Rahul M. Kohli

TET enzymes, TDG and the dynamics of DNA demethylation

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.

Biomimetic synthesis and optimization of cyclic peptide antibiotics

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.

AID/APOBEC deaminases disfavor modified cytosines implicated in DNA demethylation

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.

Type II Thioesterase Restores Activity of a NRPS Module Stalled with an Aminoacyl-S-enzyme that Cannot Be Elongated

Nonribosomal peptide synthetases (NRPSs) carry out the biosynthesis of numerous peptide natural products, including many with important clinical applications. The NRPS, organized into a series of modules, is an efficient, high-fidelity assembly line for the production of a particular peptide. Each module consists of domains, whose activities contribute to the accuracy of these assembly-line systems. The activation (A) domain uses ATP to selectively load an amino acid onto the module through formation of a thioester bond to the pantetheine arm of the thiolation (T) domain. Peptide-bond formation, catalyzed by the condensation (C) domain, is stringent for both sidechain identity and stereochemistry. The C domain accepts an aminoacylor peptidylthioester from the preceding module for nucleophilic addition by the amine of the loaded amino acid; this generates the elongated peptide attached to the downstream module. The peptide product is synthesized one amino acid at a time until it reaches the final module. There, the fully synthesized chain is released by a type I thioesterase (TEI), the terminal domain of the NRPS assembly. Despite the high fidelity of this process, an error in any step of the assembly-line synthesis severely impacts the efficiency of the system and creates a bottleneck that results in a buildup of unprocessed intermediates. For example, an error by the A domain, which can load amino acids other than that normally accepted by the C domain, would prevent peptide-bond formation. The loaded module would be blocked until the incorrect amino acid was hydrolyzed (Figure 1). A type II thioesterase (TEII), whose gene is associated with the gene cluster of many NRPSs and related polyketide synthases (PKSs), improves the efficiency of product formation in these systems and has been proposed to edit modules through hydrolysis of acyl groups. In the surfactin NRPS, TEII was shown to regenerate misacylated modules resulting from priming of the apomodule with acyl-CoA groups. In this study we provide evidence to expand the editing function of TEIIs to include restoring the activity of modules stalled by loaded amino acids that cannot be processed. N-acetylcysteamine (SNAC) thioesters have been used previously to assay NRPS domain activities. 13–15] Hydrolysis of SNAC substrates was used here to explore the specificity of the TEII from the tyrocidine biosynthetic operon, TycF. TycF accepted a broad variety of aminoacyl-SNACs of different sidechain identity and stereochemistry with a 20-fold kcat/Km range between the mostand least-active substrate (Table 1). A series of peptidyl-SNACs derived from the tyrocidine sequence was

Enzymology of acyl chain macrocyclization in natural product biosynthesis.

Polyketides and nonribosomal peptides constitute a large and diverse set of natural products with biological activity in microbial survival and pathogenesis, as well as broad pharmacological utility as antineoplastics, antibiotics or immunosupressants. These molecules are biosynthesized by the ordered condensation of monomer building blocks, acyl-CoAs or amino acids, leading to construction of linear acyl chains. Many of these natural products are constrained to their bioactive conformations by macrocyclization whereby, in one of the terminal steps of biosynthesis, parts of the molecule distant in the constructed linear acyl chain are covalently linked to one another. Typically, macrocyclization is catalyzed by a thioesterase domain at the C-terminal end of the biosynthetic assembly line, although alternative strategies are known. The enzymology of these macrocyclization catalysts, their structure, mechanism, and catalytic versatility, is the subject of this review. The diversity of macrocyclic structures accessed by enzyme catalyzed cyclization of linear acyl chains as well as their inherent substrate tolerance suggests their potential utility in reprogramming natural product biosynthesis pathways or accessing novel macrocyclic structures.

Chain Termination Steps in Nonribosomal Peptide Synthetase Assembly Lines : Directed Acyl-S-Enzyme Breakdown in Antibiotic and Siderophore Biosynthesis

A large number of therapeutically useful natural peptides areproduced nonribosomally by assembly line enzymology, involv-ing multidomain and multimodular catalysts that activate andassemble constituent amino acid monomers into oligopeptides.The peptide chains released (Scheme 1) can be final productssuch as cyclosporin (1)

Demystifying DNA Demethylation

DNA modifying and repair enzymes make a new connection in the mechanism of DNA demethylation. Variability and adaptability are necessary for overcoming the challenges of multicellular life. To address this need, nature has evolved a substantial enzymatic toolbox for altering cytosine within the genome. Methylation of the nucleotide cytosine (C) at the 5-position of the base has profound impacts on gene expression and cellular identity. The reverse of this process, DNA demethylation, is equally important for cleaning the genomic slate during embryogenesis or achieving rapid reactivation of previously silenced genes. Although the mechanism of DNA methylation has been rigorously established, active DNA demethylation in mammals has remained enigmatic, as disparate observations have failed to coalesce into a consistent model. Cytosine deamination, oxidation, and base excision repair enzymes have been proposed in a dizzying variety of combinations (1). Against this backdrop, two reports in this issue, by Ito et al. (2) on page 1300 and He et al. on page 1303 (3), help bring new clarity to the mechanistic model for DNA demethylation.

