Stereospecific hydrogenation of the CC double bond of enones by Escherichia coli overexpressing an enone reductase of Nicotiana tabacum

Abstract

We examined the biotransformation of enantiomeric pairs of enones such as pulegone and carvone in recombinant Escherichia coli expressing Nicotiana tabacum pulegone reductase. It was found that recombinant E. coli cells acquired the ability for stereospecific hydrogenation of the exocyclic C=C double bond of pulegone. However, stereospecificity in hydrogenation with the recombinant E. coli cells was opposite to that in hydrogenation with N. tabacum cells. On the other hand, the isolated recombinant pulegone reductase (rPRase) from the recombinant E. coli cells catalyzed hydrogenation of the exocyclic C=C double bond of pulegone; the hydrogen atoms participating in the reduction at C-8 and C-4 of pulegone originate from the pro-4R hydrogen of NADPH and the medium (H 2 O), respectively. Stereospecificity was lost in the hydrogenation of pulegone with the isolated rPRase, but was recovered when bovine serum albumin was added to the enzymatic reaction as an auxiliary factor.

Biocatalytic Asymmetric Alkene Reduction: Crystal Structure and Characterization of a Double Bond Reductase from Nicotiana tabacum

“…Surprisingly, the pH optimum was found to be 5.4 with 1-nitrocyclohexene ( 1a ) ( Supporting Information Figure S1), in contrast to prior reported reaction conditions of pH 7–8. 9 , 12 , 13 Given that typical biotransformation reactions proceed around neutral pH, we determined the steady-state kinetic parameters of NtDBR at both pH 5.4 and 7.3 (Table 1 ). In many cases, full steady-state kinetic parameters could not be determined because of either limited alkene solubility ( trans -2-nonenal, 4a ), significant alkene absorbance at 340/365 nm at higher substrate concentrations [ 1a and trans -cinnamaldehyde ( 6a )], or the detection of near-zero-order kinetics with respect to substrate concentration down to 10–20 μM [1-octen-3-one ( 5a ) and NADPH at pH 7.3].…”

“…NtDBR showed a higher preference for NADPH as the hydride donor 13 compared with NADH (>1600-fold). This is not surprising, given the significant number of specific interactions observed between the enzyme and the adenosyl phosphate group in the binary and tertiary structures (vide infra: Structural Analysis of NtDBR) (Figure 2 B).…”

“…The poor reactivity with ( R )-pulegone was unexpected, given the detection of significant activity in prior studies by others ( k cat = 3.3 s –1 , K m = 1.4 mM); 9 however, the earlier studies were carried out under biotransformation conditions (pH 7.0, 3 mM NADPH, 1 mM dithiothreitol, 0.2% Triton X-100), and the presence of Triton-X-100 is known to dramatically enhance the catalytic rate of NtDBR. 13 In addition, previous comparative studies of alkene reduction by pentaerythritol tetranitrate reductase and thermostable Old Yellow Enzyme (TOYE) showed that significant alkane product could be detected in biotransformations with substrates poorly reduced under steady-state conditions. 19a , 23 Therefore, longer biotransformation reactions (24 h; 5 mM alkene) allow a more comprehensive investigation of the substrate range of alkene reduction.…”

“… 9 NtDBR was later described as a pulegone reductase because of its ability to hydrogenate the exocyclic C=C bond of ( R )- and ( S )-pulegone to produce menthone and isomenthone isomers (Scheme 1 c). 13 In light of the moderate yields achieved for the latter conversion, we have chosen to refer to the enzyme as a generalized double bond reductase (NtDBR). Because menthone and isomenthone have important industrial applications in the production of synthetic peppermint oils and bases, we decided to further investigate the biocatalytic potential of recombinant NtDBR.…”

Platinum Nanoparticles Stabilized by Glycerodendrimers: Synthesis and Application to the Hydrogenation of α,β‐Unsaturated Ketones under Mild Conditions

“…The results are comparable to those obtained in our previously study concerning the efficiency of PtNPs stabilized by glycodendrimers,15b with the difference that the glycerodendrimers prepared herein are much easier to prepare and are less costly. Furthermore, this hydrogenation process is much more competitive to the hydrogenation of carvone and pulegone involving enzymatic catalysis,21 s ‐CO 2 extraction22 and electrochemistry 23…”