Prof. Hening Lin’s research is centered on nicotinamide adenine dinucleotide (NAD)-consuming enzymes and enzymes involved in diphthamide biosynthesis. Among the NAD-consuming enzymes, the focus is on sirtuins, which were known as NAD-dependent deacetylases. Sirtuins regulate aging, transcription, and metabolism, and are important targets for treating several human diseases. There are seven sirtuins in humans, Sirt1-7. Four of them (Sirt4-7) have very weak deacetylase activity, which have caused confusions and debates in the biological community. Lin and coworkers demonstrated that Sirt5 catalyzes the hydrolysis of malonyl and succinyl lysine efficiently. Furthermore, malonylated and succinylated proteins were identified from mammalian and bacterial cells, demonstrating that they are common protein posttranslational modifications. Similarly, Lin and coworkers demonstrated that mammalian Sirt6 can efficiently hydrolyze long chain fatty acyl groups from protein lysine residues. One of the substrate proteins is tumor necrosis factor α (TNFα). Sirt6 promotes the secretion of TNFα by removing long chain fatty acyl groups from TNFα. Utilizing the fact that different sirtuins have different acyl group preferences, Lin and coworker developed inhibitors that are specific for particular sirtuins. On the diphthamide biosynthesis project, Lin and coworkers discovered a novel radical SAM enzyme (PhDph2) that generates a 3-amino-3-carboxypropyl radical. Diphthamide is a modified histidine residue in translation elongation factor 2 and the target of diphtheria toxin. Its biosynthesis has been a puzzle for several decades. Lin and coworkers demonstrated that the biosynthetic enzyme PhDph2 uses a [4Fe-4S] cluster to cleave S-adenosylmethionine (SAM) to generate a 3-amino-3-carboxypropyl radical, which then reacts with the histidine residue in route to form diphthamide. Radical SAM enzymes are known to use [4Fe-4S] clusters to cleave SAM. However, classical radical SAM enzymes generate a 5’-deoxyadenosyl radical, in contrast to the 3-amino-3-carboxypropyl radical formed by PhDph2. Therefore, PhDph2 represents a novel [4Fe-4S] enzyme that uses unprecedented chemistry. This offers new opportunities to understand how different radical SAM enzymes control the reactivity of the bound [4Fe-4S] cluster to cleave SAM at different positions to carry out chemically challenging reactions. In addition, Lin and coworkers also identified the enzyme required for the last step of diphthamide biosynthesis, completing the last piece of the puzzle in the biosynthesis pathway.