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Biosynthesis of thienamycin and activity of ThnK. Two enzymes, ThnE and ThnM, form the (3 S, 5 S )-carbapenam 2 , which is then acted on by other enzymes, likely including ThnK, ThnL, and ThnP, to form thienamycin. Correspondingly, CarB and CarA can also form 2 , which CarC can convert to the simple carbapenem 3 . The demonstrated activity of ThnK is highlighted in gray.
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The antibacterial effectiveness of penicillins and cephalosporins is increasingly challenged by bacterial resistance, highlighting the increasing clinical importance of the carbapenems as the most potent, broad-spectrum class of β-lactam antibiotics. The carbapenems are produced commercially by total synthesis, rather than by lower-cos...
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... intermediates in carbapenem biosynthesis, more clearly defining the enigmatic central steps of the pathway and exemplifying an unprecedented reaction sequence among RS enzymes. pathway by identifying substrates for ThnK and establishing that this enzyme performs two consecutive methylations to generate the C6-ethyl side chain stereospecifically (Fig. 1). Although other RS cobalamin-dependent enzymes that act on carbon have been investigated previously (9)(10)(11)(12), to our knowledge this is the first in vitro characterization of a RS methylase acting sequentially to form a side ...
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... conducted in ethanolamine-M9 medium, which facilitates uptake of externally supplied hydroxocobalamin (HOCbl) into E. coli, and with con- current expression of the Azotobacter vinelandii isc operon, encoded on plasmid pDB1282. ThnK was purified under strictly anaerobic conditions. Its UV-visible spectrum revealed a 420-nm shoulder (SI Appendix, Fig. S1) typical of a bound Fe/S cluster. The relative stoichiometry of iron and sulfide bound by the as-isolated protein was determined to be 7.4 ± 1.4 and 3.7 ± 0.8 per polypeptide, respectively, consistent with the presence of a [4Fe-4S] cluster. Apparent excess iron has been observed with other RS enzymes (11,14), and was similarly seen in ...
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Radical S-adenosylmethionine (SAM) (RS) methylases perform methylation reactions at unactivated carbon and phosphorus atoms. RS enzymes typically abstract a hydrogen from their substrates, generating a substrate-centered radical; class B RS methylases catalyze methyl transfer from SAM to cobalamin and then to a substrate-centered carbon or phosphor...
The members of the radical S-adenosylmethionine (SAM) enzyme superfamily are responsible for catalyzing a diverse set of reactions in a multitude of biosynthetic pathways. Many members of this superfamily accomplish their transformations using the catalytic power of a 5'-deoxyadenosyl radical (5'-dAdo•), but there are also enzymes within this super...
![Figure 1. (A) Reductive cleavage of SAM by a [4Fe−4S] cluster typical...](publication/355225815/figure/fig1/AS:1079283816636420@1634332722888/A-Reductive-cleavage-of-SAM-by-a-4Fe-4S-cluster-typical-of-radical-SAM-enzymes-B_Q320.jpg)
While bioinformatic evidence of cobalamin-dependent radical S-adenosylmethionine (SAM) enzymes has existed since the naming of the radical SAM superfamily in 2001, none were biochemically characterized until 2011. In the past decade, the field has flourished as methodological advances have facilitated study of the subfamily. Because of the ingenuit...
Citations
... However, irrespective of length, the C6 alkyl chain is constructed by a single Cbl-dependent rSAM methylase. Two of these sequential methylases have been characterized in detail, namely, ThnK (22) and TokK (23)(24)(25), which are involved in the biosynthesis of thienamycin (2) and asparenomycin (5), respectively. This finding rendered it probable that the other two Cbl-dependent rSAM enzymes in thienamycin biosynthesis, ThnL and ThnP, have nonmethylase function. ...
