Investigation of the donor and acceptor range for chiral carboligation catalyzed by the E1 component of the 2-oxoglutarate dehydrogenase complex

https://doi.org/10.1016/j.molcatb.2013.09.010Get rights and content

Highlights

  • Synthesis of chiral compounds using E1 of 2-oxoglutarate dehydrogenase is reported.

  • Chiral 2-hydroxyketones are synthesized varying donor and acceptor substrates.

  • Chiral products with (R) or (S) enantiomers were produced with 60–95% ee.

  • Use of an ester and a 2-oxoaldehyde as acceptors for the enamine is accomplished.

  • 2-Oxovalerate and 2-oxoisovalerate are also accepted in carboligation as donors.

Abstract

The potential of thiamin diphosphate (ThDP)-dependent enzymes to catalyze Csingle bondC bond forming (carboligase) reactions with high enantiomeric excess has been recognized for many years. Here we report the application of the E1 component of the Escherichia coli 2-oxoglutarate dehydrogenase multienzyme complex in the synthesis of chiral compounds with multiple functional groups in good yield and high enantiomeric excess, by varying both the donor substrate (different 2-oxo acids) and the acceptor substrate (glyoxylate, ethyl glyoxylate and methyl glyoxal). Major findings include the demonstration that the enzyme can accept 2-oxovalerate and 2-oxoisovalerate in addition to its natural substrate 2-oxoglutarate, and that the tested acceptors are also acceptable in the carboligation reaction, thereby very much expanding the repertory of the enzyme in chiral synthesis.

Introduction

While carboligation (Csingle bondC bond formation) is catalyzed by a number of important ThDP enzymes [1] (including transketolases [2], glyoxylate carboligase [3] and 1-deoxy-d-xylulose-5-phosphate synthase [4], benzaldehyde lyase [5]) as the main reaction, it is a side reaction for nearly all ThDP-dependent 2-oxoacid decarboxylases. This property of ThDP-dependent enzymes has been exploited for purposes of chiral synthesis for a number of years [1], [6], [7]. We have explored for such purposes the E1 component (E1o) of the Escherichia coli 2-oxoglutarate dehydrogenase multienzyme complex (OGDHc): 2-oxoglutarate undergoes E1o-catalyzed decarboxylation to the nucleophilic enamine, which then adds to an aldehyde acceptor, analogously to the reaction mechanism of a number of ThDP enzymes. Our synthetic program was initiated by making substitutions of the enzyme at the putative binding site of the γ-carboxyl group of the substrate so that the enzyme would accept substrate analogs lacking the charged γ-carboxyl group [8]. The Rutgers group has previously constructed several active site variants in yeast pyruvate decarboxylase (YPDC) from Saccharomyces cerevisiae and in the E1 component of the E. coli pyruvate dehydrogenase complex (E1p) which were capable of catalyzing such reactions [9]. The E477Q YPDC variant was an effective acetoin synthase, while the D28A or D28N YPDC variants catalyze acetolactate formation [10]. On the other hand, the E636Q and E636A E1p active site variants also became acetolactate synthases [11]. Note, YPDC and E1p produced the opposite enantiomers of acetoin in excess.

The E1o component of OGDHc also catalyzes carboligation reactions. The central ThDP-bound enamine intermediate reacts with the electrophilic acceptor substrate, typically an aldehyde, which results in the formation of acetoin-like or acetolactate-like ligated products (Scheme 1). In our initial report on this topic, we observed that E1o has a broad substrate range, making it an excellent candidate for protein engineering. Indeed, saturation mutagenesis experiments carried out at histidine-260 and histidine-298 [selected on the basis of the X-ray structure which suggested that these residues are near the γ-carboxylate binding site of 2-oxoglutarate (2-OG)] revealed that while H260 is important for catalysis, H298 could be substituted by a number of hydrophobic residues with little loss of activity [8]. We here report important extensions of the carboligation studies with E1o, where both the 2-oxoacid and the acceptor aldehyde could be varied over a wide range of reactivity, greatly adding to the versatility of E1o for carboligase reactions (Fig. 1). The products and enantiomeric excess (ee) were confirmed by circular dichroism (CD), 1H nuclear magnetic resonance (NMR), and chiral gas chromatography (GC). This work adds to the power of E1o as a chiral synthetic tool by demonstrating that (a) this enzyme can also accept 2-oxovalerate (2-OV) and 2-oxoisovalerate (2-OiV) as substrates, in addition to its natural substrate 2-OG, and (b) that ethyl glyoxylate and surprisingly methylglyoxal can also serve as aldehyde acceptors, in addition to glyoxylate and other straight chain aldehydes.

Section snippets

Materials

2-Oxoglutarate, 2-oxovalerate, 2-oxoisovalerate, glyoxylate, ethyl glyoxylate, and methylglyoxal were from Sigma–Aldrich. E. coli strain JW0715 containing the plasmid pCA24N encoding the OGDHc-E1 (E1o) component [ASKA clone (−)] was obtained from National Bio Resource Project (NIG, Japan). Amicon® Ultra-4 Centrifugal Filter Units are purchased from EMD Millipore. The enzyme E1o was purified as reported previously [8].

CD spectroscopy

CD experiments were carried out on a Chirascan CD spectrometer (Applied

Results and discussion

As with all ThDP-dependent decarboxylations, E1o catalyzes the initial formation of a pre-decarboxylation covalent ThDP-bound intermediate by reaction at the C2 thiazolium atom of the enzyme-bound ThDP with the substrate's keto carbon. Decarboxylation of this intermediate leads to a strongly nucleophilic enamine, which, in the absence of the other components of the complex (the E2o-E3 sub-complex), may react with electrophilic compounds leading to the so-called carboligase products (Scheme 1).

Conclusions

The E1o component of OGDHc catalyzes the formation of acetoin-like products for all of the reactions tested in good enantiomeric excess. In addition, the products produced by E1o have different enantioselectivities, depending on both substrate and acceptor being used. For example, E1o yields the (R)-enantiomer with 2-OG as the substrate and glyoxylate acceptor. On the other hand, the (S)-enantiomer is produced with 2-OV as the substrate and glyoxylate as acceptor. Two other issues impact on the

Acknowledgements

Supported at Rutgers by NIH GM050380 (F.J.) and at NJIT by NSF MCB0746078 (E.T.F.).

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    d) We then hypothesized that the presence of an alternate electrophilic acceptor for the putative enamine would change the kinetics of build-up and/or depletion of the CD band corresponding to the intermediate. The following potential enamine acceptors were tested: (i) as a mimic of the physiological reaction, the E2o-ec-derived didomain was used (E2o-ec(1–176), comprising the N terminus, the lipoyl domain, the peripheral subunit binding domain, and linkers); (ii) DCPIP was used as an external oxidizing agent; and finally (iii) glyoxylate was used as a known enamine acceptor in a so-called “carboligase” reaction (33, 70, 71). As control, a stopped-flow CD experiment of E1o-ec with OG was carried out and displayed relatively good stability of the 365 nm signal (Fig. 7a).

1

Contributed equally to this research.

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