Transsulfurase‐Catalyzed Reversible Modification of C‐Terminal Cysteine and Application for Orthogonal Dual Labeling of Proteins

Protein thiol bioconjugation in combination with reversible modification of cysteine is a powerful strategy for orthogonal dual labeling of protein cysteine residues. Here, we utilize recombinant Escherichia coli ( E. coli ) cultivated in high‐phosphate minimal medium for the expression of ubiquitin G76C, and discovered that cystathionine‐γ‐synthase (CGS), an enzyme in bacterial methionine biosynthesis, catalyzes both in cells and in vitro a site‐specific and high‐efficient γ‐replacement reaction of the flexible C‐terminal cysteine with O‐succinyl‐homoserine, leading to the addition of a 3‐amino‐3‐carboxypropyl (acp) group. Mechanistic studies using high‐resolution NMR reveal a very weak ternary association among cystathionine‐γ‐synthase, O‐succinyl‐homoserine, and ubiquitin G76C. Cystathionine‐γ‐lyase (CGL), involved in cysteine biosynthesis, specifically catalyzes the α,γ‐elimination reaction of the modified unit, thereby effectively removing the acp unit in vitro. Their catalytic efficiency and selectivity of both enzymes were evaluated, and each one shows unidirectional catalytic activity for proteins. Reversible modification of C‐terminal cysteines across a broad range of proteins can be achieved by CGS and CGL. Finally, we demonstrated the feasibility of using this two‐enzyme system for orthogonal dual labeling of proteins in combination with thiol bioconjugation techniques. This discovery significantly broadens the toolkit for protein thiol modifications and holds substantial application for the dual site‐specific functionalization of proteins.