“On-the-Fly” Kinetics of Enzymatic Racemization Using Deuterium NMR in DNA-Based Chiral Oriented Media

Monique Chan-Huot,N Suryaprakash,Philippe Lesot,Philippe Pelupessy,Geoffrey Bodenhausen,Patricia Duchambon,U Venkateswara Reddy,Michael D Toney,Luminita Duma

doi:10.1021/ac4004002

Abstract

We report the in situ and real-time monitoring of the interconversion of L- and D-alanine-d3 by alanine racemase from Bacillus stearothermophilus directly observed by (2)H NMR spectroscopy in anisotropic phase. The enantiomers are distinguished by the difference of their (2)H quadrupolar splittings in a chiral liquid crystal containing short DNA fragments. The proof-of-principle, the reliability, and the robustness of this new method is demonstrated by the determination of the turnover rates of the enzyme using the Michaelis-Menten model.

Highlights

Have focused attention on recombinant alanine racemase from Bacillus stearothermophilus, which can be expressed and purified and is stable over a wide range of temperatures up to 60 °C.10,11
Dissolved in water or organic solvents, chiral polymers such as polypeptides, polysaccharides, or polynucleotides are known to form lyotropic chiral liquid crystals (CLCs) at concentrations that depend on their phase diagrams.[8,9,12−14] Among watercompatible aligning media, CLCs made of short-length doublestranded DNA helices can impose a different orientational order on enantiomers of various α-amino acids.[9]
The difference in linewidths of the outer doublet due to L-Ala-d3 (Δ1/2 ≈ 20 Hz) and of the inner doublet of D-Ala-d3 (Δ1/2 ≈ 11 Hz) results from 2H−2H dipolar couplings that are larger for LAla-d3.9 The 2H doublet of HOD does not show any significant variation in either splitting or linewidth as a function of time, indicating that there is no degradation of the CLC during the overnight experiments

Summary

Introduction

Have focused attention on recombinant alanine racemase from Bacillus stearothermophilus, which can be expressed and purified and is stable over a wide range of temperatures up to 60 °C.10,11. 100−300 base pairs) can impose a different orientational order on enantiomers of various α-amino acids.[9] For selectively deuterated amino acids, one can observe distinct quadrupolar splittings for the two isomers in 2H−{1H} spectra.[9,15,16] nuclei such as 1H or 13C could be observed in chiral oriented solvents, 2H nuclei (I = 1) have three attractive features:[15,16] (i) their residual quadrupolar splittings ΔνQ (specific for nuclei I > 1/2) are very sensitive to differences in orientational ordering; (ii) the analysis of 2H−{1H} 1D spectra (Supporting Information) is trivial for mono- or polydeuterated enantiomers since for each isotopically enriched site the two isomers lead to two distinct quadrupolar doublets centered on nearly the same chemical shift δaniso(2H);[15] and (iii) since Overhauser effects are negligible, the integrals in 2H quadrupolar doublets are directly proportional to the concentrations of the enantiomers, provided the recovery delays are long enough.[16] In the case of Ala-d3, both enantiomers have the same T1.9 In this study, we have chosen Ala-d3, but it would be possible to use alanine deuterated on the stereogenic Cα carbon. Details about 2H−{1H} NMR in chiral anisotropic phases can be found in Supporting Information

Methods

Results

Conclusion