of vitamins and pharmaceuticals, suffers from several drawbacks in terms of reaction conditions and selectivity. Herein, the first biocatalytic one‐pot double oxidation of the readily available α‐isophorone to ketoisophorone is described. Variants of the self‐sufficient P450cam‐RhFRed with improved activity have been identified to perform the first step of the designed cascade (regio‐ and enantioselective allylic oxidation of α‐isophorone to 4‐hydroxy‐α‐isophorone). For the second step, the screening of a broad panel of alcohol dehydrogenases (ADHs) led to the identification of Cm‐ADH10 from Candida magnoliae. The crystal structure of Cm‐ADH10 was solved and docking experiments confirmed the preferred position and geometry of the substrate for catalysis. The synthesis of ketoisophorone was demonstrated both as a one‐pot two‐step process and as a cascade process employing designer cells co‐expressing the two biocatalysts, with a productivity of up to 1.4 g L−1 d−1.

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Section: Resultsmentioning

confidence: 99%

One‐Pot Biocatalytic Double Oxidation of α‐Isophorone for the Synthesis of Ketoisophorone

et al. 2017

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“…Semi-rational approaches also delivered several success stories [75,76]. CM-GTL, constructed based on the SSM on the substrate contact sites of the active site amino acids in GTL, also showed improved selectivity, however, the improvement was not as significant as that of DM-GTL.…”

Section: Discussionmentioning

confidence: 99%

Substrate structure and computation guided engineering of a lipase for omega-3 fatty acid selectivity

Moharana

Rao

2020

PLoS ONE

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Enrichment of omega-3 fatty acids (ω-3 FAs) in natural oils is important to realize their health benefits. Lipases are promising catalysts to perform this enrichment, however, fatty acid specificity of lipases is poor. We attempted to improve the fatty acid selectivity of a lipase from Geobacillus thermoleovorans (GTL) by two approaches. In a semi-rational approach, amino acid positions critical for binding were identified by docking the substrate to the GTL and best substitutes at these positions were identified by site saturation mutagenesis followed by screening to obtain a variant of GTL (CM-GTL). In the second approach based on rational design, a variant of GTL was designed (DM-GTL) wherein the active site was narrowed by incorporating two heavier amino acids in the lining of acyl-binding pocket to hinder access to bulky ω-3 FAs. The affinities DM-GTL with designed substrates were evaluated in silico. Both, CM-GTL and DM-GTL have shown excellent ability to discriminate against the ω-3 FAs during hydrolysis of oils. Engineering the binding pocket of an enzyme of a complex substrate, such as a triglyceride, by incorporating the information on substrate structure and computationally derived binding modes, has resulted in designing two efficient lipase variants with improved substrate selectivity.

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“…Semi-rational approaches also delivered several success stories [74,75]. CM-GTL, constructed based on the SSM on the substrate contact sites of the active site amino acids in GTL, also showed improved selectivity, however, the improvement was not as significant as that of DM-GTL.…”

Section: Discussionmentioning

confidence: 99%

Substrate structure and computation guided engineering of a Lipase for Omega-3 fatty acid selectivity

Moharana

Rao

2019

Preprint

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AbstractOptimum health benefits of omega-3 fatty acids (ω-3 FAs) require it to be concentrated in its natural sources. Fatty acid selectivity of lipase governs the efficacy of the production of lipase-mediated ω-3 FAs concentrates. We attempted to improve the fatty acid selectivity of a lipase from thermophilic bacteriumGeobacillus thermoleovorans(GTL) by two approaches. In a semi-rational approach, six amino acid positions of GTL interacting with the substrate, were identified by docking and were subjected to site-saturation mutagenesis. Three best substitutions were incorporated into GTL(CM-GTL). Hydrolysis of oil by lipase was monitored in a pH-Stat and the fatty acids released at various time points were analyzed by GC-MS.CM-GTL showed a significant improvement in discrimination against DHA during hydrolysis. In the second approach based on rational design, the active site was narrowed by incorporating heavier amino acids in the lining of acyl-binding pocket to hinder access to bulky ω-3 FAs. For this purpose, two amino acids surrounding the opening of the acyl pocket were replaced with the next heavier amino acids and the affinities were evaluatedin silico.The double mutant, thus deigned, was found to be excellent in discriminating the ω-3 FAs during hydrolysis of triglycerides. Engineering the binding pocket of a complex substrate, such as a triglyceride, with the supportive information on substrate structure and its binding modes with the enzyme provided by computational methods, has resulted in designing two efficient lipase variants with improved substrate selectivity.

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Changing the substrate specificity of P450cam towards diphenylmethane by semi-rational enzyme engineering

Cited by 20 publications

References 33 publications

One‐Pot Biocatalytic Double Oxidation of α‐Isophorone for the Synthesis of Ketoisophorone

One‐Pot Biocatalytic Double Oxidation of α‐Isophorone for the Synthesis of Ketoisophorone

Substrate structure and computation guided engineering of a lipase for omega-3 fatty acid selectivity

Substrate structure and computation guided engineering of a Lipase for Omega-3 fatty acid selectivity

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