Tuning and Switching Enantioselectivity of Asymmetric Carboligation in an Enzyme through Mutational Analysis of a Single Hot Spot

Abstract

Enantioselective bond making and breaking is a hallmark of enzyme action, yet switching the enantioselectivity of the reaction is a difficult undertaking, and typically requires extensive screening of mutant libraries and multiple mutations. Here, we demonstrate that mutational diversification of a single catalytic hot spot in the enzyme pyruvate decarboxylase gives access to both enantiomers of acyloins acetoin and phenylacetylcarbinol, important pharmaceutical precursors, in the case of acetoin even starting from the unselective wild-type protein. Protein crystallography was used to rationalize these findings and to propose a mechanistic model of how enantioselectivity is controlled. In a 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.

Biocatalytic Aldol Addition of Simple Aliphatic Nucleophiles to Hydroxyaldehydes

“…6 , 9 , 10 Enzymatic C–C bond formation mediated by aldolases is a very attractive alternative for this purpose, because of its unparalleled high stereoselectivity, avoidance of extensive protective group chemistry, and operation under mild conditions. 11 − 14 A typical disadvantage of these enzymes is their rather narrow scope of acceptable nucleophilic substrates, whereas they tolerate a broad variety of aldehydes as the electrophilic components.…”

Crystal structure of pyruvate decarboxylase fromZymobacter palmae

“…To date, only six bacterial PDCs have been described, including that found in Z. palmae (ZpPDC; PDB entry 5euj; this study). The Z. mobilis enzyme (ZmPDC) has been extensively studied, with a variety of structural variants published [PDB entries 1zpd (Dobritzsch et al, 1998), 2wva, 2wvg and 2wvh (Pei et al, 2010), 3oei (Meyer et al, 2010) and 4zp1 (Wechsler et al, 2015)]. Other bacterial PDCs include those from Acetobacter pasteurianus (ApPDC; PDB entry 2vbi; D. Gocke, C. L. Berthold, G. Schneider & M. Pohl, unpublished work), Gluconoacetobacter diazotrophicus (GdPDC; PDB entry 4cok; van Zyl, Schubert et al, 2014) and Gluconobacter oxydans (GoPDC; van Zyl, Taylor et al, 2014), and from the only known Gram-positive species possessing a PDC, Sarcina ventriculi (SvPDC; Lowe & Zeikus, 1992).…”

Thiamine‐Diphosphate‐Dependent Enzymes as Catalytic Tools for the Asymmetric Benzoin‐Type Reaction

“…In contrast, exchange for very small residues allowed the production of ( S )‐PAC. The best results (Table , Entries 5 and 6) were obtained with the Zm PDC variants Glu473Phe ( ee 99.6 % R ) and Glu473Gly ( ee 76 % S ) . The enzymatic synthesis of ( R )‐PAC has also been carried out in the presence of the acetohydroxyacid synthase (isoenzyme I) from E. coli ( Ec AHAS‐I), an enzyme with physiological carboligase activity that consists of the synthesis of ( S )‐acetolactate [( S )‐ 6 ] or ( S )‐2‐ethyl‐2‐hydroxy‐3‐oxobutyrate through the homocoupling of pyruvate or the cross‐coupling of pyruvate with 2‐oxobutyrate, respectively.…”

“…The corresponding gene from Bacillus licheniformis was cloned and overexpressed in E. coli , and the enzyme showed an impressive substrate scope, accepting all the aromatic aldehydes already used with CDH [Table , Columns 4 and 5; Table , values (b)] and giving even higher yields than CDH with the difficult hydroxy‐ and nitrobenzaldehyde substrates (Table , Entries 13–17). The real novelty highlighted by our work, however, has been the discovery of an S ‐specific wild‐type enzyme for the synthesis of PAC derivatives; the few other enzymes reported to be specific for the formation of ( S )‐PAC are engineered variants of R ‐specific wt enzymes (Table , Entries 6 and 7) , …”