Consecutive radical S -adenosylmethionine methylations form the ethyl side chain in thienamycin biosynthesis

Abstract

Despite their broad anti-infective utility, the biosynthesis of the paradigm carbapenem antibiotic, thienamycin, remains largely unknown. Apart from the first two steps shared with a simple carbapenem, the pathway sharply diverges to the more structurally complex members of this class of β-lactam antibiotics, such as thienamycin. Existing evidence points to three putative cobalamindependent radical S-adenosylmethionine (RS) enzymes, ThnK, ThnL, and ThnP, as potentially being responsible for assembly of the ethyl side chain at C6, bridgehead epimerization at C5, installation of the C2-thioether side chain, and C2/3 desaturation. The C2 substituent has been demonstrated to be derived by stepwise truncation of CoA, but the timing of these events with respect to C2-S bond formation is not known. We show that ThnK of the three apparent cobalamin-dependent RS enzymes performs sequential methylations to build out the C6-ethyl side chain in a stereocontrolled manner. This enzymatic reaction was found to produce expected RS methylase coproducts S-adenosylhomocysteine and 5′-deoxyadenosine, and to require cobalamin. For double methylation to occur, the carbapenam substrate must bear a CoA-derived C2-thioether side chain, implying the activity of a previous sulfur insertion by an as-yet unidentified enzyme. These insights allow refinement of the central steps in complex carbapenem biosynthesis.β-lactam antibiotics | carbapenem | radical SAM | cobalamin | methylase

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

“…Lastly, when ThnK is overproduced in E. coli harboring pBAD42-BtuCEDFB, its yield (7.1 mg/L) is enhanced dramatically over that obtained in previous isolations. 35 …”

“…33 Using this strategy, low milligram quantities of homogeneous enzyme could be purified from 32 L of E. coli culture, allowing for spectroscopic studies of the enzyme to be conducted. 34 A similar strategy proved successful for purifying soluble ThnK 35 —a class B RS methylase involved in the biosynthesis of thienomycin—and PoyC, a class B RS methylase involved in the biosynthesis of polytheonamide. 18, 36 …”

Vitamin B 12 in the spotlight again

“…Of particular interest is the reaction catalyzed by PhpK, which is responsible for the formation of a C-P bond linkage in a bialaphos precursor. This challenging methylation as well as other methylation reactions catalyzed by members of this enzyme class have been hypothesized to require both Cbl and AdoMet as shown in Figure 4a [36,37,39–41,48,49,51,52]. Unlike the traditional Cbl-dependent methyltransferase reactions, these enzymes are hypothesized to catalyze methylation of a substrate following homolytic Co-C bond cleavage of MeCbl; precedent for transfer of a methyl radical species comes from Cbl model chemistry [53].…”

A B12-dependent radical SAM enzyme involved in oxetanocin A biosynthesis

“…With over 7,000 AdoMet radical enzymes now annotated as Cbl-dependent, these dual cofactor enzymes are emerging as a new superfamily 7–16 . Characterized Cbl-dependent AdoMet radical enzymes catalyze methylation of unactivated C- and P-centers 10,12–14,17–20 , but not all reactions attributed to this family involve methylation 21 . Here we describe the first characterization of a non-methylating Cbl-dependent AdoMet radical enzyme and the first structure of a superfamily member.…”