Unactivated Carbon Centers Research Articles

The Atypical Cobalamin-Dependent S-Adenosyl-l-Methionine Nonradical Methylase TsrM and Its Radical Counterparts.

Cobalamin (Cbl)-dependent S-adenosyl-l-methionine (AdoMet) radical methylases are known for their use of a dual cofactor system to perform challenging radical methylation reactions at unactivated carbon and phosphorus centers. These enzymes are part of a larger subgroup of Cbl-dependent AdoMet radical enzymes that also perform difficult ring contractions and radical rearrangements. This subgroup is a largely untapped reservoir of diverse chemistry that requires steady efforts in biochemical and structural characterization to reveal its complexity. In this Perspective, we highlight the significant efforts over many years to elucidate the function, mechanism, and structure of TsrM, an unexpected nonradical methylase in this subgroup. We also discuss recent achievements in characterizing radical methylase subgroup members that exemplify how key tools in mechanistic enzymology are valuable time and again. Finally, we identify recent enzyme activity studies that have made use of bioinformatic analyses to expand our definition of the subgroup. Additional breakthroughs in radical (and nonradical) enzymatic chemistry and challenging transformations from the unexplored space of this subgroup are undoubtedly on the horizon.

Distribution and diversity of dimetal-carboxylate halogenases in cyanobacteria

BackgroundHalogenation is a recurring feature in natural products, especially those from marine organisms. The selectivity with which halogenating enzymes act on their substrates renders halogenases interesting targets for biocatalyst development. Recently, CylC – the first predicted dimetal-carboxylate halogenase to be characterized – was shown to regio- and stereoselectively install a chlorine atom onto an unactivated carbon center during cylindrocyclophane biosynthesis. Homologs of CylC are also found in other characterized cyanobacterial secondary metabolite biosynthetic gene clusters. Due to its novelty in biological catalysis, selectivity and ability to perform C-H activation, this halogenase class is of considerable fundamental and applied interest. The study of CylC-like enzymes will provide insights into substrate scope, mechanism and catalytic partners, and will also enable engineering these biocatalysts for similar or additional C-H activating functions. Still, little is known regarding the diversity and distribution of these enzymes.ResultsIn this study, we used both genome mining and PCR-based screening to explore the genetic diversity of CylC homologs and their distribution in bacteria. While we found non-cyanobacterial homologs of these enzymes to be rare, we identified a large number of genes encoding CylC-like enzymes in publicly available cyanobacterial genomes and in our in-house culture collection of cyanobacteria. Genes encoding CylC homologs are widely distributed throughout the cyanobacterial tree of life, within biosynthetic gene clusters of distinct architectures (combination of unique gene groups). These enzymes are found in a variety of biosynthetic contexts, which include fatty-acid activating enzymes, type I or type III polyketide synthases, dialkylresorcinol-generating enzymes, monooxygenases or Rieske proteins. Our study also reveals that dimetal-carboxylate halogenases are among the most abundant types of halogenating enzymes in the phylum Cyanobacteria.ConclusionsOur data show that dimetal-carboxylate halogenases are widely distributed throughout the Cyanobacteria phylum and that BGCs encoding CylC homologs are diverse and mostly uncharacterized. This work will help guide the search for new halogenating biocatalysts and natural product scaffolds.

Catalytic C–H Oxidation Enhances Polyethylene Bonding

Selectively adding functionality to commodity plastics is an abiding challenge in polymer science. In this issue of Chem, Hartwig and co-workers describe a selective, catalytic oxidation of polyethylene C–H bonds by using high-valent ruthenium-oxo species. The transformation increases polyethylene’s adhesive properties without significantly modifying its thermal stability or mechanical strength.

A Radical Exploration of the Cobalamin-Dependent Radical SAM Enzyme CysS

The cobalamin-dependent radical SAM enzyme CysS performs methylation reactions at an unactivated carbon center using what is predicted to be an unprecedented radical substitution mechanism. Herein, we highlight significant progress made by Wang and Begley in providing support for the enzymatic relevance of this proposal.

Aliphatic Ether Bond Formation Expands the Scope of Radical SAM Enzymes in Natural Product Biosynthesis.

