Cofactor Binding Dynamics Influence the Catalytic Activity and Selectivity of an Artificial Metalloenzyme

Abstract

We present an artificial metalloenzyme based on the transcriptional regulator LmrR that exhibits dynamics involving the positioning of its abiological metal cofactor. The position of the cofactor, in turn, was found to be related to the preferred catalytic reactivity, which is either the enantioselective Friedel–Crafts alkylation of indoles with β-substituted enones or the tandem Friedel–Crafts alkylation/enantioselective protonation of indoles with α-substituted enones. The artificial metalloenzyme could be specialized for one of these catalytic reactions introducing a single mutation in the protein. The relation between cofactor dynamics and activity and selectivity in catalysis has not been described for natural enzymes and, to date, appears to be particular for artificial metalloenzymes.

In Vivo Assembly of Artificial Metalloenzymes and Application in Whole‐Cell Biocatalysis**

“…From previous work on the LmrR protein we know that it binds Hoechst 33342 and ethidium bromide with high affinity in its hydrophobic pocket and that they can inhibit catalysis by competing for binding with Cu II ‐Phen and the substrates [24] . Hoechst 33342 is cell permeable, whereas ethidium has difficulties crossing the double membrane barrier of E. coli and is normally used to stain dead E. coli cells, which have permeable cell membranes.…”

“…LmrR_A92E was chosen as the starting point for directed evolution because it was shown to be significantly more active than LmrR [24] . Site‐saturation libraries with NDT codon degeneracy were designed for positions 8, 14, 88, 89, 93 and 100, which were identified in the alanine scan as positions of interest (Figure 4 a).…”

In Vivo Assembly of Artificial Metalloenzymes and Application in Whole‐Cell Biocatalysis**

“…From previous work on the LmrR protein we know that it binds Hoechst 33342 and ethidium bromide with high affinity in its hydrophobic pocket and that they can inhibit catalysis by competing for binding with Cu II -Phen and the substrates. [24] Hoechst 33342 is cell permeable, whereas ethidium has difficulties crossing the double membrane barrier of E. coli and is normally used to stain dead E. coli cells, which have permeable cell membranes. Fluorescence microscopy confirmed that Hoechst 33342 was readily taken up by the LmrR expressing cells, whereas ethidium bromide was not (Figures S4-S6).…”

“…LmrR_A92E was chosen as the starting point for directed evolution because it was shown to be significantly more active than LmrR. [24] Site-saturation libraries with NDT codon degeneracy were designed for positions 8, 14, 88, 89, 93 and 100, which were identified in the alanine scan as positions of interest (Figure 4 a). From one round of screening (in total ca.…”

“…Understanding of the structural effect of these different mutations and their role in catalysis is not easily rationalized and will require molecular dynamics studies, as we performed before for the A92E mutation. [24]…”