Stereochemical Course of the Reaction Catalyzed by RimO, a Radical SAM Methylthiotransferase

RimO is a member of the growing radical S-adenosylmethionine (SAM) superfamily of enzymes, which use a reduced [4Fe–4S] cluster to effect reductive cleavage of the 5′ C–S bond of SAM to form a 5′-deoxyadenosyl 5′-radical (5′-dA^•) intermediate. RimO uses this potent oxidant to catalyze the attachment of a methylthio group (−SCH₃) to C3 of aspartate 89 of protein S12, one of 21 proteins that compose the 30S subunit of the bacterial ribosome. However, the exact mechanism by which this transformation takes place has remained elusive. Herein, we describe the stereochemical course of the RimO reaction. Using peptide mimics of the S12 protein bearing deuterium at the 3 <i>pro-R</i> or 3 <i>pro-S</i> positions of the target aspartyl residue, we show that RimO from <i>Bacteroides thetaiotaomicron</i> (<i>Bt</i>) catalyzes abstraction of the <i>pro-S</i> hydrogen atom, as evidenced by the transfer of deuterium into 5′-deoxyadenosine (5′-dAH). The observed kinetic isotope effect on H atom versus D atom abstraction is ∼1.9, suggesting that this step is at least partially rate determining. We also demonstrate that <i>Bt</i> RimO can utilize the flavodoxin/flavodoxin oxidoreductase/NADPH reducing system from <i>Escherichia coli</i> as a source of requisite electrons. Use of this <i>in vivo</i> reducing system decreases, but does not eliminate, formation of 5′-dAH in excess of methylthiolated product

Investigation of Solvent Hydron Exchange in the Reaction Catalyzed by the Antibiotic Resistance Protein Cfr

Cfr is a radical <i>S</i>-adenosylmethionine (RS) methylase that appends methyl groups to C8 and C2 of adenosine 2503 in 23S rRNA. Methylation of C8 confers resistance to several classes of antibiotics that bind in or near the peptidyltransferase center of the bacterial ribosome, including the synthetic antibiotic linezolid. The Cfr reaction requires the action of five conserved cysteines, three of which ligate a required [4Fe-4S] cluster cofactor. The two remaining cysteines play a more intricate role in the reaction; one (Cys338) becomes transiently methylated during catalysis. The function of the second (Cys105) has not been rigorously established; however, in the related RlmN reaction, it (Cys118) initiates resolution of a key protein–nucleic acid cross-linked intermediate by abstracting the proton from the carbon center (C2) undergoing methylation. We previously proposed that, unlike RlmN, Cfr would utilize a polyprotic base during resolution of the protein–nucleic acid cross-linked intermediate during C8 methylation and, like RlmN, use a monoprotic base during C2 methylation. We based this proposal on the fact that solvent hydrons could exchange into the product during C8 methylation, but not during C2 methylation. Herein, we show that Cys105 of Cfr has a function similar to that of Cys118 of RlmN while methylating C8 of A2503 and provide evidence for one molecule of water that is in close contact with it, which provides the exchangeable protons during catalysis

Identification of an Intermediate Methyl Carrier in the Radical <i>S</i>‑Adenosylmethionine Methylthiotransferases RimO and MiaB

RimO and MiaB are radical <i>S</i>-adenosylmethionine (SAM) enzymes that catalyze the attachment of methylthio (−SCH₃) groups to macromolecular substrates. RimO attaches a methylthio group at C3 of aspartate 89 of protein S12, a component of the 30<i>S</i> subunit of the bacterial ribosome. MiaB attaches a methylthio group at C2 of <i>N</i>⁶-(isopentenyl)­adenosine, found at nucleotide 37 in several prokaryotic tRNAs. These two enzymes are prototypical members of a subclass of radical SAM enzymes called methylthiotransferases (MTTases). It had been assumed that the sequence of steps in MTTase reactions involves initial sulfur insertion into the organic substrate followed by capping of the inserted sulfur atom with a SAM-derived methyl group. In this work, however, we show that both RimO and MiaB from Thermotoga maritima catalyze methyl transfer from SAM to an acid/base labile acceptor on the protein in the absence of their respective macromolecular substrates. Consistent with the assignment of the acceptor as an iron–sulfur cluster, denaturation of the SAM-treated protein with acid results in production of methanethiol. When RimO or MiaB is first incubated with SAM in the absence of substrate and reductant and then incubated with excess <i>S</i>-adenosyl-l-[<i>methyl</i>-<i>d</i>₃]­methionine in the presence of substrate and reductant, production of the unlabeled product precedes production of the deuterated product, showing that the methylated species is chemically and kinetically competent to be an intermediate

Electrochemical Resolution of the [4Fe-4S] Centers of the AdoMet Radical Enzyme BtrN: Evidence of Proton Coupling and an Unusual, Low-Potential Auxiliary Cluster

