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.

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

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.

Vancomycin is a potent glycopeptide antibiotic that has evolved to specifically bind to the D-Ala-D-Ala dipeptide termini of nascent peptidoglycans. Although this mode of action is well established, several studies indicate that vancomycin and analogues exploit noncanonical target sites. In order to address all vancomycin targets in clinically relevant Staphylococcus aureus and Enterococcus faecalis strains we developed a series of small-molecule photoaffinity probes based on vancomycin. Proteomic profiling revealed the specific labeling of two previously unknown vancomycin targets that are likely to contribute to its antibiotic activity. The specific inhibition of the major staphylococcal autolysin Atl confirms previous observations that vancomycin alters S. aureus cell morphology by interaction with the autolytic machinery. Moreover, in E. faecalis the vancomycin photoprobe specifically binds to an ABC transporter protein, which likely impedes the uptake of essential nutrients such as sugars and peptides. The labeling of these two prominent membrane targets in living cells reveals a thus far unexplored mode of vancomycin binding and inhibition that could allow a rational design of variants with improved activity.

Microtubules (mt) are highly dynamic polymers composed of alpha-and beta-tubulin monomers that are present in all dividing and non-dividing cells. A broad variety of natural products exists that are known to interfere with the microtubule network, by either stabilizing or de-stabilizing these rope-like polymers. Among those tubulysins represent a new and potent class of cytostatic tetrapeptides originating from myxobacteria. Early studies suggested that tubulysins interact with the eukaryotic cytoskeleton by inhibition of tubulin polymerization with EC 50 values in the picomolar range. Recently, pretubulysins have been described to retain the high tubulindegradation activity of their more complex tubulysin relatives and represent an easier synthetic target with an efficient synthesis already in place. Although tubulin has been suggested as the dedicated target of tubulysin a comprehensive molecular target analysis of pretubulysin in the context of the whole proteome has not been carried out so far. Here we utilize synthetic chemistry to develop two pretubulysin photoaffinity probes which were applied in cellular activity-based protein profiling and imaging studies in order to unravel and visualize dedicated targets. Our results clearly show a remarkable selectivity of pretubulysin for beta-tubulin which we independently confirmed by a mass-spectrometry based proteomic profiling platform as well as by tubulin antibody based co-staining on intact cells.

Resistance to chemotherapeutic agents represents a major challenge in cancer research. One approach to this problem is combination therapy, the application of a toxic chemotherapeutic drug together with a sensitizing compound that addresses the vulnerability of cancer cells to induce apoptosis. Here we report the discovery of a new compound class (T8) that sensitizes various cancer cells towards etoposide treatment at subtoxic concentrations. Proteomic analysis revealed protein disulfide isomerase (PDI) as the target of the T8 class. In-depth chemical and biological studies such as the synthesis of optimized compounds, molecular docking analyses, cellular imaging, and apoptosis assays confirmed the unique mode of action through reversible PDI inhibition.

VC generated transgenic plants. XC, ET, JR, SM and MU performed western blot analyses. UA and SM performed co-immunoprecipitations. MU generated cTP-YFP fusions and localized fusions using confocal microscopy. SM performed BN-PAGE analysis. JE and IF performed MS analysis on BN slices. MU produced recombinant protein in E. coli and performed size exclusion chromatography together with ML and AS. ES and AS performed MS analysis of recombinant protein. SM, MU and ET performed Chl a fluorescence measurements during light fluctuations. MAS measured 77K Chl a fluorescence emission spectra, ECS kinetics and Cytf redox state. MAS and UA performed Chl a fluorescence and P700 light response curves and steady state measurements, respectively. TvB carried out simultaneous CO2 assimilation and Chl a fluorescence measurements. UA wrote manuscript with help from all authors.

N a-terminal acetylation (NTA) is a prevalent protein modification in eukaryotes. In plants, the biological function of NTA remains enigmatic. The dominant N-acetyltransferase (Nat) in Arabidopsis (Arabidopsis thaliana) is NatA, which cotranslationally catalyzes acetylation of ;40% of the proteome. The core NatA complex consists of the catalytic subunit NAA10 and the ribosome-anchoring subunit NAA15. In human (Homo sapiens), fruit fly (Drosophila melanogaster), and yeast (Saccharomyces cerevisiae), this core NatA complex interacts with NAA50 to form the NatE complex. While in metazoa, NAA50 has N-acetyltransferase activity, yeast NAA50 is catalytically inactive and positions NatA at the ribosome tunnel exit. Here, we report the identification and characterization of Arabidopsis NAA50 (AT5G11340). Consistent with its putative function as a cotranslationally acting Nat, AtNAA50-EYFP localized to the cytosol and the endoplasmic reticulum but also to the nuclei. We demonstrate that purified AtNAA50 displays N a-terminal acetyltransferase and lysine-«-autoacetyltransferase activity in vitro. Global N-acetylome profiling of Escherichia coli cells expressing AtNAA50 revealed conservation of NatE substrate specificity between plants and humans. Unlike the embryo-lethal phenotype caused by the absence of AtNAA10 and AtNAA15, loss of NAA50 expression resulted in severe growth retardation and infertility in two Arabidopsis transfer DNA insertion lines (naa50-1 and naa50-2). The phenotype of naa50-2 was rescued by the expression of HsNAA50 or AtNAA50. In contrast, the inactive ScNAA50 failed to complement naa50-2. Remarkably, loss of NAA50 expression did not affect NTA of known NatA substrates and caused the accumulation of proteins involved in stress responses. Overall, our results emphasize a relevant role of AtNAA50 in plant defense and development, which is independent of the essential NatA activity.