... To further validate this result, an analogous strain containing ThnE, ThnM, and ThnK was constructed (SI Appendix, Fig S3C). ThnK catalyzes sequential methylations only on C2-PantSHcontaining substrates and does not methylate carbapenam 3 (22). As expected, the TK24-EMK strain produced levels of 3 comparable to the TK24-EM strain regardless of OHCbl supplementation, as visualized by the bioassay (Fig. 2). ...
... The installation of PantSH onto carbapenam 3 suggests that thioether bond formation by ThnL is the only enzymatic step between bicycle formation by ThnM and C6 alkylation by ThnK. This conclusion is in accord with the fact that ThnK methylation is more efficient with the 2R diastereomer than its endo 2S counterpart (22). ...
Complex carbapenems are important clinical antibiotics used to treat recalcitrant infections. Their biosynthetic gene clusters contain three essential B12-dependent radical S-adenosylmethionine (rSAM) enzymes. The majority of characterized enzymes in this subfamily catalyze methyl transfer, but only one is required to sequentially install all methionine-derived carbons in complex carbapenems. Therefore, it is probable that the other two rSAM enzymes have noncanonical functions. Through a series of fermentation and in vitro experiments, we show that ThnL uses radical SAM chemistry to catalyze thioether bond formation between C2 of a carbapenam precursor and pantetheine, uniting initial bicycle assembly common to all carbapenems with later tailoring events unique to complex carbapenems. ThnL also catalyzes reversible thiol/disulfide redox on pantetheine. Neither of these functions has been observed previously in a B12-dependent radical SAM enzyme. ThnL expands the known activity of this subclass of enzymes beyond carbon-carbon bond formation or rearrangement. It is also the only radical SAM enzyme currently known to catalyze carbon-sulfur bond formation with only an rSAM Fe-S cluster and no additional auxiliary clusters.
... This enzyme family canonically uses SAM, methylcobalamin, and a [4Fe-4S] 2+ cluster to initiate methyl transfer onto nonnucleophilic phosphorous or carbon atoms (15). Only a few B 12 -dependent rSAM methylation enzymes have been characterized in vitro (16)(17)(18)(19)(20)(21)(22)(23)(24). TmoD was expressed aerobically in E. coli as a recombinant His 6 -fusion protein that did not contain cobalamin and a [4Fe-4S] 2+ cluster. ...
... These findings support the structure of peptide 4 as having an isobutyl chain connected to the sulfur atom (Fig. 4B). Other B 12 -dependent rSAM enzymes that methylate their substrates consecutively in vitro include CysS (20,27), ThnK (18), and TokK (24, 28) (SI Appendix, Fig. S14). CysS methylates the methoxy group of its substrate up to three times to generate a tert-butoxy group, and ThnK installs the C6-ethyl chain of thienamycin. ...
A subset of natural products, such as polyketides and nonribosomal peptides, is biosynthesized while tethered to a carrier peptide via a thioester linkage. Recently, we reported that the biosyntheses of 3-thiaglutamate and ammosamide, single amino acid-derived natural products, employ a very different type of carrier peptide to which the biosynthetic intermediates are bound via an amide linkage. During their biosyntheses, a peptide aminoacyl-transfer ribonucleic acid (tRNA) ligase (PEARL) first loads an amino acid to the C terminus of the carrier peptide for subsequent modification by other enzymes. Proteolytic removal of the modified C-terminal amino acid yields the mature product. We termed natural products that are biosynthesized using such pathways pearlins. To investigate the diversity of pearlins, in this study we experimentally characterized another PEARL-encoding biosynthetic gene cluster (BGC) from Tistrella mobilis (tmo). The enzymes encoded in the tmo BGC transformed cysteine into 3-thiahomoleucine both in vitro and in Escherichia coli. During this process, a cobalamin-dependent radical S-adenosylmethionine (SAM) enzyme catalyzes C-isopropylation. This work illustrates that the biosynthesis of amino acid-derived natural products on a carrier peptide is a widespread strategy in nature and expands the spectrum of thiahemiaminal analogs of amino acids that may serve a broader, currently unknown function.
