Biosynthesis of Branched Alkoxy Groups: Iterative Methyl Group Alkylation by a Cobalamin-Dependent Radical SAM Enzyme

Wang, Yuanyou; Schnell, Bastien; Baumann, Sascha; Müller, Rolf; Begley, Tadhg P.

doi:10.1021/jacs.6b10901

Cited by 55 publications

(64 citation statements)

References 23 publications

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“…It must be mentioned, however, that not all cobalamin-dependent RS enzymes are difficult to purify from soluble crude lysate, as has been shown from in vitro studies of CysS 70 and OxsB. 71 Indeed, Sato et al were able to purify Fom3 from S. wedmorenesis ; however, their yields were not reported.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2018

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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, B, C, and 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 coexpressed 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 cobalamin uptake into the cytoplasm and improves the solubility of the target enzymes significantly.

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Section: Resultsmentioning

confidence: 99%

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

et al. 2018

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“…Y. Kim et al, 2008), clorobiocin (Anderle et al, 2007), fortimicin (Dairi, Ohta, Hashimoto, & Hasegawa, 1992; Kuzuyama, Seki, Dairi, Hidaka, & Seto, 1995), thiostrepton (Kelly, Pan, & Li, 2009), chondrochloren (Rachid, Scharfe, Blocker, Weissman, & Muller, 2009), polytheonamide (Parent et al, 2016), cystobactamids (Baumann et al, 2014; Wang, Schnell, Baumann, Muller, & Begley, 2017), watasemycin (Inahashi et al, 2017), and bialaphos (Hidaka, Hidaka, Kuzuyama, & Seto, 1995; Kamigiri, Hidaka, Imai, Murakami, & Seto, 1992). However, mechanistic investigations of these enzymes have been limited, due in large part to their inherent insolubility upon overproduction in E. coli .…”

Section: Introductionmentioning

confidence: 99%

“…Three other Class B methylases, PoyC, ThnK, and TsrM, were amenable to purification from soluble crude lysate in an active form that contained both cofactors (Blaszczyk et al, 2016; Marous et al, 2015; Parent et al, 2016), but only when overproduced in a specialized medium containing ethanolamine, which has been suggested to increase cobalamin uptake in E. coli (Bandarian & Matthews, 2004). Lastly, another Class B RS methylase, CysS, has been purified from soluble crude lysate and reconstituted with cobalamin to yield an active form of the enzyme that contains the Fe-S cluster as well as cobalamin (Wang et al, 2017). …”

Section: Introductionmentioning

confidence: 99%

TsrM as a Model for Purifying and Characterizing Cobalamin-Dependent Radical S -Adenosylmethionine Methylases

Blaszczyk¹,

Wang²,

Booker³

2017

Methods in Enzymology

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Cobalamin-dependent radical S-adenosylmethionine (SAM) methylases play vital roles in the de novo biosynthesis of many antibiotics, cofactors, and other important natural products, yet remain an understudied subclass of radical SAM (RS) enzymes. In addition to a [4Fe-4S] cluster that is ligated by three cysteine residues, these enzymes also contain an N-terminal cobalamin-binding domain. In vitro studies of these enzymes have been severely limited because many are insoluble or sparingly soluble upon their overproduction in E. coli. This solubility issue has led a number of groups either to purify the protein from inclusion bodies or to purify soluble protein that often lacks proper cofactor incorporation. Herein, we use TsrM as a model to describe methods that we have used to generate soluble protein that is purified in an active form with both cobalamin and [4Fe-4S] cluster cofactors bound. Additionally, we highlight the methods that we developed to characterize the enzyme following purification.

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“…18 Class A includes the RNA-modifying enzymes RlmN and Cfr. 19,20 Class B comprises cobalamin-dependent methyltransferases, such as TsrM, 21,22 Fom3, 23,24 GenK, 25 CysS, 26 and PoyC. 27 Class C are cobalamin-independent and found in a variety of natural product BGCs.…”

mentioning

confidence: 99%

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

et al. 2017

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Thiomuracin is a thiopeptide antibiotic with potent activity towards 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.

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Biosynthesis of Branched Alkoxy Groups: Iterative Methyl Group Alkylation by a Cobalamin-Dependent Radical SAM Enzyme

Cited by 55 publications

References 23 publications

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

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

TsrM as a Model for Purifying and Characterizing Cobalamin-Dependent Radical S -Adenosylmethionine Methylases

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

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