Mechanistic Diversity of Radical S-Adenosylmethionine (SAM)-dependent Methylation

107

All of the heme-degrading enzymes that have been characterized to date require molecular oxygen as a cosubstrate. Escherichia coli O157:H7 has been shown to express heme uptake and transport proteins, as well as use heme as an iron source. This enteric pathogen colonizes the anaerobic space of the lower intestine in mammals, yet no mechanism for anaerobic heme degradation has been reported. Herein we provide evidence for an oxygen-independent heme-degradation pathway. Specifically, we demonstrate that ChuW is a radical S-adenosylmethionine methyltransferase that catalyzes a radical-mediated mechanism facilitating iron liberation and the production of the tetrapyrrole product we termed “anaerobilin.” We further demonstrate that anaerobilin can be used as a substrate by ChuY, an enzyme that is coexpressed with ChuW in vivo along with the heme uptake machinery. Our findings are discussed in terms of the competitive advantage this system provides for enteric bacteria, particularly those that inhabit an anaerobic niche in the intestines.

Section: Significancementioning

confidence: 99%

Radical new paradigm for heme degradation in Escherichia coli O157:H7

LaMattina

Nix

Lanzilotta

2016

107

“…To determine whether any of our library compounds was a substrate for ThnK, we developed an LC-MS analytical screen whereby enzymatic reactions were monitored for appearance of the expected RS methylase coproducts, S-adenosylhomocysteine (SAH) and 5′-deoxyadenosine (5′-dA) (15,16). Initial screens were conducted with ThnK, S-adenosylmethionine (SAM), and potential substrates in the presence of methyl viologen and NADPH as the Fe/S cluster reductants (10).…”

Section: Significancementioning

confidence: 99%

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

Marous

Lloyd

Buller

et al. 2015

Self Cite

113

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

“…This feature sets the NifB proteins apart from the RlmN and Cfr proteins (Fig. S5A), which are emerging as a distinct class (class A) of a larger subset of radical SAM methyltransferases (RSMTs) that use conserved protein residues to facilitate methyltransfer (21,22). The three NifB homologs are further distinguished from the other known classes of RSMTs (classes B-D), with classes B, C, and D carrying a cobalamin-binding domain, a HemN domain, and a methylenetetrahydrofolate domain, respectively, in addition to the canonical radical SAM domain (Fig.…”

mentioning

confidence: 99%

Identification and characterization of functional homologs of nitrogenase cofactor biosynthesis protein NifB from methanogens

Fay

Wiig

Lee

et al. 2015

Nitrogenase biosynthesis protein NifB catalyzes the radical S-adenosyl-L-methionine (SAM)-dependent insertion of carbide into the M cluster, the cofactor of the molybdenum nitrogenase from Azotobacter vinelandii. Here, we report the identification and characterization of two naturally "truncated" homologs of NifB from Methanosarcina acetivorans (NifB Ma ) and Methanobacterium thermoautotrophicum (NifB Mt ), which contain a SAM-binding domain at the N terminus but lack a domain toward the C terminus that shares homology with NifX, an accessory protein in M cluster biosynthesis. NifB Ma and NifB Mt are monomeric proteins containing a SAM-binding [Fe 4 S 4 ] cluster (designated the SAM cluster) and a [Fe 4 S 4 ]-like cluster pair (designated the K cluster) that can be processed into an [Fe 8 S 9 ] precursor to the M cluster (designated the L cluster). Further, the K clusters in NifB Ma and NifB Mt can be converted to L clusters upon addition of SAM, which corresponds to their ability to heterologously donate L clusters to the biosynthetic machinery of A. vinelandii for further maturation into the M clusters. Perhaps even more excitingly, NifB Ma and NifB Mt can catalyze the removal of methyl group from SAM and the abstraction of hydrogen from this methyl group by 5′-deoxyadenosyl radical that initiates the radical-based incorporation of methyl-derived carbide into the M cluster. The successful identification of NifB Ma and NifB Mt as functional homologs of NifB not only enabled classification of a new subset of radical SAM methyltransferases that specialize in complex metallocluster assembly, but also provided a new tool for further characterization of the distinctive, NifB-catalyzed methyl transfer and conversion to an iron-bound carbide.nitrogenase | NifB | methanogens | radical SAM | homologs N itrogenase biosynthesis protein NifB is a radical S-adenosyl-L-methionine (SAM) enzyme that plays an essential role in the biosynthesis of the M cluster, a [MoFe 8 S 9 C-homocitrate] cluster that serves as the cofactor of the molybdenum (Mo) nitrogenase from Azotobacter vinelandii (1-7). Carrying a signature CxxxCxxC motif at its N terminus that houses the SAM-binding [Fe 4 S 4 ] cluster (designated the SAM cluster), NifB also contains a number of additional ligands that could accommodate coordination of the entire complement of iron (Fe) atoms of the M cluster (Fig. S1). Moreover, it shares sequence homology with NifX, an accessory protein in M-cluster biosynthesis (8), toward its C terminus (Fig. S1). Characterization of the NifB protein from A. vinelandii had long been hampered by the instability of NifB in aqueous solutions until this protein was expressed as part of a NifEN-B fusion protein, wherein NifB was fused with and protected by NifEN, the biosynthetic apparatus immediately downstream of NifB along the M-cluster assembly pathway (9). Expression of the NifEN-B fusion protein in A. vinelendii was "modeled" after a naturally occurring NifEN-B fusion protein in Clostridium pasteurianum, which has the N terminus o...