Stereoselective Oxidation of Aryl-Substituted Vicinal Diols into Chiral α-Hydroxy Aldehydes by Re-Engineered Propanediol Oxidoreductase

α-Hydroxy aldehydes are chiral building blocks used in synthesis of natural products and synthetic drugs. One route to their production is by regioselective oxidation of vicinal diols and, in this work, we aimed to perform the oxidation of 3-phenyl-1,2-propanediol into the corresponding α-hydroxy aldehyde applying enzyme catalysis. Propanediol oxidoreductase from Escherichia coli efficiently catalyzes the stereoselective oxidation of S-1,2-propanediol into S-lactaldehyde. The enzyme, however, shows no detectable activity with aryl-substituted or other bulky alcohols. We conducted ISM-driven directed evolution on FucO and were able to isolate several mutants that were active with S-3-phenyl-1,2-propanediol. The most efficient variant displayed a kcat/KM of 40 s–1 M–1 and the most enantioselective variant an E-value (S/R) of 80. Furthermore, other isolated variants showed up to 4400-fold increased activity with another bulky substrate, phenylacetaldehyde. The results with engineered propanediol oxidoreducta...

[1]  Frances H Arnold,et al.  Directed enzyme evolution: climbing fitness peaks one amino acid at a time. , 2009, Current opinion in chemical biology.

[2]  L. Elfström,et al.  Catalysis of potato epoxide hydrolase, StEH1. , 2005, The Biochemical journal.

[3]  R. Adams,et al.  SIMPLIFICATION OF THE GATTERMANN SYNTHESIS OF HYDROXY ALDEHYDES , 1923 .

[4]  Robert Kourist,et al.  Complete inversion of enantioselectivity towards acetylated tertiary alcohols by a double mutant of a Bacillus subtilis esterase. , 2008, Angewandte Chemie.

[5]  Manfred T Reetz,et al.  Addressing the Numbers Problem in Directed Evolution , 2008, Chembiochem : a European journal of chemical biology.

[6]  L-1,2-propanediol exits more rapidly than L-lactaldehyde from Escherichia coli , 1989, Journal of bacteriology.

[7]  D. Enders,et al.  Enantioselective synthesis of protected α-hydroxy aldehydes and ketones via hydroxylation of metalated chiral hydrazones , 1988 .

[8]  Gheorghe-Doru Roiban,et al.  Achieving Regio‐ and Enantioselectivity of P450‐Catalyzed Oxidative CH Activation of Small Functionalized Molecules by Structure‐Guided Directed Evolution , 2012, Chembiochem : a European journal of chemical biology.

[9]  E. Eliel,et al.  ASYMMETRIC SYNTHESES BASED ON 1,3-OXATHIANES. 2. SYNTHESIS OF CHIRAL TERTIARY α-HYDROXY ALDEHYDES, α-HYDROXY ACIDS, GLYCOLS (R1R2C(OH)CH2OH), AND CARBINOLS (R1R2C(OH)ME) IN HIGH ENANTIOMERIC PURITY. , 1984 .

[10]  D. Monti,et al.  Redox reactions catalyzed by isolated enzymes. , 2011, Chemical reviews.

[11]  Bernd Nidetzky,et al.  Identification of Candida tenuis xylose reductase as highly selective biocatalyst for the synthesis of aromatic alpha-hydroxy esters and improvement of its efficiency by protein engineering. , 2007, Chemical communications.

[12]  M. James,et al.  Crystal structure of scytalidoglutamic peptidase with its first potent inhibitor provides insights into substrate specificity and catalysis. , 2007, Journal of molecular biology.

[13]  T. Blundell,et al.  Comparative protein modelling by satisfaction of spatial restraints. , 1993, Journal of molecular biology.

[14]  Yosephine Gumulya,et al.  Iterative saturation mutagenesis accelerates laboratory evolution of enzyme stereoselectivity: rigorous comparison with traditional methods. , 2010, Journal of the American Chemical Society.

[15]  Zhilei Chen,et al.  Rapid creation of a novel protein function by in vitro coevolution. , 2005, Journal of molecular biology.

