X-ray analysis of butirosin biosynthetic enzyme BtrN redefines structural motifs for AdoMet radical chemistry

Goldman, Peter; Grove, Tyler L.; Booker, Squire J.; Drennan, Catherine L.

doi:10.1073/pnas.1312228110

Cited by 68 publications

(92 citation statements)

References 45 publications

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“…S3) and are similar to the binding modes observed in high-resolution crystal structures of other radical SAM enzymes (28,(38)(39)(40)(41)(42)(43) (Fig. S4).…”

Section: Resultssupporting

confidence: 80%

Structural studies of viperin, an antiviral radical SAM enzyme

Fenwick

Cresswell

et al. 2017

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

112

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Viperin is an IFN-inducible radical S-adenosylmethionine (SAM) enzyme that inhibits viral replication. We determined crystal structures of an anaerobically prepared fragment of mouse viperin (residues 45-362) complexed with S-adenosylhomocysteine (SAH) or 5′-deoxyadenosine (5′-dAdo) and L-methionine (L-Met). Viperin contains a partial (βα) 6 -barrel fold with a disordered N-terminal extension (residues 45-74) and a partially ordered C-terminal extension (residues 285-362) that bridges the partial barrel to form an overall closed barrel structure. Cys84, Cys88, and Cys91 located after the first β-strand bind a [4Fe-4S] cluster. The active site architecture of viperin with bound SAH (a SAM analog) or 5′-dAdo and L-Met (SAM cleavage products) is consistent with the canonical mechanism of 5′-deoxyadenosyl radical generation. The viperin structure, together with sequence alignments, suggests that vertebrate viperins are highly conserved and that fungi contain a viperin-like ortholog. Many bacteria and archaebacteria also express viperin-like enzymes with conserved active site residues. Structural alignments show that viperin is similar to several other radical SAM enzymes, including the molybdenum cofactor biosynthetic enzyme MoaA and the RNA methyltransferase RlmN, which methylates specific nucleotides in rRNA and tRNA. The viperin putative active site contains several conserved positively charged residues, and a portion of the active site shows structural similarity to the GTPbinding site of MoaA, suggesting that the viperin substrate may be a nucleoside triphosphate of some type.radical SAM | IFN-stimulated gene | antiviral cellular factor | free radical | S-adenosyl methionine V iruses exploit the metabolic machinery of host cells to replicate and spread to other cells. Although cytotoxic T cells and antibody-producing B cells can ultimately be produced in an adaptive response to the virus, innate immune mechanisms are used to respond to infection rapidly. Upon infection, cells can sense the presence of virus via pattern recognition receptors (1, 2) and produce IFNs that limit the spread of infection to other cells (3). IFNs induce the expression of hundreds of IFN-stimulated genes (ISGs), many of which are involved in various antiviral processes, including antigen presentation, apoptosis, and inhibition of viral replication (4-7).Viperin, the product of rsad-2, was first identified as a protein induced by exposure of human macrophages to IFN-γ and by infection of primary human fibroblasts with human cytomegalovirus (8,9). Early studies showed that viperin is induced in various cell types by IFN-α and IFN-β, associates with the cytosolic face of the endoplasmic reticulum (ER), and inhibits human cytomegalovirus replication when preexpressed in human fibroblasts (8). Since then, viperin has been shown to be induced by several factors, including lipopolysaccharide (10-12), and to inhibit a broad range of viruses, including HIV-1 (13), West Nile virus (14), hepatitis C virus (15, 16), dengue virus type 2 (17), influ...

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“…S3) and are similar to the binding modes observed in high-resolution crystal structures of other radical SAM enzymes (28,(38)(39)(40)(41)(42)(43) (Fig. S4).…”

Section: Resultssupporting

confidence: 80%

Structural studies of viperin, an antiviral radical SAM enzyme

Fenwick

Cresswell

et al. 2017

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

112

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“…The phenolic side chain occupies a pocket between the side chains of Phe95 and Phe369. This model positions the tyrosine near to SAM, with a distance between the 5′ carbon of SAM and the amino group of tyrosine of 5.1 Å, only slightly longer than that observed (3.7-4.1 Å) in substrate complex structures of other radical SAM enzymes (23,26,40,41). This arrangement would enable a tyrosine cleavage reaction, in which the SAM-derived 5′-deoxyadenosyl radical abstracts an α-amino hydrogen atom (by analogy to a recent mechanistic proposal for NosL) (21) followed by homolysis of the tyrosine Cα-Cβ bond (Fig.…”

