Structure‐Guided Mutagenesis Reveals the Catalytic Residue that Controls the Regiospecificity of C6‐Indole Prenyltransferases

Abstract

Indole is a significant structural moiety and functionalization of the C−H bond in indole‐containing molecules expands their chemical space, and modifies their properties and/or activities. Indole prenyltransferases (IPTs) catalyze the direct regiospecific installation of prenyl moieties on indole‐derived compounds. IPTs have shown relaxed substrate flexibility enabling them to be used as tools for indole functionalization. However, the mechanism by which certain IPTs target a specific carbon position is not fully understood. Herein, we use structure‐guided site‐directed mutagenesis, in vitro enzymatic reactions, kinetics and structural‐elucidation of analogs to verify the key catalytic residues that control the regiospecificity of all characterized regiospecific C6 IPTs. The presented results also demonstrate that substitution of PriB_His312 to Tyr leads to the synthesis of analogs prenylated at different positions than C6. This work contributes to understanding of how certain IPTs can access a challenging position in indole‐derived compounds.

Promiscuous Enzymes for Residue‐Specific Peptide and Protein Late‐Stage Functionalization

“…Within this context, there has been an enormous progress in the characterization of the three-dimensional structure methods of enzymes with over 204,000 enzyme structures deposited on the protein databank (Protein Data Bank, accessed 05/2023) which can help in structural-guided functional characterization of enzymes [ 259 , 260 ] and identification of their mechanisms. [ 205 ]…”

“…The regiospecificity (position of modification) of IPTs seems to be controlled by key catalytic residues such as Lys, [ 202 ] Gln, [ 203 ] Glu, [ 204 ] or His. [ 205 ] Three-dimensional structural analysis and engineering of IPTs have revealed insights into the promiscuity of several members of this class. The size of the active site as well as the presence and absence of specific residues control substrate specificity.…”

“…The size of the active site as well as the presence and absence of specific residues control substrate specificity. [ 198 , 204 , 206 ] In addition, rational design of IPTs has led to the generation of variants with increased promiscuity, [ 207 ] change of regiospecificity, [ 203 , 205 , 208 ] or altered donor preference. [ 209 ] Mutation of the gateway residue from a bulky residue to Gly has allowed incorporation of larger donors.…”

Selective Late-Stage Functionalization of Tryptophan-Containing Peptides To Facilitate Bioorthogonal Tetrazine Ligation

“…In addition, the installed handle enabled downstream click chemistry via an interaction between the installed olefin handle and a tetrazine–biotin substrate. IPT engineering efforts to alter promiscuity, , donor substrate preference, or regiospecificity will facilitate future improvement of desired activities. This approach opens the door for the capture and imaging of TCPs and other applications in chemical biology as well as the development of drug leads.…”