... 39 To further characterize the multiple methylations of TokK, time course experiments were performed to compare the relative rates of methylation between TokK and another carbapenem Cbldependent AdoMet radical methylase (ThnK) that accepts the same substrate but only performs two methylations to form an ethyl substituent en route to thienamycin. 40 The individually methylated intermediates from both TokK and ThnK were detected by UPLC-HRMS and a kinetic model and simulation curves were created with the tools COPASI and VCell. 39,41,42 The authors concluded that TokK performs its first and second methylation events 2-fold and 10-fold faster than ThnK based on modeling of the experimental data to obtain relative rate constants. ...
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.
... In addition, a hydrophobic pocket prevents water from converting the pentacoordinated MeCbl into its more stabilized hexacoordinated counterpart, which is a strategy conserved in other B 12 -dependent radical SAM enzymes. These enzymes thus appear to have evolved unique structures and mechanisms to alkylate sp 2 -and sp 3 -hybridized carbon atoms using the twin catalytic power of the cobalamin and SAM cofactors 8,13,14,[16][17][18][19]21,50,51 . In contrast to catalysis by known radical SAM enzymes, catalysis by Mmp10 requires active site reorganization and SAM flexibility within the active site. ...
By catalysing the microbial formation of methane, methyl-coenzyme M reductase has a central role in the global levels of this greenhouse gas1,2. The activity of methyl-coenzyme M reductase is profoundly affected by several unique post-translational modifications3–6, such as a unique C-methylation reaction catalysed by methanogenesis marker protein 10 (Mmp10), a radical S-adenosyl-l-methionine (SAM) enzyme7,8. Here we report the spectroscopic investigation and atomic resolution structure of Mmp10 from Methanosarcina acetivorans, a unique B12 (cobalamin)-dependent radical SAM enzyme9. The structure of Mmp10 reveals a unique enzyme architecture with four metallic centres and critical structural features involved in the control of catalysis. In addition, the structure of the enzyme–substrate complex offers a glimpse into a B12-dependent radical SAM enzyme in a precatalytic state. By combining electron paramagnetic resonance spectroscopy, structural biology and biochemistry, our study illuminates the mechanism by which the emerging superfamily of B12-dependent radical SAM enzymes catalyse chemically challenging alkylation reactions and identifies distinctive active site rearrangements to provide a structural rationale for the dual use of the SAM cofactor for radical and nucleophilic chemistry. Structural and spectroscopic studies show how a B12-dependent radical SAM enzyme catalyses unique and challenging alkylation chemistry, including protein post-translational modification required for methane biosynthesis.
... ThnK and TokK share 79.3% sequence identity and act on the same substrate, (2R)-pantetheinylated carbapenam ( Supplementary Fig. 7), but ThnK performs two sequential methylations whereas TokK catalyses three 18,27 . Nearly all residues in proximity to the active site are identical in the two orthologues. ...
... Synthesis of the TokK substrate, (2R,3R,5R)-3-((2-(3-((R)-2,4-dihydroxy-3, 3-dimethylbutanamido)propanamido)ethyl)thio)-7-oxo-1-azabicyclo [3.2.0]heptane-2-carboxylic acid (1), was carried out as previously described 27 . Characterization matched that previously reported. ...
