DNA methylation has a profound impact on genome stability, transcription and development. Although enzymes that catalyse DNA methylation have been well characterized, those that are involved in methyl group removal have remained elusive, until recently. The transformative discovery that ten-eleven translocation (TET) family enzymes can oxidize 5-methylcytosine has greatly advanced our understanding of DNA demethylation. 5-Hydroxymethylcytosine is a key nexus in demethylation that can either be passively depleted through DNA replication or actively reverted to cytosine through iterative oxidation and thymine DNA glycosylase (TDG)-mediated base excision repair. Methylation, oxidation and repair now offer a model for a complete cycle of dynamic cytosine modification, with mounting evidence for its significance in the biological processes known to involve active demethylation.

Cancer immunotherapy based on genetically redirecting T cells has been used successfully to treat B cell malignancies. In this strategy, the T cell genome is modified by integration of viral vectors or transposons encoding chimaeric antigen receptors (CARs) that direct tumour cell killing. However, this approach is often limited by the extent of expansion and persistence of CAR T cells. Here we report mechanistic insights from studies of a patient with chronic lymphocytic leukaemia treated with CAR T cells targeting the CD19 protein. Following infusion of CAR T cells, anti-tumour activity was evident in the peripheral blood, lymph nodes and bone marrow; this activity was accompanied by complete remission. Unexpectedly, at the peak of the response, 94% of CAR T cells originated from a single clone in which lentiviral vector-mediated insertion of the CAR transgene disrupted the methylcytosine dioxygenase TET2 gene. Further analysis revealed a hypomorphic mutation in this patient's second TET2 allele. TET2-disrupted CAR T cells exhibited an epigenetic profile consistent with altered T cell differentiation and, at the peak of expansion, displayed a central memory phenotype. Experimental knockdown of TET2 recapitulated the potency-enhancing effect of TET2 dysfunction in this patient's CAR T cells. These findings suggest that the progeny of a single CAR T cell induced leukaemia remission and that TET2 modification may be useful for improving immunotherapies.

In the biosynthesis of many macrocyclic natural products by multidomain megasynthases, a carboxy-terminal thioesterase (TE) domain is involved in cyclization and product release; however, it has not been determined whether TE domains can catalyse macrocyclization (and elongation in the case of symmetric cyclic peptides) independently of upstream domains. The inability to decouple the TE cyclization step from earlier chain assembly steps has precluded determination of TE substrate specificity, which is important for the engineered biosynthesis of new compounds. Here we report that the excised TE domain from tyrocidine synthetase efficiently catalyses cyclization of a decapeptide-thioester to form the antibiotic tyrocidine A, and can catalyse pentapeptide-thioester dimerization followed by cyclization to form the antibiotic gramicidin S. By systematically varying the decapeptide-thioester substrate and comparing cyclization rates, we also show that only two residues (one near each end of the decapeptide) are critical for cyclization. This specificity profile indicates that the tyrocidine synthetase TE, and by analogy many other TE domains, will be able to cyclize and release a broad range of new substrates and products produced by engineered enzymatic assembly lines.

AID/APOBEC family cytosine deaminases, known to function in diverse cellular processes from antibody diversification to mRNA editing, have also been implicated in DNA demethylation, an important process for transcriptional activation. While oxidation-dependent pathways for demethylation have been described, pathways involving deamination of either 5-methylcytosine (mC) or 5-hydroxymethylcytosine (hmC) have emerged as alternatives. Here, we have addressed the biochemical plausibility of deamination-coupled demethylation. We found that purified AID/APOBECs have substantially reduced activity on mC relative to cytosine, their canonical substrate, and no detectable deamination of hmC. This finding was explained by the reactivity of a series of modified substrates, where steric bulk was increasingly detrimental to deamination. Further, upon AID/APOBEC overexpression, the deamination product of hmC was undetectable in genomic DNA, while oxidation intermediates remained detectable. Our results indicate that the steric requirements for cytosine deamination are one intrinsic barrier to the proposed function of deaminases in DNA demethylation.

Many biologically active natural peptides are synthesized by nonribosomal peptide synthetases (NRPS). Product release is accomplished by dedicated thioesterase (TE) domains, some of which catalyze an intramolecular cyclization to form macrolactone or macrolactam cyclic peptides. The excised 28 kDa SrfTE domain, a member of the alpha/beta hydrolase enzyme family, exhibits a distinctive bowl-shaped hydrophobic cavity that hosts the acylpeptide substrate and tolerates its folding to form a cyclic structure. A substrate analog confirms the substrate binding site and suggests a mechanism for substrate acylation/deacylation. Docking of the peptidyl carrier protein domain immediately preceding SrfTE positions the 4'-phosphopantheinyl prosthetic group that transfers the nascent acyl-peptide chain to SrfTE. The structure provides a basis for understanding the mechanism of acyl-PCP substrate recognition and for the cyclization reaction that results in release of the macrolactone cyclic heptapeptide.

Tyrosine 196 in Old Yellow Enzyme (OYE) was mutated to phenylalanine, and the resulting mutant enzyme was characterized to evaluate the mechanistic role of the residue. The residue demonstrates little effect on ligand binding and the reductive half-reaction, but a dramatic slowing by nearly 6 orders of magnitude of its oxidative half-reaction with 2-cyclohexenone. Observation of the oxidative half-reaction with a series of substrates allows us to propose a model describing the mechanism of the oxidative half-reaction. In addition, the curtailed reactivity with enones allows for characterization of the manner in which reduced enzyme primes the substrate for the redox reaction by observation of the Michaelis complex with reduced enzyme bound to substrate.

Molecules in nature are often brought to a bioactive conformation by ring formation (macrocyclization). A recurrent theme in the enzymatic synthesis of macrocyclic compounds by non-ribosomal and polyketide synthetases is the tethering of activated linear intermediates through thioester linkages to carrier proteins, in a natural analogy to solid-phase synthesis. A terminal thioesterase domain of the synthetase catalyses release from the tether and cyclization. Here we show that an isolated thioesterase can catalyse the cyclization of linear peptides immobilized on a solid-phase support modified with a biomimetic linker, offering the possibility of merging natural-product biosynthesis with combinatorial solid-phase chemistry. Starting from the cyclic decapeptide antibiotic tyrocidine A, this chemoenzymatic approach allows us to diversify the linear peptide both to probe the enzymology of the macrocyclizing enzyme, TycC thioesterase, and to create a library of cyclic peptide antibiotic products. We have used this method to reveal natural-product analogues of potential therapeutic utility; these compounds have an increased preference for bacterial over eukaryotic membranes and an improved spectrum of activity against some common bacterial pathogens.

A multitude of functions have evolved around cytosine within DNA, endowing the base with physiological significance beyond simple information storage. This versatility arises from enzymes that chemically modify cytosine to expand the potential of the genome. Some modifications alter coding sequences, such as deamination of cytosine by AID/APOBEC enzymes to generate immunologic or virologic diversity. Other modifications are critical to epigenetic control, altering gene expression or cellular identity. Of these, cytosine methylation is well understood, in contrast to recently discovered modifications, such as oxidation by TET enzymes to 5-hydroxymethylcytosine. Further complexity results from cytosine demethylation, an enigmatic process that impacts cellular pluripotency. Recent insights help us to propose an integrated DNA demethylation model, accounting for contributions from cytosine oxidation, deamination and base excision repair. Taken together, this rich medley of alterations renders cytosine a genomic “wild card”, whose context-dependent functions make the base far more than a static letter in the code of life.