Purification and characterization of sequential cobalamin-dependent radical SAM methylases ThnK and TokK in carbapenem β-lactam antibiotic biosynthesis

Sinner, Erica K.; Townsend, Craig A.

doi:10.1016/bs.mie.2021.09.014

Cited by 5 publications

(9 citation statements)

References 29 publications

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“…TokK and ThnK were expressed and purified as reported previously. 41 Each enzyme (100 μM) was incubated with each substrate (1 mM) for 90 min and the formation of products was monitored by UPLC-HRMS. By using a substrate concentration 10-fold higher than the enzyme concentration, the first methylation in the sequence is the dominant reaction for each enzyme, giving Me-PCPM (2) as the major product.…”

Section: Resultsmentioning

confidence: 99%

Stereochemical course of cobalamin-dependent radical SAM methylation by TokK and ThnK

2022

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Section: Resultsmentioning

confidence: 99%

Stereochemical course of cobalamin-dependent radical SAM methylation by TokK and ThnK

2022

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“…3 A ) at 0.2 mg/L to 0.3 mg/L of expression culture. ThnL was anaerobically purified using methods similar to those developed for TokK and ThnK ( 25 ). Protein concentration was determined by the Bradford assay with a correction factor of 1.21 as determined by amino acid analysis.…”

Section: Resultsmentioning

confidence: 99%

“…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 – 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.…”

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ThnL, a B12-dependent radical S -adenosylmethionine enzyme, catalyzes thioether bond formation in carbapenem biosynthesis

Sinner

Marous

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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Complex carbapenems are important clinical antibiotics used to treat recalcitrant infections. Their biosynthetic gene clusters contain three essential B 12 -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 B 12 -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.

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“…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. As an added benefit, protein expression can often be conducted in standard LB medium supplemented with Cbl, rather than ethanolamine-M9 …”

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confidence: 99%

“…As an added benefit, protein expression can often be conducted in standard LB medium supplemented with Cbl, rather than ethanolamine-M9. 36 With soluble, cofactor-loaded ThnK in hand, attention was directed to determining its activity. Traditionally, dithionite had been used as an Fe−S cluster reductant with rSAM enzymes, although it elicited poor results from ThnK.…”

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Evolution of Methods for the Study of Cobalamin-Dependent Radical SAM Enzymes

Sinner

Marous

Townsend

2021

ACS Bio Med Chem Au

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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.

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Purification and characterization of sequential cobalamin-dependent radical SAM methylases ThnK and TokK in carbapenem β-lactam antibiotic biosynthesis

Cited by 5 publications

References 29 publications

Stereochemical course of cobalamin-dependent radical SAM methylation by TokK and ThnK

Stereochemical course of cobalamin-dependent radical SAM methylation by TokK and ThnK

ThnL, a B12-dependent radical S -adenosylmethionine enzyme, catalyzes thioether bond formation in carbapenem biosynthesis

Evolution of Methods for the Study of Cobalamin-Dependent Radical SAM Enzymes

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