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

Abstract

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.

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

“…34, 41 When a similar strategy was applied to TsrM overproduced in the E. coli BL21 (DE3) strain rather than the RosettaBlue (DE3) strain, only ~0.5 mg/L soluble protein that was approximately 25% pure was obtained (Figure S20). To determine if additional cobalamin in the cell could increase the amount of soluble TsrM, TsrM was overproduced as a fusion protein with SUMO as previously described, 34 but under conditions in which proteins encoded by the pBAD42-BtuCEDFB plasmid are present.…”

“…Purification of the SUMO-tagged enzyme was performed as previously described. 34, 41 Briefly, harvested cells were resuspended and lysed inside an anaerobic chamber. Target proteins were purified by an initial immobilized metal affinity chromatography (IMAC) step using Ni-NTA, followed by cleavage of the SUMO tag using the Ulp1 protease overnight on ice.…”

Structural basis for carbapenamC-alkylation by TokK, a B12-dependent radical SAM enzyme

“…TokK (Uniprot ID: A0A6B9HEI0) was produced heterologously in Escherichia coli BL21 (DE3) via overexpression from a pET29b:tokKTev construct (17). To facilitate production of soluble TokK protein with maximal occupancy of [4Fe-4S] cluster and cobalamin cofactors, the strain was transformed with two additional plasmids, pDB1282 and pBAD42-BtuCEDFB (17,(27)(28)(29). A 100 mL LB starter culture with 50 µg/mL kanamycin (pET29b) containing tokK), 50 µg/mL spectinomycin (pBAD42-BtuCEDFB), and 100 µg/mL ampicillin (pDB1282) was inoculated from a single colony and incubated for 18 h at 37 °C while shaking at 250 rpm.…”

C-Methylation of S-adenosyl-L-Methionine Occurs Prior to Cyclopropanation in the Biosynthesis of 1-Amino-2-Methylcyclopropanecarboxylic Acid (Norcoronamic Acid) in a Bacterium

“…The resulting transformant, EcSufBtu_orf29, was inoculated into LB medium, containing 50 µg/mL kanamycin, 100 µg/mL ampicillin, and 5 µg/mL tetracycline. After growth overnight at 37 • C, the culture (1 mL) was inoculated into 200 mL of M9-ethanolamine medium [24], containing 50 µg/mL kanamycin, 100 µg/mL ampicillin, and 5 µg/mL tetracycline, and incubated at 37 • C with 200 rpm agitation until the OD600 = 0.2. L-Arabinose was added to a final concentration of 1 mg/mL, followed by re-cultivation until the OD600 = 0.6.…”