Carbapenems are antibiotics of last resort in the clinic. Owing to their potency and broad-spectrum activity, they are an important part of the antibiotic arsenal. The vital role of carbapenems is exemplified by the approval acquired by Merck from the US Food and Drug Administration (FDA) for the use of an imipenem combination therapy to treat the increased levels of hospital-acquired and ventilator-associated bacterial pneumonia that have occurred during the COVID-19 pandemic1. The C6 hydroxyethyl side chain distinguishes the clinically used carbapenems from the other classes of β-lactam antibiotics and is responsible for their low susceptibility to inactivation by occluding water from the β-lactamase active site2. The construction of the C6 hydroxyethyl side chain is mediated by cobalamin- or B12-dependent radical S-adenosylmethionine (SAM) enzymes3. These radical SAM methylases (RSMTs) assemble the alkyl backbone by sequential methylation reactions, and thereby underlie the therapeutic usefulness of clinically used carbapenems. Here we present X-ray crystal structures of TokK, a B12-dependent RSMT that catalyses three-sequential methylations during the biosynthesis of asparenomycin A. These structures, which contain the two metallocofactors of the enzyme and were determined in the presence and absence of a carbapenam substrate, provide a visualization of a B12-dependent RSMT that uses the radical mechanism that is shared by most of these enzymes. The structures provide insight into the stereochemistry of initial C6 methylation and suggest that substrate positioning governs the rate of each methylation event. X-ray crystal structures of TokK, a cobalamin- or B12-dependent radical SAM methylase, provide insight into how these enzymes use sequential radical-mediated methylations to assemble the C6 side chain of carbapenem antibiotics.
... Cobalamin (Cbl) is a cobalt-containing tetrapyrrole and can participate in both one-and two-electron processes since the cobalt center can accommodate oxidation states from 1 + to 3 +. While more than 9700 UniRef50 sequence clusters have been annotated as B 12 -dependent radical SAM enzymes (Oberg, Precord, Mitchell, & Gerlt, 2021), only a handful have been characterized (Allen & Wang, 2014a;Bridwell-Rabb, Zhong, Sun, Drennan, & Liu, 2017;Huang et al., 2015;Kim, Liu, McCarty, & Liu, 2017;Kim et al., 2013;Marous et al., 2015;Maruyama et al., 2020;Parent et al., 2016;Pierre et al., 2012;Radle, Miller, Laremore, & Booker, 2019;Sinner, Lichstrahl, Li, Marous, & Townsend, 2019;Wang, Schnell, Baumann, Müller, & Begley, 2017;Werner et al., 2011). Moreover, only three B 12 -dependent radical SAM enzymes, OxsB (Bridwell-Rabb et al., 2017), TsrM and TokK (Knox, Sinner, Townsend, Boal, & Booker, 2021), have been structurally characterized to date. ...
The B12-dependent radical SAM enzymes are an emerging subgroup of biological catalysts that bind a cobalamin cofactor in addition to the canonical [Fe4S4] cluster characteristic of radical SAM enzymes. Most of the B12-dependent radical SAM enzymes that have been characterized mediated methyltransfer reactions; however, a small number are known to catalyze more diverse reactions such as ring contractions. Thus, Genk is a methyltransferase from the gentamicin C biosynthetic pathway, whereas OxsB catalyzes the oxidative ring contraction of 2′-deoxyadenosine 5′-phosphates to generate an oxetane aldehyde during the biosynthesis of oxetanocin A. The preparation and in vitro characterization of such enzymes is complicated by the presence of two redox sensitive cofactors in addition to challenges in obtaining soluble protein for study. This chapter describes expression, purification and assay methodologies for GenK and OxsB highlighting the use of denaturation/refolding protocols for solubilizing inclusion bodies as well as the use of cluster assembly and cobalamin uptake machinery during in vivo expression.
... (B) Like TsrM, CloN6 and CouN6 have been proposed to use their cofactors to catalyze a non-radical-based methylation reaction. 23 (C) Analogous to TokK, ThnK, 33 CysS, 34 Swb9, 36 BchQ, 37 and PoyB 38 have been suggested to catalyze multiple radical-based methylation reactions. In the top panel, two methyl groups are added by ThnK (one to the β-lactam ring and then one to the resultant methyl group, which is shown in pink). ...
... Akin to the reaction catalyzed by TokK, enzymes that include ThnK, 33 CysS, 34 PctJ, 35 Swb9, 36 BchQ, 37 and PoyB 38 appear to catalyze multiple methylation reactions on a substrate ( Figure 2C). ThnK methylates its substrate's β-lactam ring and then methylates the initially added methyl group, 33 and CysS catalyzes iterative methylation reactions on a methyl group to form ethyl-, isopropyl-, sec-butyl-, and tertbutyl-containing cystobactamids. ...