The <i>S</i>-adenosylmethionine (AdoMet) radical superfamily of enzymes includes over 113 500 unique members, each of which contains one indispensable iron–sulfur (FeS) cluster that is required to generate a 5′-deoxyadenosyl 5′-radical intermediate during catalysis. Enzymes within several subgroups of the superfamily, however, have been found to contain one or more additional FeS clusters. While these additional clusters are absolutely essential for enzyme activity, their exact roles in the function and/or mechanism of action of many of the enzymes are at best speculative, indicating a need to develop methods to characterize and study these clusters in more detail. Here, BtrN, an AdoMet radical dehydrogenase that catalyzes the two-electron oxidation of 2-deoxy-<i>scyllo</i>-inosamine to amino-dideoxy-<i>scyllo</i>-inosose, an intermediate in the biosynthesis of 2-deoxystreptamine antibiotics, is examined through direct electrochemistry, where the potential of both its AdoMet radical and auxiliary [4Fe-4S] clusters can be measured simultaneously. We find that the AdoMet radical cluster exhibits a midpoint potential of −510 mV, while the auxiliary cluster exhibits a midpoint potential of −765 mV, to our knowledge the lowest [4Fe-4S]^2+/+ potential to be determined to date. The impact of AdoMet binding and the pH dependence of catalysis are also quantitatively observed. These data show that direct electrochemical methods can be used to further elucidate the chemistry of the burgeoning AdoMet radical superfamily in the future

Evidence for Only Oxygenative Cleavage of Aldehydes to Alk(a/e)nes and Formate by Cyanobacterial Aldehyde Decarbonylases

Cyanobacterial aldehyde decarbonylases (ADs) catalyze the conversion of C_<i>n</i> fatty aldehydes to formate (HCO₂^–) and the corresponding C_<i>n</i>‑1 alk­(a/e)­nes. Previous studies of the <i>Nostoc punctiforme</i> (<i>Np</i>) AD produced in <i>Escherichia coli</i> (<i>Ec</i>) showed that this apparently hydrolytic reaction is actually a cryptically redox oxygenation process, in which one O-atom is incorporated from O₂ into formate and a protein-based reducing system (NADPH, ferredoxin, and ferredoxin reductase; N/F/FR) provides all four electrons needed for the complete reduction of O₂. Two subsequent publications by Marsh and co-workers [Das, et al. (2011) Angew. Chem. Int. Ed. 50, 7148−7152; Eser, et al. (2011) Biochemistry 50, 10743–10750] reported that their <i>Ec</i>-expressed <i>Np</i> and <i>Prochlorococcus marinus</i> (<i>Pm</i>) AD preparations transform aldehydes to the same products more rapidly by an O₂-independent, truly hydrolytic process, which they suggested proceeded by transient substrate reduction with obligatory participation by the reducing system (they used a chemical system, NADH and phenazine methosulfate; N/PMS). To resolve this discrepancy, we re-examined our preparations of both AD orthologues by a combination of (i) activity assays in the presence and absence of O₂ and (ii) ¹⁸O₂ and H₂¹⁸O isotope-tracer experiments with <i>direct</i> mass-spectrometric detection of the HCO₂^– product. For multiple combinations of the AD orthologue (<i>Np</i> and <i>Pm</i>), reducing system (protein-based and chemical), and substrate (<i>n</i>-heptanal and <i>n</i>-octadecanal), our preparations strictly require O₂ for activity and do not support detectable hydrolytic formate production, despite having catalytic activities similar to or greater than those reported by Marsh and co-workers. Our results, especially of the ¹⁸O-tracer experiments, suggest that the activity observed by Marsh and co-workers could have arisen from contaminating O₂ in their assays. The definitive reaffirmation of the oxygenative nature of the reaction implies that the enzyme, initially designated as aldehyde decarbonylase when the C1-derived coproduct was thought to be carbon monoxide rather than formate, should be redesignated as aldehyde-deformylating oxygenase (ADO)

NosN, a Radical <i>S</i>‑Adenosylmethionine Methylase, Catalyzes Both C1 Transfer and Formation of the Ester Linkage of the Side-Ring System during the Biosynthesis of Nosiheptide

Nosiheptide, a member of the <i>e</i> series of macrocyclic thiopeptide natural products, contains a side-ring system composed of a 3,4-dimethylindolic acid (DMIA) moiety connected to Glu6 and Cys8 of the thiopeptide backbone via ester and thioester linkages, respectively. Herein, we show that NosN, a predicted class C radical <i>S</i>-adenosylmethionine (SAM) methylase, catalyzes both the transfer of a C1 unit from SAM to 3-methylindolic acid linked to Cys8 of a synthetic substrate surrogate as well as the formation of the ester linkage between Glu6 and the nascent C4 methylene moiety of DMIA. In contrast to previous studies that indicated that 5′-methylthioadenosine is the immediate methyl donor in the reaction, in our studies, SAM itself plays this role, giving rise to <i>S</i>-adenosylhomocysteine as a coproduct of the reaction

Evidence for a Catalytically and Kinetically Competent Enzyme–Substrate Cross-Linked Intermediate in Catalysis by Lipoyl Synthase

