The site-selective incorporation of a NAD+ cofactor mimic into a folded helix–loop–helix polypeptide motif

Abstract

LA-42, a polypeptide with 42 amino acid residues, has been designed to fold into a hairpin helix–loop–helix motif that can dimerise in solution to form a four-helix bundle. On the surface of the folded motif a reactive site has been introduced that contains a histidine and a lysine residue in a helical sequence. The reaction between the His-Lys site and a p-nitrophenyl ester has previously been shown to lead to the site-selective formation of an amide at the side chain of the flanking lysine residue. An N-methylnicotinoyl group has now been incorporated into LA-42, in a reaction between the peptide and the N-methylnicotinic acid ester in aqueous solution at pH 5.9, to form a template for the engineering of selective catalysts for the reduction of carbonyl compounds. The formation of an amide bond between LA-42 and the cofactor mimic was established by electro-spray mass spectrometry and NMR spectroscopy and the reduction of the N-methylnicotinoyl residue by sodium dithionite in aqueous solution at pH 6.5–7 was demonstrated by UV and NMR spectroscopy. Key problems with NAD⁺/NADH models in aqueous solution include poor solubility of the ox and/or red forms of the catalysts and short lifetimes of the red form due to hydrolysis of the enamine. Both the red and the ox forms of the peptide-linked nicotinoyl cofactor are soluble in aqueous solution, which is a necessary condition for the development of turnover systems, and the lifetime of the reduced form of the polypeptide catalyst has been increased by more than a factor of three over that of the 1-methylnicotinamide.