The biosynthetic pathways of microbial natural products provide a rich source of novel enzyme-catalyzed transformations. Using a new bioinformatic search strategy, we recently identified an abundance of gene clusters for ribosomally synthesized and post-translationally modified peptides (RiPPs) that contain at least one radical S-adenosylmethionine (RaS) metalloenzyme and are regulated by quorum sensing. In the present study, we characterize a RaS enzyme from one such RiPP gene cluster and find that it installs an aliphatic ether cross-link at an unactivated carbon center, linking the oxygen of a Thr side chain to the α-carbon of a Gln residue. This reaction marks the first ether cross-link installed by a RaS enzyme. Additionally, it leads to a new heterocyclization motif and underlines the utility of our bioinformatics approach in finding new families of RiPP modifications.

Enhanced Solubilization of Class B Radical S-Adenosylmethionine Methylases by Improved Cobalamin Uptake in Escherichia coli.

The methylation of unactivated carbon and phosphorus centers is a burgeoning area of biological chemistry, especially given that such reactions constitute key steps in the biosynthesis of numerous enzyme cofactors, antibiotics, and other natural products of clinical value. These kinetically challenging reactions are catalyzed exclusively by enzymes in the radical S-adenosylmethionine (SAM) superfamily and have been grouped into four classes (A-D). Class B radical SAM (RS) methylases require a cobalamin cofactor in addition to the [4Fe-4S] cluster that is characteristic of RS enzymes. However, their poor solubility upon overexpression and their generally poor turnover has hampered detailed in vitro studies of these enzymes. It has been suggested that improper folding, possibly caused by insufficient cobalamin during their overproduction in Escherichia coli, leads to formation of inclusion bodies. Herein, we report our efforts to improve the overproduction of class B RS methylases in a soluble form by engineering a strain of E. coli to take in more cobalamin. We cloned five genes ( btuC, btuE, btuD, btuF, and btuB) that encode proteins that are responsible for cobalamin uptake and transport in E. coli and co-expressed these genes with those that encode TsrM, Fom3, PhpK, and ThnK, four class B RS methylases that suffer from poor solubility during overproduction. This strategy markedly enhances the uptake of cobalamin into the cytoplasm and improves the solubility of the target enzymes significantly.

Cobalamin-Dependent C-Methyltransferases From Marine Microbes: Accessibility via Rhizobia Expression.

Cobalamin-dependent radical S-adenosylmethionine (rSAM) methyltransferases catalyze chemically challenging methylation reactions on diverse natural products at unactivated carbon centers. In vivo reconstitution and biosynthetic studies of natural product gene clusters encoding these enzymes are often severely limited by ineffective heterologous expression hosts, including the otherwise versatile Escherichia coli. In this chapter, we describe the use of rhizobia bacteria as effective expression hosts for cobalamin-dependent rSAM C-methyltransferases. We chose the natural product pathway encoding the heavily modified cytotoxic peptides, the polytheonamides, as our model pathway due to the presence of two methyltransferases responsible for a total of 17 C-methylations. Detailed protocols are given for vector construction, transformation, and heterologous expression in Rhizobium leguminosarum bv. viciae 3841. Additional methods pertaining to analytical separation and mass spectrometric analysis of modified peptides are also entailed. As genomics continues to uncover new enzymes and pathways from unknown and uncultivated microbes, use of metabolically distinct heterologous expression hosts like rhizobia will be a necessary tool to unravel the catalytic and metabolic diversity of marine microbial life.

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.

A new strategy for aromatic ring alkylation in cylindrocyclophane biosynthesis.

Alkylation of aromatic rings with alkyl halides is an important transformation in organic synthesis, yet an enzymatic equivalent is unknown. Here, we report that cylindrocyclophane biosynthesis in Cylindrospermum licheniforme ATCC 29412 involves chlorination of an unactivated carbon center by a novel halogenase, followed by a previously uncharacterized enzymatic dimerization reaction featuring sequential, stereospecific alkylations of resorcinol aromatic rings. Discovery of the enzymatic machinery underlying this unique biosynthetic carbon-carbon bond formation has implications for biocatalysis and metabolic engineering.

Structure-Guided Reprogramming of a Hydroxylase To Halogenate Its Small Molecule Substrate.

