Total synthesis and biochemical characterization of mirror image barnase

Abstract

In this study we synthesized and characterized mirror image barnase (B. amyloliquefaciens ribonuclease). d-Barnase was identical to l-barnase, when analyzed by liquid chromatography and mass-spectrometry. Proteolysis of the mirror image enzyme revealed that in contrast to its native counterpart, d-barnase was completely stable to digestive proteases. In enzymatic assays, d-barnase had the reciprocal chiral specificity and was fully active towards mirror image substrates. Interestingly, d-barnase also hydrolyzed the substrate of the native chirality, albeit 4000 times less efficiently. This effect was further confirmed by digesting a native 112-mer RNA with the enzyme. Additional studies revealed that barnase accommodates a range of substrates with various chiralities, but the prime requirement for guanosine remains. These studies point toward using mirror image enzymes as modern agents in biotechnology.

Fig. 1. (a) Synthetic strategy for l- and d-barnase. (b) HPLC-MS (TIC) chromatograms for the synthesized proteins with mass spectra insets for the main peaks. Maximum entropy deconvolution spectra of the MS spectra on the top are displayed on the bottom. For both proteins main identified contaminants were +32 Da (Ala → Cys, incomplete desulfurization), and –57 Da (Gly deletion).

Fig. 2. (a) Chemical structure of the A^DG^DA^DA^D fluorogenic substrate. Stereogenic centers are highlighted in blue. (b) Biochemical characterization of synthetic l- and d-barnase using the fluorogenic assay. One representative kinetic curve is shown for each enzyme. Barnase concentration was 1.0 nM in all cases.

Fig. 3. The RNA gel showing the digest of 112 nucleotide-long d-RNA by 450 nM l-barnase, 450 nM d-barnase, and 1.5 μM d-barnase over the course of four hours. The negative control (no RNase added) is shown on the left. The image is digitally modified by inverting the color scheme and adjusting the contrast.

Fig. 4. Comparison of the proteolytic stability of l- and d-barnase. Remaining catalytic activity of the enzymes, associated with the extent of the proteolytic digestion, was measured using the fluorogenic assay. Three out of six assayed proteases are not displayed for clarity. Digestion of 1.0 nM l-barnase with different proteases was assayed using A^DG^DA^DA^D as a substrate (data shown on the left). Digestion of 0.8 nM d-barnase with different proteases was assayed using A^LG^LA^LA^L as a substrate (on the right).