Graham A. Hudson

A prevalent peptide-binding domain guides ribosomal natural product biosynthesis

Ribosomally synthesized and posttranslationally modified peptides (RiPPs) are a rapidly growing natural product class. RiPP precursor peptides can undergo extensive enzymatic tailoring, yielding structurally and functionally diverse products, and their biosynthetic logic makes them attractive bioengineering targets. Recent work suggests that unrelated RiPP modifying enzymes contain structurally similar precursor peptide-binding domains. Using profile hidden Markov model comparisons, we discovered related and previously unrecognized peptide-binding domains in proteins spanning the majority of known prokaryotic RiPP classes; thus, we named this conserved domain the RiPP precursor peptide recognition element (RRE). Through binding studies, we verify the role of the RRE for three distinct RiPP classes: linear azole-containing peptides, thiopeptides, and lasso peptides. Because numerous RiPP biosynthetic enzymes act on peptide substrates, our findings have powerful predictive value as to which protein(s) drive substrate binding, laying a foundation for further characterization of RiPP biosynthetic pathways and the rational engineering of new peptide-binding activities.

In Vitro Biosynthesis of the Core Scaffold of the Thiopeptide Thiomuracin.

Thiopeptides are potent antibiotics that inhibit protein synthesis. They are made by a remarkable post-translational modification process that transforms a linear peptide into a polycyclic structure. We present here the in vitro biosynthesis of the core scaffold of thiomuracin catalyzed by six proteins. We show that cyclodehydration precedes dehydration, and that dehydration is catalyzed by two proteins in a tRNAGlu-dependent manner. The enzyme that generates the pyridine core from two dehydroalanines ejects the leader peptide as a C-terminal carboxamide. Mutagenesis studies of the enzyme TbtD identified important residues for a formal [4+2] cycloaddition process. The core structure of thiomuracin exhibits similar antimicrobial activity to other known congeners, illustrating that in vitro biosynthesis is a viable route to potent antibiotics that can be explored for the rapid and renewable generation of analogues.

Biosynthetic Timing and Substrate Specificity for the Thiopeptide Thiomuracin

The biosynthesis of the thiopeptide thiomuracin is a well-orchestrated process involving a multitude of posttranslational modifications. We show that six Cys residues of a precursor peptide are first cyclodehydrated and oxidized to thiazoles in an ordered, but nonlinear fashion that is leader-peptide-dependent. Then four alcohols are glutamylated and converted to alkenes in a C-to-N terminal directional process that is leader-peptide-independent. Finally, two of these alkenes undergo a formal [4 + 2] cycloaddition to form a trithiazole-substituted pyridine macrocycle. We describe here the factors that govern the substrate specificity and order of biosynthetic events that turn a ribosomal peptide into a powerful antibiotic.

Reconstitution and Substrate Specificity of the Radical S-Adenosyl-methionine Thiazole C-Methyltransferase in Thiomuracin Biosynthesis

Thiomuracin is a thiopeptide antibiotic with potent activity toward Gram-positive drug-resistant bacteria. Thiomuracin is biosynthesized from a precursor peptide, TbtA, by a complex array of posttranslational modifications. One of several intriguing transformations is the C-methylation of thiazole, occurring at an unactivated sp2 carbon. Herein, we report the in vitro reconstitution of TbtI, the responsible radical S-adenosyl-methionine (rSAM) C-methyltransferase, which catalyzes the formation of 5-methylthiazole at a single site. Our studies demonstrate that a linear hexazole-bearing intermediate of TbtA is a substrate for TbtI whereas macrocyclized thiomuracin GZ is not. In determining the minimal substrate for TbtI, we found that the enzyme is functional when most of the leader peptide has been removed. The in vitro reconstitution of TbtI, a class C rSAM methyltransferase, further adds to the chemical versatility of rSAM enzymes, and informs on the complexity of thiomuracin biosynthesis.

Chimeric Leader Peptides for the Generation of Non-Natural Hybrid RiPP Products

Combining biosynthetic enzymes from multiple pathways is an attractive approach for producing molecules with desired structural features; however, progress has been hampered by the incompatibility of enzymes from unrelated pathways and intolerance toward alternative substrates. Ribosomally synthesized and posttranslationally modified peptides (RiPPs) are a diverse natural product class that employs a biosynthetic logic that is highly amenable to engineering new compounds. RiPP biosynthetic proteins modify their substrates by binding to a motif typically located in the N-terminal leader region of the precursor peptide. Here, we exploit this feature by designing leader peptides that enable recognition and processing by multiple enzymes from unrelated RiPP pathways. Using this broadly applicable strategy, a thiazoline-forming cyclodehydratase was combined with enzymes from the sactipeptide and lanthipeptide families to create new-to-nature hybrid RiPPs. We also provide insight into design features that enable control over the hybrid biosynthesis to optimize enzyme compatibility and establish a general platform for engineering additional hybrid RiPPs.