Enzymatic installation of chlorine/bromine into unactivated carbon centers provides a versatile, selective, and environmentally friendly alternative to chemical halogenation. Iron(II) and 2-(oxo)-glutarate (FeII/2OG)-dependent halogenases are powerful biocatalysts that are capable of cleaving aliphatic C-H bonds to introduce useful functional groups, including halogens. Using the structure of the Fe/2OG halogenase, WelO5, in complex with its small molecule substrate, we identified a similar N-acyl amino acid hydroxylase, SadA, and reprogrammed it to halogenate its substrate, thereby generating a new chiral haloalkyl center. The work highlights the potential of FeII/2OG enzymes as platforms for development of novel stereospecific catalysts for late-stage C-H functionalization.

Transformations of the FeS Clusters of the Methylthiotransferases MiaB and RimO, Detected by Direct Electrochemistry.

The methylthiotransferases (MTTases) represent a subfamily of the S-adenosylmethionine (AdoMet) radical superfamily of enzymes that catalyze the attachment of a methylthioether (-SCH3) moiety on unactivated carbon centers. These enzymes contain two [4Fe-4S] clusters, one of which participates in the reductive fragmentation of AdoMet to generate a 5′-deoxyadenosyl 5′-radical and the other of which, termed the auxiliary cluster, is believed to play a central role in constructing the methylthio group and attaching it to the substrate. Because the redox properties of the bound cofactors within the AdoMet radical superfamily are so poorly understood, we have examined two MTTases in parallel, MiaB and RimO, using protein electrochemistry. We resolve the redox potentials of each [4Fe-4S] cluster, show that the auxiliary cluster has a potential higher than that of the AdoMet-binding cluster, and demonstrate that upon incubation of either enzyme with AdoMet, a unique low-potential state of the enzyme emerges. Our results are consistent with a mechanism whereby the auxiliary cluster is transiently methylated during substrate methylthiolation.

Enzymology: A radical finding.

A new family of radical halogenases has been discovered that regio- and stereoselectively chlorinates the unactivated carbon center of indolemonoterpenoid substrates without the prerequisite for the substrate to be bound to a protein carrier.

Cylindrocyclophane Biosynthesis Involves Functionalization of an Unactivated Carbon Center

The cylindrocyclophanes are a family of natural products that share a remarkable paracyclophane carbon scaffold. Using genome sequencing and bioinformatic analyses, we have discovered a biosynthetic gene cluster involved in the assembly of cylindrocyclophane F. Through a combination of in vitro enzyme characterization and feeding studies, we confirm the connection between this gene cluster and cylindrocyclophane production, elucidate the chemical events involved in initiating and terminating an unusual type I polyketide synthase assembly line, and discover that macrocycle assembly involves functionalization of an unactivated carbon center.

Escherichia coli Allows Efficient Modular Incorporation of Newly Isolated Quinomycin Biosynthetic Enzyme into Echinomycin Biosynthetic Pathway for Rational Design and Synthesis of Potent Antibiotic Unnatural Natural Product

Natural products display impressive activities against a wide range of targets, including viruses, microbes, and tumors. However, their clinical use is hampered frequently by their scarcity and undesirable toxicity. Not only can engineering Escherichia coli for plasmid-based pharmacophore biosynthesis offer alternative means of simple and easily scalable production of valuable yet hard-to-obtain compounds, but also carries a potential for providing a straightforward and efficient means of preparing natural product analogs. The quinomycin family of nonribosomal peptides, including echinomycin, triostin A, and SW-163s, are important secondary metabolites imparting antibiotic antitumor activity via DNA bisintercalation. Previously we have shown the production of echinomycin and triostin A in E. coli using our convenient and modular plasmid system to introduce these heterologous biosynthetic pathways into E. coli. However, we have yet to develop a novel biosynthetic pathway capable of producing bioactive unnatural natural products in E. coli. Here we report an identification of a new gene cluster responsible for the biosynthesis of SW-163s that involves previously unknown biosynthesis of (+)-(1S, 2S)-norcoronamic acid and generation of aliphatic side chains of various sizes via iterative methylation of an unactivated carbon center. Substituting an echinomycin biosynthetic gene with a gene from the newly identified SW-163 biosynthetic gene cluster, we were able to rationally re-engineer the plasmid-based echinomycin biosynthetic pathway for the production of a novel bioactive compound in E. coli.