... Akin to the reaction catalyzed by TokK, enzymes that include ThnK, 33 CysS, 34 PctJ, 35 Swb9, 36 BchQ, 37 and PoyB 38 appear to catalyze multiple methylation reactions on a substrate ( Figure 2C). ThnK methylates its substrate's β-lactam ring and then methylates the initially added methyl group, 33 and CysS catalyzes iterative methylation reactions on a methyl group to form ethyl-, isopropyl-, sec-butyl-, and tertbutyl-containing cystobactamids. 34 These enzymes, along with PctJ and Swb9, cluster together in the SSN ( Figure 1B). ...
The members of the radical S-adenosylmethionine (SAM) enzyme superfamily are responsible for catalyzing a diverse set of reactions in a multitude of biosynthetic pathways. Many members of this superfamily accomplish their transformations using the catalytic power of a 5'-deoxyadenosyl radical (5'-dAdo•), but there are also enzymes within this superfamily that bind auxiliary cofactors and extend the catalytic repertoire of SAM. In particular, the cobalamin (Cbl)-dependent class synergistically uses Cbl to facilitate challenging methylation and radical rearrangement reactions. Despite identification of this class by Sofia et al. 20 years ago, the low sequence identity between members has led to difficulty in predicting function of uncharacterized members, pinpointing catalytic residues, and elucidating reaction mechanisms. Here, we capitalize on the three recent structures of Cbl-dependent radical SAM enzymes that use common cofactors to facilitate ring contraction as well as radical-based and non-radical-based methylation reactions. With these three structures as a framework, we describe how the Cbl-dependent radical SAM enzymes repurpose the traditional SAM- and Cbl-binding motifs to form an active site where both Cbl and SAM can participate in catalysis. In addition, we describe how, in some cases, the classic SAM- and Cbl-binding motifs support the diverse functionality of this enzyme class, and finally, we define new motifs that are characteristic of Cbl-dependent radical SAM enzymes.
... These enzymes present myriad experimental challenges, including oxygen sensitivity and low solubility upon heterologous expression (Lanz et al., 2018). We have made significant progress in developing experimental methods that allow the study of one of the three enzymes, ThnK, which is involved in thienamycin (1) biosynthesis (Marous et al., 2015), and it's ortholog TokK, which plays a similar role in asparenomycin (2) biosynthesis (Kawamura, Yasuda, Mayama, & Tanaka, 1982;Sinner, Lichstrahl, Li, Marous, & Townsend, 2019) (Fig. 1). Both enzymes have the distinctive ability to sequentially transfer multiple methyl groups onto their substrates, a trait shared by another recently characterized Cbl-dependent rSAM CysS (Wang, Schnell, Baumann, Müller, & Begley, 2017). ...
... The MeCbl species is then attacked by the substrate radical producing a methylated product and Cob(II)alamin, which requires a one-electron reduction to return to its highly nucleophilic Co(I) form for another turnover (Bridwell-Rabb, Grell, & Drennan, 2018;Kim et al., 2013). Both ThnK and TokK produce alkylated product, S-adenosylhomocysteine (SAH), and 5′-deoxyadenosine (5′dAH) in an approximately 1:1:1 ratio (Marous et al., 2015;Sinner et al., 2019), consistent with this mechanism. Perhaps because of the complexity of the cycle, catalysis by ThnK and TokK is slow, but we have developed conditions where the enzymes remain active for up to 48 h, enabling time-course analysis of the sequential methylation reactions. ...
