title: 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 cobalamin-dependent 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.
authors.affiliation: Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205;
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Marous
authors.affiliation: Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218;
authors.forename: Evan P
authors.initials: EP
authors.lastname: Lloyd
authors.affiliation: Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218;
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Buller
authors.affiliation: Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218;
authors.forename: Kristos A
authors.initials: KA
authors.lastname: Moshos
authors.affiliation: Department of Chemistry, The Pennsylvania State University, University Park, PA 16802;
authors.forename: Tyler L
authors.initials: TL
authors.lastname: Grove
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Blaszczyk
authors.affiliation: Department of Chemistry, The Pennsylvania State University, University Park, PA 16802; Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802.
authors.forename: Squire J
authors.initials: SJ
authors.lastname: Booker
authors.affiliation: Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218; ctownsend@jhu.edu.
authors.forename: Craig A
authors.initials: CA
authors.lastname: Townsend
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry; D015780:Carbapenems / Q000737:chemistry*; D002384:Catalysis; D002511:Cephalosporins / Q000737:chemistry; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D019175:DNA Methylation; D015195:Drug Design; D004926:Escherichia coli; D005285:Fermentation; D008745:Methylation; D010406:Penicillins / Q000737:chemistry; D012436:S-Adenosylmethionine / Q000737:chemistry; D013302:Streptomyces; D053719:Tandem Mass Spectrometry; D013845:Thienamycins / Q000096:biosynthesis* / Q000737:chemistry; D014805:Vitamin B 12 / Q000737:chemistry; D047090:beta-Lactams / Q000737:chemistry
chemical_list: D000900:Anti-Bacterial Agents; D015780:Carbapenems; D002511:Cephalosporins; D010406:Penicillins; D013845:Thienamycins; D047090:beta-Lactams; D012436:S-Adenosylmethionine; D014805:Vitamin B 12; C015537:thienamycin
keywords: carbapenem; cobalamin; methylase; radical SAM; β-lactam antibiotics