Radical S-Adenosylmethionine Enzymes Involved in RiPP Biosynthesis

Ribosomally synthesized and post-translationally modified peptides (RiPPs) display a diverse range of structures and continue to expand as a natural product class. Accordingly, RiPPs exhibit a wide array of bioactivities, acting as broad and narrow spectrum growth suppressors, antidiabetics, and antinociception and anticancer agents. Because of these properties, and the complex repertoire of post-translational modifications (PTMs) that give rise to these molecules, RiPP biosynthesis has been intensely studied. RiPP biosynthesis often involves enzymes that perform unique chemistry with intriguing reaction mechanisms, which attract chemists and biochemists alike to study and re-engineer these pathways. One particular type of RiPP biosynthetic enzyme is the so-called radical S-adenosylmethionine (rSAM) enzyme, which utilizes radical-based chemistry to install several distinct PTMs. Here, we describe the rSAM enzymes characterized over the past decade that catalyze six reaction types from several RiPP biosynthetic pathways. We present the current state of mechanistic understanding and conclude with possible directions for future characterization of this enzyme family.

RiPP antibiotics: biosynthesis and engineering potential

The threat of antibiotic resistant bacterial infections continues to underscore the need for new treatment options. Historically, small molecule metabolites from microbes have provided a rich source of antibiotic compounds, and as a result, significant effort has been invested in engineering the responsible biosynthetic pathways to generate novel analogs with attractive pharmacological properties. Unfortunately, biosynthetic stringency has limited the capacity of non-ribosomal peptide synthetases and polyketide synthases from producing substantially different analogs in large numbers. Another class of natural products, the ribosomally synthesized and post-translationally modified peptides (RiPPs), have rapidly expanded in recent years with many natively displaying potent antibiotic activity. RiPP biosynthetic pathways are modular and intrinsically tolerant to alternative substrates. Several prominent RiPPs with antibiotic activity will be covered in this review with a focus on their biosynthetic plasticity. While only a few RiPP enzymes have been thoroughly investigated mechanistically, this knowledge has already been harnessed to generate new-to-nature compounds. Through the use of synthetic biology approaches, on-going efforts in RiPP engineering hold great promise in unlocking the potential of this natural product class.

Mechanism of a Class C Radical S-Adenosyl-l-methionine Thiazole Methyl Transferase

The past decade has seen the discovery of four different classes of radical S-adenosylmethionine (rSAM) methyltransferases that methylate unactivated carbon centers. Whereas the mechanism of class A is well understood, the molecular details of methylation by classes B–D are not. In this study, we present detailed mechanistic investigations of the class C rSAM methyltransferase TbtI involved in the biosynthesis of the potent thiopeptide antibiotic thiomuracin. TbtI C-methylates a Cys-derived thiazole during posttranslational maturation. Product analysis demonstrates that two SAM molecules are required for methylation and that one SAM (SAM1) is converted to 5′-deoxyadenosine and the second SAM (SAM2) is converted to S-adenosyl-l-homocysteine (SAH). Isotope labeling studies show that a hydrogen is transferred from the methyl group of SAM2 to the 5′-deoxyadenosine of SAM1 and the other two hydrogens of the methyl group of SAM2 appear in the methylated product. In addition, a hydrogen appears to be transferred from the β-position of the thiazole to the methyl group in the product. We also show that the methyl protons in the product can exchange with solvent. A mechanism consistent with these observations is presented that differs from other characterized radical SAM methyltransferases.

In Vitro Biosynthetic Studies of Bottromycin Expand the Enzymatic Capabilities of the YcaO Superfamily

The bottromycins belong to the ribosomally synthesized and posttranslationally modified peptide (RiPP) family of natural products. Bottromycins exhibit unique structural features, including a hallmark macrolactamidine ring and thiazole heterocycle for which divergent members of the YcaO superfamily have been biosynthetically implicated. Here we report the in vitro reconstitution of two YcaO proteins, BmbD and BmbE, responsible for the ATP-dependent cyclodehydration reactions that yield thiazoline- and macrolactamidine-functionalized products, respectively. We also establish the substrate tolerance for BmbD and BmbE and systematically dissect the role of the follower peptide, which we show serves a purpose similar to canonical leader peptides in directing the biosynthetic enzymes to the substrate. Lastly, we leverage the expanded capabilities of YcaO proteins to conduct an extensive bioinformatic survey to classify known YcaO chemistry. This analysis predicts new functions remain to be uncovered within the superfamily.

Bioinformatic Expansion and Discovery of Thiopeptide Antibiotics

Thiopeptides are members of the ribosomally synthesized and post-translationally modified peptide family of natural products. Most characterized thiopeptides display nanomolar potency toward Gram-positive bacteria by blocking protein translation with several being produced at the industrial scale for veterinary and livestock applications. Employing our custom bioinformatics program, RODEO, we expand the thiopeptide family of natural products by a factor of four. This effort revealed many new thiopeptide biosynthetic gene clusters with products predicted to be distinct from characterized thiopeptides and identified gene clusters for previously characterized molecules of unknown biosynthetic origin. To further validate our data set of predicted thiopeptide biosynthetic gene clusters, we isolated and characterized a structurally unique thiopeptide featuring a central piperidine and rare thioamide moiety. Termed saalfelduracin, this thiopeptide displayed potent antibiotic activity toward several drug-resistant Gram-positive pathogens. A combination of whole-genome sequencing, comparative genomics, and heterologous expression experiments confirmed that the thioamide moiety of saalfelduracin is installed post-translationally by the joint action of two proteins, TfuA and YcaO. These results reconcile the previously unknown origin of the thioamide in two long-known thiopeptides, thiopeptin and Sch 18640. Armed with these new insights into thiopeptide chemical-genomic space, we provide a roadmap for the discovery of additional members of this natural product family.