Fused radical SAM and αKG-HExxH domain proteins contain a distinct structural fold and catalyse cyclophane formation and β-hydroxylation

Abstract

Two of nature’s recurring binding motifs in metalloproteins are the CxxxCxxC motif in radical SAM enzymes and the 2-His-1-carboxylate motif found both in zincins and α-ketoglutarate and non-haem iron enzymes. Here we show the confluence of these two domains in a single post-translational modifying enzyme containing an N-terminal radical S-adenosylmethionine domain fused to a C-terminal 2-His-1-carboxylate (HExxH) domain. The radical SAM domain catalyses three-residue cyclophane formation and is the signature modification of triceptides, a class of ribosomally synthesized and post-translationally modified peptides. The HExxH domain is a defining feature of zinc metalloproteases. Yet the HExxH motif-containing domain studied here catalyses β-hydroxylation and is an α-ketoglutarate non-haem iron enzyme. We determined the crystal structure for this HExxH protein at 2.8 Å, unveiling a distinct structural fold, thus expanding the family of α-ketoglutarate non-haem iron enzymes with a class that we propose to name αKG-HExxH. αKG-HExxH proteins represent a unique family of ribosomally synthesized and post-translationally modified peptide modifying enzymes that can furnish opportunities for genome mining, synthetic biology and enzymology. Radical SAM (rSAM) and 2-His-1-carboxylate enzymes are known to co-occur in RiPP biosynthetic pathways. Here we show the fusion of these enzymes in a single protein where the rSAM enzyme catalyzes cyclophane formation. The 2-His-1-carboxylate enzymes—termed αKG-HExxH—are α-ketoglutarate non-haem iron enzymes that harbour a distinct fold and catalyse β-hydroxylation.