CD and MCD of CytC3 and Taurine Dioxygenase: Role of the Facial Triad in α-KG-Dependent Oxygenases

The alpha-ketoglutarate (alpha-KG)-dependent oxygenases are a large and diverse class of mononuclear non-heme iron enzymes that require FeII, alpha-KG, and dioxygen for catalysis with the alpha-KG cosubstrate supplying the additional reducing equivalents for oxygen activation. While these systems exhibit a diverse array of reactivities (i.e., hydroxylation, desaturation, ring closure, etc.), they all share a common structural motif at the FeII active site, termed the 2-His-1-carboxylate facial triad. Recently, a new subclass of alpha-KG-dependent oxygenases has been identified that exhibits novel reactivity, the oxidative halogenation of unactivated carbon centers. These enzymes are also structurally unique in that they do not contain the standard facial triad, as a Cl- ligand is coordinated in place of the carboxylate. An FeII methodology involving CD, MCD, and VTVH MCD spectroscopies was applied to CytC3 to elucidate the active-site structural effects of this perturbation of the coordination sphere. A significant decrease in the affinity of FeII for apo-CytC3 was observed, supporting the necessity of the facial triad for iron coordination to form the resting site. In addition, interesting differences observed in the FeII/alpha-KG complex relative to the cognate complex in other alpha-KG-dependent oxygenases indicate the presence of a distorted 6C site with a weak water ligand. Combined with parallel studies of taurine dioxygenase and past studies of clavaminate synthase, these results define a role of the carboxylate ligand of the facial triad in stabilizing water coordination via a H-bonding interaction between the noncoordinating oxygen of the carboxylate and the coordinated water. These studies provide initial insight into the active-site features that favor chlorination by CytC3 over the hydroxylation reactions occurring in related enzymes.

Two interconverting Fe(IV) intermediates in aliphatic chlorination by the halogenase CytC3

Enzymatic incorporation of a halogen atom is a common feature in the biosyntheses of more than 4,500 natural products. Halogenation of unactivated carbon centers in the biosyntheses of several compounds of nonribosomal peptide origin is carried out by a class of mononuclear nonheme iron enzymes that require alpha-ketoglutarate (alphaKG, 1), chloride and oxygen. To investigate the ability of these enzymes to functionalize unactivated methyl groups, we characterized the chlorination of the gamma-methyl substituent of L-2-aminobutyric acid (L-Aba, 2) attached to the carrier protein CytC2 by iron halogenase (CytC3) from soil Streptomyces sp. We identified an intermediate state comprising two high-spin Fe(IV) complexes in rapid equilibrium. At least one of the Fe(IV) complexes abstracts hydrogen from the substrate. The demonstration that chlorination proceeds through an Fe(IV) intermediate that cleaves a C-H bond reveals the mechanistic similarity of aliphatic halogenases to the iron- and alphaKG-dependent hydroxylases.

Trichlorination of l-Leucin by Fe(II) Halogenases

Barbamide 1 was isolated from marine bacterium Lyngbya majuscule (J. Orjala et al. J. Nat. Prod. 1996, 59, 427-430). It shows some activity as molluscicidal agent. Trichlorination of unactivated carbon centers cannot be achieved easily. Herein, l-[14C]Leu-S-BarA (2) or monohalogenated 3 were transformed to dichloroleucine 4 with BarB2 in the presence of O2, chloride and α-ketoglutarate (α-KG). Incubating this mixture with BarB1 gave trichlorinated product 5. These two enzymes complement each other; employing only one of them or adding them in the opposite order did not yield the desired product. Peptidyl carrier protein and nonheme Fe(II) halogenases BarB1and BarB2 could be produced from E. coli.

Halogenation of Unactivated Carbon Centers in Natural Product Biosynthesis: Trichlorination of Leucine during Barbamide Biosynthesis

The in vitro reconstitution of leucine halogenation during barbamide biosynthesis has been accomplished. It has been demonstrated that the triple chlorination of the unactivated pro-R methyl group of the peptidyl carrier protein-tethered l-Leu substrate is carried out by the tandem action of two nonheme iron(II)-dependent halogenases, BarB1 and BarB2. Investigation of the substrate specificities of each of the halogenating enzymes revealed their complementary roles in the generation of trichloroleucine.