Methylations in complex carbapenem biosynthesis are catalyzed by a single cobalamin-dependent radicalS-adenosylmethionine enzyme

Abstract

TokK and ThnK are cobalamin-dependent radical SAM enzymes that methylate a carbapenem precursor three or two times, respectively, in a sequential, non-processive fashion.

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

“…C6 of 1 is located 4.2 Å above the axial ligand of Cbl (Figure 2D) at an angle of ~85° relative to the 5'-carbon of 5'-dAH. This arrangement suggests that the methyl group adds to the bottom face of the b-lactam ring, consistent with the absolute configurations observed in the TokK products and thienamycin (17,18). The distance and orientation of reactant functional groups in TokK also compares favorably to other enzymes that catalyze radical-mediated activation and functionalization of a substrate C-H bond, such as iron-dependent hydroxylases in the cytochrome P450 and 2-oxo-glutarate-(2OG)-dependent superfamilies (Figure S12).…”

“…The interactions between TokK and substrate 1 position the b-lactam appropriately both for activation of C6 by 5'-dA• and subsequent methyl addition by the Cbl cofactor (Figure 2D) (17). C6 of 1 is located directly in front of the 5'-carbon of 5'-dAH, 3.7 Å away, similar to other RS enzyme-substrate complexes that initiate reactions by H-atom abstraction.…”

“…ThnK and TokK share 79.3% sequence identity and act on the same substrate, (2R)pantetheinylated carbapenam (Figure S15), but ThnK performs two sequential methylations while TokK catalyzes three (17,26). Nearly all of the residues in proximity to the active site are identical in the two orthologs.…”

“…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.…”

“…Methylation Assays of TokK Variants. Expression and purification of TokK variants was carried out as previously described for wild-type TokK (17), except reconstitution (29) was done concurrently with overnight TEV protease cleavage, and an additional buffer exchange was done using an Econo-Pac 10DG column (Bio-Rad) to remove excess reconstitution reagents.…”