Timing of epimerization and condensation reactions in nonribosomal peptide assembly lines: kinetic analysis of phenylalanine activating elongation modules of tyrocidine synthetase B.

The cyclic decapeptide antibiotic tyrocidine has D-Phe residues at positions 1 and 4, produced during peptide chain growth from L-Phe residues by 50 kDa epimerase (E) domains embedded, respectively, in the initiation module (TycA) and the TycB3 module of the three-subunit (TycABC), 10-module nonribosomal peptide synthetase. While the initiation module clearly epimerizes the aminoacyl thioester Phe1-S-TycA intermediate, the timing of epimerization versus peptide bond condensation at internal E domains has been less well characterized in nonribosomal peptide synthetases. In this study, we use rapid quench techniques to evaluate a three-domain (ATE) and a four-domain version (CATE) of the TycB3 module and a six-domain fragment (ATCATE) of the TycB2(-3) bimodule to measure the ability of the E domain in the TycB3 module to epimerize the aminoacyl thioester Phe-S-TycB3 and the dipeptidyl-S-enzyme (L-Phe-L-Phe-S-TycB3 if L-Phe-D-Phe-S-TycB3). The chiralities of the Phe-S-enzyme and Phe-Phe-S-enzyme species over time were determined by hydrolysis and chiral TLC separations, allowing for the clear conclusion that epimerization in the internal TycB3 module occurs preferentially on the peptidyl-S-enzyme rather than the aminoacyl-S-enzyme, by a factor of about 3000/1. In turn, this imposes constraints on the chiral selectivity of the condensation (C) domains immediately upstream and downstream of E domains. The stereoselectivity of the upstream C domain was shown to be L-selective at both donor and acceptor sites ((L)C(L)) by site-directed mutagenesis studies of an E domain active site residue and using the small-molecule surrogate D-Phe-Pro-L-Phe-N-acetylcysteamine thioester (D-Phe-Pro-L-Phe-SNAC) and D-Phe-Pro-D-Phe-SNAC as donor probes.

The thioesterase domain from a nonribosomal peptide synthetase as a cyclization catalyst for integrin binding peptides

Nonribosomal peptide synthetases responsible for the production of macrocyclic compounds often use their C-terminal thioesterase (TE) domain for enzymatic cyclization of a linear precursor. The excised TE domain from the nonribosomal peptide synthetase responsible for the production of the cyclic decapeptide tyrocidine A, TycC TE, retains autonomous ability to catalyze head-to-tail macrocyclization of a linear peptide thioester with the native sequence of tyrocidine A and can additionally cyclize peptide analogs that incorporate limited alterations in the peptide sequence. Here we show that TycC TE can catalyze macrocyclization of peptide substrates that are dramatically different from the native tyrocidine linear precursor. Several peptide thioesters that retain a limited number of elements of the native peptide sequence are shown to be substrates for TycC TE. These peptides were designed to integrate an Arg-Gly-Asp sequence that confers potential activity in the inhibition of ligand binding by integrin receptors. Although enzymatic hydrolysis of the peptide thioester substrates is preferred over cyclization, TycC TE can be used on a preparative scale to generate both linear and cyclic peptide products for functional characterization. The products are shown to be inhibitors of ligand binding by integrin receptors, with cyclization and Nα-methylation being important contributors to the nanomolar potency of the best inhibitors of fibrinogen binding to αIIbβ3 integrin. This study provides evidence for TycC TE as a versatile macrocyclization catalyst and raises the prospect of using TE catalysis for the generation of diverse macrocyclic peptide libraries that can be probed for novel biological function.

Targets for Combating the Evolution of Acquired Antibiotic Resistance

Bacteria possess a remarkable ability to rapidly adapt and evolve in response to antibiotics. Acquired antibiotic resistance can arise by multiple mechanisms but commonly involves altering the target site of the drug, enzymatically inactivating the drug, or preventing the drug from accessing its target. These mechanisms involve new genetic changes in the pathogen leading to heritable resistance. This recognition underscores the importance of understanding how such genetic changes can arise. Here, we review recent advances in our understanding of the processes that contribute to the evolution of antibiotic resistance, with a particular focus on hypermutation mediated by the SOS pathway and horizontal gene transfer. We explore the molecular mechanisms involved in acquired resistance and discuss their viability as potential targets. We propose that additional studies into these adaptive mechanisms not only can provide insights into evolution but also can offer a strategy for potentiating our current antibiotic arsenal.