Rational substrate and enzyme engineering of transketolase for aromatics (original) (raw)
Related papers
Chemical Communications, 2010
Transketolase mutants have been identified that accept aromatic acceptors with good stereoselectivities, in particular benzaldehyde for which the wild type enzyme showed no activity. The advantages of using biocatalysts as a sustainable resource 10 in synthesis are well established and includes their potential to achieve high regio-and stereoselectivities. 1,2 Transketolase (TK) (E.C.2.2.1.1) is a thiamine diphosphate (ThDP) dependent carbon-carbon bond forming enzyme. 3 In vivo it reversibly transfers a two carbon ketol unit to D-erythrose-4-65 Notes and references
A colorimetric assay for screening transketolase activity
Bioorganic & Medicinal Chemistry, 2006
A tetrazolium red-based colorimetric assay has been devised to screen for transketolase activity with a range of aldehyde acceptors. The colorimetric TK assay is able to detect >8% bioconversion using non-a-hydroxylated aldehydes as acceptor substrates and is significantly faster and more convenient to use than chromatographic procedures. Ó 2006 Elsevier Ltd. All rights reserved.
Development of benign synthesis of some terminal α-hydroxy ketones and aldehydes
2009
The synthesis of α-hydroxy aldehydes and hydroxymethyl ketones as well as their interconversion to each other are discussed in this thesis. The literature survey of the monograph reviews the synthetic methods for the preparation of 1,2-bifunctionalized hydroxy aldehydes and ketones. The keto-aldehyde isomerisation reaction catalyzed by Triosephosphate isomerase enzyme (TIM) and organic compounds that interact with the TIM are also introduced. In addition, the microwave heating techniques in organic syntheses are reviewed. The practical work consists of two entities: The synthesis of new substrate candidates and transition state analogues for a mutated monomeric TIM. These compounds are model compounds for the catalytic activity and the structural studies of the mutated monomeric TIM. The synthesis of the sulphonyl α-hydroxy ketone-based substrate candidates consists of four successive syntheses. The microwave-activation was utilized in the preparation of a carbon-sulphur bond and th...
α, α -Dihydroxy Ketones and 2-Amino-1,3-diols: Synthetic and Process Strategies Using Biocatalysts
Current Organic Chemistry, 2010
There is increasing interest in the use of biocatalysts in synthetic applications due to their ability to achieve highly stereoselective and atom efficient conversions. Recent developments in molecular biology techniques and synthetic biology have also enhanced potential applications using non-natural substrates. Here we review strategies to , '-dihydroxy ketones and 2-amino-1,3-diols via the use of transketolase, a carbon-carbon bond forming biocatalyst, and the enzyme transaminase which converts aldehydes and ketones to amines. Using an integrated strategy we have investigated new chemistries and assays, identified novel biocatalysts and used directed evolution strategies, together with miniaturization studies and modelling to achieve rapid and predictive process scale-up.
Structural Analysis of an Evolved Transketolase Reveals Divergent Binding Modes
Scientific Reports, 2016
The S385Y/D469T/R520Q variant of E. coli transketolase was evolved previously with three successive smart libraries, each guided by different structural, bioinformatical or computational methods. Substrate-walking progressively shifted the target acceptor substrate from phosphorylated aldehydes, towards a non-phosphorylated polar aldehyde, a non-polar aliphatic aldehyde, and finally a nonpolar aromatic aldehyde. Kinetic evaluations on three benzaldehyde derivatives, suggested that their active-site binding was differentially sensitive to the S385Y mutation. Docking into mutants generated in silico from the wild-type crystal structure was not wholly satisfactory, as errors accumulated with successive mutations, and hampered further smart-library designs. Here we report the crystal structure of the S385Y/D469T/R520Q variant, and molecular docking of three substrates. This now supports our original hypothesis that directed-evolution had generated an evolutionary intermediate with divergent binding modes for the three aromatic aldehydes tested. The new active site contained two binding pockets supporting π-π stacking interactions, sterically separated by the D469T mutation. While 3-formylbenzoic acid (3-FBA) preferred one pocket, and 4-FBA the other, the less well-accepted substrate 3-hydroxybenzaldehyde (3-HBA) was caught in limbo with equal preference for the two pockets. This work highlights the value of obtaining crystal structures of evolved enzyme variants, for continued and reliable use of smart library strategies. Transketolase (TK) (EC 2.2.1.1) is a ubiquitous thiamine diphosphate (ThDP)-dependent enzyme linking the non-oxidative pentose phosphate pathway and the Calvin cycle 1. It catalyses the reversible transfer of a two-carbon moiety from a ketol donor to an aldehyde acceptor 2,3. Transketolase has been used to form asymmetric carbon-carbon bonds in biocatalytic organic synthesis 4-6 , where the use of β-hydroxypyruvate (HPA) as the ketol donor renders the donor half-reaction irreversible. The stereoselective synthesis of α ,α '-dihydroxyketones can be directed to the synthesis of ketosugars 6 , chiral aminodiols 7,8 and other high-value synthons 9-12. Besides the biocatalytic potential, TK has proven to be a suitable model to evaluate new directed-evolution approaches 13-15 , in which small libraries were found to be effective with several targeting strategies, such that by altering even highly-conserved sites, the substrate specificity of an enzyme could be modified significantly. Using relatively small libraries, 100-fold improvements in efficiency can be achieved compared to error-prone PCR (EP-PCR) 16 approaches. Such methods have been successfully applied to lipases, for example, to expand substrate-range via combinatorial active-site saturation test (CASTing) or improve thermostability via iterative saturation mutagenesis (ISM) 17,18. Likewise, constrained alphabet approaches have proven successful tools to maximise directed evolution efficiency by creating smaller and/or smarter libraries 14,19-21. For E. coli TK a series of smart library approaches were combined previously in an overall substrate-walking approach, to shift the substrate specificity from phosphorylated to non-phosphorylated polar acceptors 13 , then to non-polar aliphatic substrates 22-24 , and on to hetero-aromatic 25 and non-polar aromatic substrates 15,26. As shown in Fig. 1, a different library design strategy was used at each stage to guide saturation mutagenesis, including the use of structural and phylogenetic information 13,22 , statistical coupling analysis (SCA) methods to target networks for re-stabilisation 14 , and substrate docking 15. In the latter stage, saturation mutagenesis of two TK active-site
Enzymatic activation of alkanes: constraints and prospective
Applied Catalysis A-general, 2004
The alkane activation by biological catalysts (enzymes and whole cells) is reviewed in order to identify their potential of application in the petrochemical industry. Enzymes such as cytochrome P450, methane monooxygenase and alkane hydroxylase are able to catalyze the alkane oxidation to the corresponding alcohol. The range of alkanes recognized as substrates covers up to C24, depending on the enzymatic system used. The enzymatic alkane activation is identified as a selective and efficient method to produce more valuable products as alcohols, aldehydes and acids. The limitations detected are the low catalytic activity for this type of enzymes, mainly due to the slow electron transfer velocity; and the continuous demand for electrons, which means a usually expensive stoichiometric supply of cofactors. Strategies to overcome these problems are mentioned. The use of molecular biology techniques and the discovery of new enzymes with better characteristics are the main tools to obtain suitable biocatalysts for industrial purposes.