Structure and reactivity of intermediates in the carboligation of ThDP-dependent enzymes
Final Report Abstract
The enantioselective bond making and breaking of enzyme catalysts is central to their application in biocatalysis. The tuning and switching of enantioselectivities is notoriously difficult to achieve, and often requires extensive screening campaigns of mutant libraries and introduction of multiple mutations. Our work in the context of the FOR 1296 demonstrates that mutational diversification of a single catalytic hot spot in the enzyme pyruvate decarboxylase, glutamate 473, provides chemoenzymatic access to both enantiomers of acyloins acetoin and phenylacetylcarbinol, important pharmaceutical precursors, in case of acetoin even starting from the unselective wild-type protein. We used structural analysis by means of protein crystallography and molecular modelling to rationalize these findings and to propose a general mechanistic model of how enantioselectivity is controlled in ThDP enzymes. In broader context, our studies highlight the efficiency of mechanism-inspired and structure-guided rational protein design for enhancing and switching enantioselectivity of enzymatic reactions by systematically exploring the biocatalytic potential of a single hot spot. Our comparative structural analysis of the central Breslow intermediate in two prototypical members of the ThDP enzyme superfamily showcases that the active sites specifically stabilize different forms of the intermediate, either the enamine as in transketolase with a nucleophilic center, or the ketothiazoline tautomer with an electrophilic center as observed in pyruvate oxidase. Transketolase variants with greatly diminished activities were discovered, in which the enamine had undergone tautomerization. Our findings thus suggest that thiamin enzymes control the chemical fate of the central Breslow intermediate through opening or blockage of tautomerization proton transfer pathways. The chemical and kinetic mechanism of the ThDP enzyme phosphoketolase was studied in great detail and allowed us to disclose important aspects regarding the role of co-substrate phosphate for the required tautomerization of a critical enol-acetyl-ThDP intermediate. Experimental findings conjointly with quantum-chemical calculations strongly support a mechanism, in which tautomerization is facilitated by phosphate that acts as acid-base catalyst. Exploiting the accumulation of the long-lived enol in phosphoketolase, we could establish a novel aldol reaction for the class of ThDP enzymes, which might be a springboard for a whole new class of carboligation reactions (acylations, annulations).
Publications
- Conversion of Pyruvate Decarboxylase into an Enantioselective Carboligase with Biosynthetic Potential. J Am Chem Soc 2011, 133: 3609–3616
Meyer D, Walter L, Kolter G, Pohl M, Müller M, Tittmann K
(See online at https://doi.org/10.1021/ja110236w) - Unexpected tautomeric equilibria of the carbanion-enamine intermediate in pyruvate oxidase highlight unrecognized chemical versatility of the thiamin cofactor. Proc. Natl. Acad. Sci. USA 2012, 109: 10867-72
Meyer D, Neumann P, Koers E, Sjuts H, Lüdtke S, Sheldrick G M, Ficner R, Tittmann K
(See online at https://doi.org/10.1073/pnas.1201280109) - Observation of a stable carbene at the active site of a thiamin enzyme. Nature Chem. Biol. 2013, 9: 488-490
Meyer D, Neumann P, Ficner R, Tittmann K
(See online at https://doi.org/10.1038/nchembio.1275) - Tuning and switching enantioselectivity of asymmetric carboligation in an enzyme through mutational analysis of a single hot spot. ChemBioChem 2015, 16: 2580-4
Wechsler C, Meyer D, Loschonsky S, Funk LM, Neumann P, Ficner R, Brodhun F, Müller M, Tittmann K
(See online at https://doi.org/10.1002/cbic.201500529) - Mechanistic and Structural Insight to an Evolved Benzoylformate Decarboxylase with Enhanced Pyruvate Decarboxylase Activity. Catalysts 2016, 6: 190
Andrews FH, Wechsler C, Rogers MP, Meyer D, Tittmann K, McLeish MJ
(See online at https://doi.org/10.3390/catal6120190) - Structural Analysis of an Evolved Transketolase Reveals Divergent Binding Modes. Scientific Reports 2016, 6: 35716
Affaticati P, Dai S, Payongsri P, Hailes H, Tittmann K, Dalby P
(See online at https://doi.org/10.1038/srep35716)