ThnK and TokK are cobalamin-dependent radical S-adenosylmethionine enzymes that catalyze sequential methylations of a common carbapenem biosynthetic intermediate. ThnK was an early characterized member of the subfamily of cobalamin-dependent radical S-adenosylmethionine enzymes. Since initial publication of the ThnK function, the field has progressed, and we have made methodological strides in the expression and purification of this enzyme and its ortholog TokK. An optimized protocol for obtaining the enzymes in pure and active form has enabled us to characterize their reactions and gain greater insight into the kinetic behavior of the sequential methylations they catalyze. We share here the methods and strategy that we have developed through our study of these enzymes.
... Therefore, the construction of chassis strains that express heterologous FeS maturation pathways can be a strategy to restore the function of foreign FeS enzymes. For instance, the over-expression of the ISC operon from Azetobacter vinelandii is a common approach used by biochemistry groups to ensure functional expression of foreign FeS enzymes [16,47,49]. Yet, using the A. vinelandii pathway is not sufficient to deliver FeS clusters to all FeS. ...
Microbes are routinely engineered to synthesize high-value chemicals from renewable materials through synthetic biology and metabolic engineering. Microbial biosynthesis often relies on expression of heterologous biosynthetic pathways, i.e., enzymes transplanted from foreign organisms. Metallocluster enzymes are one of the most ubiquitous family of enzymes involved in natural product biosynthesis and are of great biotechnological importance. However, the functional expression of recombinant metallocluster enzymes in live cells is often challenging and represents a major bottleneck. The activity of metallocluster enzymes requires essential supporting pathways, involved in protein maturation, electron supply, and/or enzyme stability. Proper function of these supporting pathways involves specific protein-protein interactions that remain poorly characterized and are often overlooked by traditional synthetic biology approaches. Consequently, engineering approaches that focus on enzymatic expression and carbon flux alone often overlook the particular needs of metallocluster enzymes. This review highlights the biotechnological relevance of metallocluster enzymes and discusses novel synthetic biology strategies to advance their industrial application, with a particular focus on iron-sulfur cluster enzymes. Strategies to enable functional heterologous expression and enhance recombinant metallocluster enzyme activity in industrial hosts include: (1) optimizing specific maturation pathways; (2) improving catalytic stability; and (3) enhancing electron transfer. In addition, we suggest future directions for developing microbial cell factories that rely on metallocluster enzyme catalysis.
... 10,11 Coexpression of pDB1282 in ethanolamine-M9 medium ultimately was sufficient to obtain active ThnK. 34 In another pivotal step forward, the Booker lab has recently developed the plasmid pBAD42-BtuCEDFB, 35 which encodes a Cbl-uptake system. Coexpression of pBAD42-BtuCEDFB significantly improves the solubility of several Cbl-dependent rSAM enzymes and will likely enable the study of previously inaccessible members of this protein family. ...
... In the end, we employed a screening approach to deduce the substrate and catalytic activity of ThnK. 34 Initially, a library of potential substrates was synthesized and assessed in batches. LC-MS detection was focused on the SAM coproducts, 5′-dAH and SAH, rather than the reaction product. ...
... 31,39 We used the appearance of 5′-dAH and SAH to deduce the function and substrate preference of ThnK and upon closer inspection found that approximately one equivalent of each coproduct is formed during each methyl transfer event. 34 This 1:1:1 stoichiometry was first observed during the initial characterization of GenK. 22 It is a nearly unifying characteristic of Cbl-dependent rSAM methylases and supports the mechanism shown in Figure 1C. ...
While bioinformatic evidence of cobalamin-dependent radical S-adenosylmethionine (SAM) enzymes has existed since the naming of the radical SAM superfamily in 2001, none were biochemically characterized until 2011. In the past decade, the field has flourished as methodological advances have facilitated study of the subfamily. Because of the ingenuity and perseverance of researchers in this field, we now have functional, mechanistic, and structural insight into how this class of enzymes harnesses the power of both the cobalamin and radical SAM cofactors to achieve catalysis. All of the early characterized enzymes in this subfamily were methylases, but the activity of these enzymes has recently been expanded beyond methylation. We anticipate that the characterized functions of these enzymes will become both better understood and increasingly diverse with continued study.
