Rational Protein Design of ThDP‐Dependent Enzymes—Engineering Stereoselectivity

Gocke, Dörte; Walter, Lydia; Gauchenova, Ekaterina; Kolter, Geraldine; Knöll, Martin; Berthold, C.L.; Schneider, Günter; Pleiss, Jürgen; Müller, Michael; Pohl, Martina

doi:10.1002/cbic.200700598

Cited by 70 publications

(96 citation statements)

References 25 publications

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“…As expected, the S selectivity increased from 92 (S) to 98%ee (S) upon formation of 2-HPP with acetaldehyde as the acceptor, and a reversal of stereoselectivity was observed from 21 (R) to 93 % ee (S) for the formation of 2-hydroxy-1-phenylbutan-1-one with propanal as the acceptor. [32] The L461G variant showed an even more pronounced effect.…”

Section: Case Study 1: Engineering the Regioselectivity Of Cytochromementioning

confidence: 93%

“…Upon carboligation of benzaldehyde and propanal, (R)-2-hydroxy-1-phenylbutan-1-one is preferably formed with 21 % ee. [32] This model was validated by designing variants with shifted stereoselectivity. In PfBAL, the binding pocket for the acceptor molecule is mainly formed by the side chain of T481, as well as by G27, A28, A480, and F484.…”

Section: Case Study 1: Engineering the Regioselectivity Of Cytochromementioning

confidence: 99%

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Systematic Analysis of Large Enzyme Families: Identification of Specificity‐ and Selectivity‐Determining Hotspots

Pleiss

2014

ChemCatChem

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A general strategy is described that integrates data mining of enzyme families and molecular modeling of enzyme–substrate interactions to identify selectivity hotspots and to design focused variant libraries for changed regio‐ and stereoselectivity. This strategy is demonstrated for two case studies; the design of cytochrome P450 monooxygenases with improved regioselectivity and of thiamine diphosphate dependent enzymes with improved stereoselectivity. In both families, two selectivity hotspots are found in almost all sequences, and simple, generic rules are established to predict the effect of mutations at these positions on selectivity. The crucial role of the hotspot positions is validated for an increasing number of enzymes by designing variants with improved or switched selectivity.

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Section: Case Study 1: Engineering the Regioselectivity Of Cytochromementioning

confidence: 93%

Section: Case Study 1: Engineering the Regioselectivity Of Cytochromementioning

confidence: 99%

Systematic Analysis of Large Enzyme Families: Identification of Specificity‐ and Selectivity‐Determining Hotspots

Pleiss

2014

ChemCatChem

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“…As discussed before, the residues in positions 460 and 464 are important for the stereoselectivity of BFD. A further important residue in this part of the structure is leucine 461, which mainly defines the so-called (S)-pocket responsible for the formation of (S)-products (Gocke et al, 2008;Knoll et al, 2006). All three residues are part of an a-helix located next to the cofactor ThDP in the active site (Polovnikova et al, 2003), which may provide a good structural-based explanation for the pressure-induced change in stereoselectivity.…”

Section: Reversibility Of the Pressure-induced Changes In Enantioselementioning

confidence: 98%

Influence of the hydrostatic pressure and pH on the asymmetric 2‐hydroxyketone formation catalyzed by Pseudomonas putida benzoylformate decarboxylase and variants thereof

Berheide

Peper

Kara

et al. 2010

Biotech & Bioengineering

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Benzoylformate decarboxylase (BFD) from Pseudomonas putida is a thiamine diphosphate-dependent (ThDP) enzyme that catalyzes the asymmetric C--C bond formation to (S)-2-hydroxypropiophenone [(S)-HPP] starting from benzaldehyde and acetaldehyde. The enantioselectivity of BFD was shown to be a function of temperature and substrate concentration. It can additionally be changed by site-directed mutagenesis on hot spot positions in the active site. In this article, we present the effect of hydrostatic pressure up to 250 MPa on the enantioselectivity for the recombinant wtBFD as well as for the variants BFD F464I, BFD A460I, and BFD A460I-F464I. A general tendency toward lower amounts of (S)-HPP could be observed at increasing pressures. For two of these variants an increase in pressure even caused an inversion in the enantioselectivity and thus increasing enantiomeric excesses, respectively. A pressure-induced increase in enantioselectivity could therefore be observed for the first time in biocatalysis to the best of our knowledge. Furthermore, the pH is shown to be a parameter that also significantly influences the enantioselectivity of the reaction mentioned above.

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“…In literature, possible mechanisms of the considered enantioselective carboligation catalyzed by the enzyme BFD at atmospheric pressure are discussed [31][32][33]. It was found that the residues in positions 460, 464 and 461 are important for the stereoselectivity of BFD whereby the latter mainly defines the so-called (S)-pocket responsible for the formation of the (S)-product [31,32]. Thereby all residues are part of an a-helix located next to the cofactor ThDP in the active site [33].…”

Section: Stability Of the Silica-immobilized Bfd A460i-f464i And Kinementioning

confidence: 99%

Immobilization and characterization of benzoylformate decarboxylase from Pseudomonas putida on spherical silica carrier

Peper

Kara

Long

et al. 2011

Bioprocess Biosyst Eng

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If an adequate biocatalyst is identified for a specific reaction, immobilization is one possibility to further improve its properties. The immobilization allows easy recycling, improves the enzyme performance, and it often enhances the stability of the enzyme. In this work, the immobilization of the benzoylformate decarboxylase (BFD) variant, BFD A460I-F464I, from Pseudomonas putida was accomplished on spherical silica. Silicagel is characterized by its high mechanical stability, which allows its application in different reactor types without restrictions. The covalently bound enzyme was characterized in terms of its activity, stability, and kinetics for the formation of chiral 2-hydroxypropiophenone (2-HPP) from benzaldehyde and acetaldehyde. Moreover, temperature as well as pressure dependency of immobilized BFD A460I-F464I activity and enantioselectivity were analyzed. The used wide-pore silicagel shows a good accessibility of the immobilized enzyme. The activity of the immobilized BFD A460I-F464I variant was determined to be 70% related to the activity of the free enzyme. Thereby, the enantioselectivity of the enzyme was not influenced by the immobilization. In addition, a pressure-induced change in stereoselectivity was found both for the free and for the immobilized enzyme. With increasing pressure, the enantiomeric excess (ee) of (R)-2-HPP can be increased from 44% (0.1 MPa) to 76% (200 MPa) for the free enzyme and from 43% (0.1 MPa) to 66% (200 MPa) for the immobilized enzyme.

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Rational Protein Design of ThDP‐Dependent Enzymes—Engineering Stereoselectivity

Cited by 70 publications

References 25 publications

Systematic Analysis of Large Enzyme Families: Identification of Specificity‐ and Selectivity‐Determining Hotspots

Systematic Analysis of Large Enzyme Families: Identification of Specificity‐ and Selectivity‐Determining Hotspots

Influence of the hydrostatic pressure and pH on the asymmetric 2‐hydroxyketone formation catalyzed by Pseudomonas putida benzoylformate decarboxylase and variants thereof

Immobilization and characterization of benzoylformate decarboxylase from Pseudomonas putida on spherical silica carrier

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