[16]  J. Aguilar,et al.  Evolution of l-1,2-Propanediol Catabolism in Escherichia coli by Recruitment of Enzymes for l-Fucose and l-Lactate Metabolism , 1974, Journal of bacteriology.

[17]  C. R. Soccol,et al.  Improving Cry8Ka toxin activity towards the cotton boll weevil (Anthonomus grandis) , 2011, BMC biotechnology.

[18]  J. Hajdu,et al.  Visualization of dioxygen bound to copper during enzyme catalysis. , 1999, Science.

[19]  G. Huisman,et al.  Engineering the third wave of biocatalysis , 2012, Nature.

[20]  Dan S. Tawfik,et al.  Enzyme promiscuity: evolutionary and mechanistic aspects. , 2006, Current opinion in chemical biology.

[21]  D. Rice,et al.  Glycerol dehydrogenase. structure, specificity, and mechanism of a family III polyol dehydrogenase. , 2001, Structure.

[22]  A. Bommarius,et al.  Enantioenriched compounds via enzyme-catalyzed redox reactions. , 2011, Chemical reviews.

[23]  F. Molinari,et al.  Biocatalytic strategies for the asymmetric synthesis of alpha-hydroxy ketones. , 2010, Accounts of chemical research.

[24]  M. Widersten,et al.  Obtaining optical purity for product diols in enzyme-catalyzed epoxide hydrolysis: contributions from changes in both enantio- and regioselectivity. , 2012, Biochemistry.

[25]  Andreas Vogel,et al.  Iterative saturation mutagenesis on the basis of B factors as a strategy for increasing protein thermostability. , 2006, Angewandte Chemie.

[26]  Peter Meinhold,et al.  Structure-guided engineering of Lactococcus lactis alcohol dehydrogenase LlAdhA for improved conversion of isobutyraldehyde to isobutanol. , 2013, Journal of biotechnology.

[27]  Paul N. Devine,et al.  Biocatalytic Asymmetric Synthesis of Chiral Amines from Ketones Applied to Sitagliptin Manufacture , 2010, Science.

[28]  Palle Schneider,et al.  Directed evolution of a fungal peroxidase , 1999, Nature Biotechnology.

[29]  Xiong Wang,et al.  Construction of "small-intelligent" focused mutagenesis libraries using well-designed combinatorial degenerate primers. , 2012, BioTechniques.

[30]  L. Blank,et al.  Redox biocatalysis and metabolism: molecular mechanisms and metabolic network analysis. , 2010, Antioxidants & redox signaling.

[31]  M. Widersten,et al.  Functional characterization of a stereospecific diol dehydrogenase, FucO, from Escherichia coli: Substrate specificity, pH dependence, kinetic isotope effects and influence of solvent viscosity , 2010 .

[32]  M. Widersten,et al.  Modification of Substrate Specificity Resulting in an Epoxide Hydrolase with Shifted Enantiopreference for (2,3‐Epoxypropyl)benzene , 2010, Chembiochem : a European journal of chemical biology.

[33]  Manfred T Reetz,et al.  Reducing codon redundancy and screening effort of combinatorial protein libraries created by saturation mutagenesis. , 2013, ACS synthetic biology.

[34]  Hein J Wijma,et al.  Directed Evolution Strategies for Enantiocomplementary Haloalkane Dehalogenases: From Chemical Waste to Enantiopure Building Blocks , 2012, Chembiochem : a European journal of chemical biology.

[35]  Adam Godzik,et al.  Structural genomics of the Thermotoga maritima proteome implemented in a high-throughput structure determination pipeline , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[36]  Juan Aguilar,et al.  Crystal Structure of an Iron-Dependent Group III Dehydrogenase That Interconverts l-Lactaldehyde and l-1,2-Propanediol in Escherichia coli , 2005, Journal of bacteriology.

[37]  C. A. Fewson,et al.  Molecular characterization of microbial alcohol dehydrogenases. , 1994, Critical reviews in microbiology.