Lipoyl synthase (LS) catalyzes the final step in lipoyl cofactor biosynthesis: the insertion of two sulfur atoms at C6 and C8 of an (<i>N</i>⁶-octanoyl)-lysyl residue on a lipoyl carrier protein (LCP). LS is a member of the radical SAM superfamily, enzymes that use a [4Fe–4S] cluster to effect the reductive cleavage of <i>S</i>-adenosyl-l-methionine (SAM) to l-methionine and a 5′-deoxyadenosyl 5′-radical (5′-dA^•). In the LS reaction, two equivalents of 5′-dA^• are generated sequentially to abstract hydrogen atoms from C6 and C8 of the appended octanoyl group, initiating sulfur insertion at these positions. The second [4Fe–4S] cluster on LS, termed the auxiliary cluster, is proposed to be the source of the inserted sulfur atoms. Herein, we provide evidence for the formation of a covalent cross-link between LS and an LCP or synthetic peptide substrate in reactions in which insertion of the second sulfur atom is slowed significantly by deuterium substitution at C8 or by inclusion of limiting concentrations of SAM. The observation that the proteins elute simultaneously by anion-exchange chromatography but are separated by aerobic SDS-PAGE is consistent with their linkage through the auxiliary cluster that is sacrificed during turnover. Generation of the cross-linked species with a small, unlabeled (<i>N</i>⁶-octanoyl)-lysyl-containing peptide substrate allowed demonstration of both its chemical and kinetic competence, providing strong evidence that it is an intermediate in the LS reaction. Mössbauer spectroscopy of the cross-linked intermediate reveals that one of the [4Fe–4S] clusters, presumably the auxiliary cluster, is partially disassembled to a 3Fe-cluster with spectroscopic properties similar to those of reduced [3Fe–4S]⁰ clusters

Rerouting the Pathway for the Biosynthesis of the Side Ring System of Nosiheptide: The Roles of NosI, NosJ, and NosK

Nosiheptide (NOS) is a highly modified thiopeptide antibiotic that displays formidable in vitro activity against a variety of Gram-positive bacteria. In addition to a central hydroxypyridine ring, NOS contains several other modifications, including multiple thiazole rings, dehydro-amino acids, and a 3,4-dimethylindolic acid (DMIA) moiety. The DMIA moiety is required for NOS efficacy and is synthesized from l-tryptophan in a series of reactions that have not been fully elucidated. Herein, we describe the role of NosJ, the product of an unannotated gene in the biosynthetic operon for NOS, as an acyl carrier protein that delivers 3-methylindolic acid (MIA) to NosK. We also reassign the role of NosI as the enzyme responsible for catalyzing the ATP-dependent activation of MIA and MIA’s attachment to the phosphopantetheine moiety of NosJ. Lastly, NosK catalyzes the transfer of the MIA group from NosJ-MIA to a conserved serine residue (Ser102) on NosK. The X-ray crystal structure of NosK, solved to 2.3 Å resolution, reveals that the protein is an α/β-fold hydrolase. Ser102 interacts with Glu210 and His234 to form a catalytic triad located at the bottom of an open cleft that is large enough to accommodate the thiopeptide framework

Characterization of a Radical Intermediate in Lipoyl Cofactor Biosynthesis

Lipoyl synthase (LipA) catalyzes the final step in the biosynthesis of the lipoyl cofactor, the insertion of two sulfur atoms at C6 and C8 of an <i>n</i>-octanoyl chain. LipA is a member of the radical <i>S</i>-adenosylmethionine (SAM) superfamily of enzymes and uses two [4Fe–4S] clusters to catalyze its transformation. One cluster binds in contact with SAM and donates the requisite electron for the reductive cleavage of SAM to generate two 5′-deoxyadenosyl 5′-radicals, which abstract hydrogen atoms from C6 and C8 of the substrate. By contrast, the second, auxiliary [4Fe–4S] cluster, has been hypothesized to serve as the sulfur donor in the reaction. Such a sacrificial role for an iron–sulfur cluster during catalysis has not been universally accepted. Use of a conjugated 2,4-hexadienoyl-containing substrate analogue has allowed the substrate radical to be trapped and characterized by continuous-wave and pulsed electron paramagnetic resonance methods. Here we report the observation of a ⁵⁷Fe hyperfine coupling interaction with the paramagnetic signal, which indicates that the iron–sulfur cluster of LipA and its substrate are within bonding distance

Enhanced Solubilization of Class B Radical <i>S</i>‑Adenosylmethionine Methylases by Improved Cobalamin Uptake in <i>Escherichia coli</i>

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 <i>S</i>-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 <i>in vitro</i> studies of these enzymes. It has been suggested that improper folding, possibly caused by insufficient cobalamin during their overproduction in <i>Escherichia coli</i>, 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 <i>E. coli</i> to take in more cobalamin. We cloned five genes (<i>btuC</i>, <i>btuE</i>, <i>btuD</i>, <i>btuF</i>, and <i>btuB</i>) that encode proteins that are responsible for cobalamin uptake and transport in <i>E. coli</i> 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