Highlights d Engineering tRNA-synthetase interactions generates mutually orthogonal PylT/RS pairs d Combination of optimized pairs allows for efficient dual nonsense suppression d Site-specific incorporation of two orthogonal chemical handles in the same protein d Dual-color labeling and crosslinking of surface receptors on live mammalian cells

Ribonucleotide reductase (RNR) catalyzes the only known de-novo pathway for production of all four deoxyribonucleotides required for DNA synthesis1,2. It is essential for all organisms with DNA as genetic material and a current drug target3,4. Since the discovery that iron is required for function in the aerobic, class I RNR found in all eukaryotes and many bacteria, a di-nuclear metal site has been viewed as a requirement for generating and stabilizing a catalytic radical, essential for RNR activity5,6,7. Here, we describe a new group of RNR proteins in Mollicutes, including Mycoplasma pathogens, which possesses a metal-independent stable radical residing on a modified tyrosyl residue. Structural, biochemical and spectroscopic characterization reveal an unprecedented stable DOPA radical species that directly supports ribonucleotide reduction in vitro and in vivo. This observation overturns the presumed requirement of a dinuclear metal site in aerobic RNR. The metal-independent radical compels completely novel mechanisms for radical generation and stabilization, processes that are targeted by RNR inhibitors. Conceivably, this RNR variant provides an advantage under metal starvation induced by the immune system. Organisms encoding this type of RNR are involved in diseases of the respiratory, urinary and genital tracts, some with developing resistance to antibiotics. Further characterization of this novel RNR family and its mechanism for cofactor generation will provide insight into new enzymatic chemistry and be of value to devise strategies to combat the pathogens that utilize it. We propose that the new RNR subclass is denoted class Ie.

Protein acetylation is a highly frequent protein modification. However, comparatively little is known about its enzymatic machinery. N-a-acetylation (NTA) and e-lysine acetylation (KA) are known to be catalyzed by distinct families of enzymes (NATs and KATs, respectively), although the possibility that the same GCN5-related N-acetyltransferase (GNAT) can perform both functions has been debated. Here, we discovered a new family of plastid-localized GNATs, which possess a dual specificity. All characterized GNAT family members display a number of unique features. Quantitative mass spectrometry analyses revealed that these enzymes exhibit both distinct KA and relaxed NTA specificities. Furthermore, inactivation of GNAT2 leads to significant NTA or KA decreases of several plastid proteins, while proteins of other compartments were unaffected. The data indicate that these enzymes have specific protein targets and likely display partly redundant selectivity, increasing the robustness of the acetylation process in vivo. In summary, this study revealed a new layer of complexity in the machinery controlling this prevalent modification and suggests that other eukaryotic GNATs may also possess these previously underappreciated broader enzymatic activities.