Section: Resultsmentioning

confidence: 70%

X-ray crystallographic and EPR spectroscopic analysis of HydG, a maturase in [FeFe]-hydrogenase H-cluster assembly

Dinis

Suess

Fox

et al. 2015

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

162

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Hydrogenases use complex metal cofactors to catalyze the reversible formation of hydrogen. In [FeFe]-hydrogenases, the H-cluster cofactor includes a diiron subcluster containing azadithiolate, three CO, and two CN − ligands. During the assembly of the H cluster, the radical S-adenosyl methionine (SAM) enzyme HydG lyses the substrate tyrosine to yield the diatomic ligands. These diatomic products form an enzyme-bound Fe(CO) x (CN) y synthon that serves as a precursor for eventual H-cluster assembly. To further elucidate the mechanism of this complex reaction, we report the crystal structure and EPR analysis of HydG. At one end of the HydG (βα) 8 triosephosphate isomerase (TIM) barrel, a canonical [4Fe-4S] cluster binds SAM in close proximity to the proposed tyrosine binding site. At the opposite end of the active-site cavity, the structure reveals the auxiliary Fe-S cluster in two states: one monomer contains a [4Fe-5S] cluster, and the other monomer contains a [5Fe-5S] cluster consisting of a [4Fe-4S] cubane bridged by a μ 2 -sulfide ion to a mononuclear Fe 2+ center. This fifth iron is held in place by a single highly conserved protein-derived ligand: histidine 265. EPR analysis confirms the presence of the [5Fe-5S] cluster, which on incubation with cyanide, undergoes loss of the labile iron to yield a [4Fe-4S] cluster. We hypothesize that the labile iron of the [5Fe-5S] cluster is the site of Fe (CO) x (CN) y synthon formation and that the limited bonding between this iron and HydG may facilitate transfer of the intact synthon to its cognate acceptor for subsequent H-cluster assembly.radical SAM enzyme | tyrosine lyase | H-cluster biosynthesis T he assembly of the [FeFe]-hydrogenase diiron subcluster (1, 2) requires three maturase proteins, HydE, HydF, and HydG (3), and in vitro, they can assemble an active hydrogenase (4). The sequence and structure of the maturase HydE (5) indicates that it is a member of the radical S-adenosyl methionine (SAM) superfamily, although the biochemical function of HydE has not been experimentally determined. The GTPase HydF (6, 7) has been shown to transfer synthetic (8) or biologically derived (7, 9) diiron subclusters into apo-hydrogenase, suggesting that HydF functions as a template for diiron subcluster assembly. The tyrosine lyase HydG is also a member of the radical SAM superfamily and uses SAM and a reductant (such as dithionite) to cleave the Cα-Cβ bond of tyrosine, yielding p-cresol as the side chain-derived byproduct (10) and fragmenting the amino acid moiety into cyanide (CN − ) (11) and carbon monoxide (CO) (12), which are ultimately incorporated as ligands in the H cluster of the [FeFe]-hydrogenase HydA (4). Two site-differentiated [4Fe-4S] clusters in HydG have been identified using a combination of spectroscopy and site-directed mutagenesis (12-16). The cluster bound close to the N terminus ([4Fe-4S] RS ) by the CX 3 CX 2 C cysteine triad motif (SI Appendix, Fig. S1) is typical of the radical SAM superfamily (17, 18) and has been shown to catalyze the reductive cl...

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“…anSME was also the first structure of a SAM radical enzyme with dehydrogenase activity, and it, along with the structures of butirosin biosynthetic enzyme BtrN from Bacillus circulans, and molybdenum cofactor biosynthetic enzyme MoaA (12) (Fig. 2), revealed unexpected structural homology between SPASM and non-SPASM enzymes (13). In particular, the comparison of anSME, BtrN, and MoaA led to the identification of a truncated SPASM domain used for binding a single auxiliary [4Fe-4S] cluster, which has been termed a Twitch domain (Fig.…”

Section: Members Of the S-adenosylmethionine (Sam)mentioning

confidence: 95%

“…In particular, the comparison of anSME, BtrN, and MoaA led to the identification of a truncated SPASM domain used for binding a single auxiliary [4Fe-4S] cluster, which has been termed a Twitch domain (Fig. 1B) (11,13). This review discusses the function and architecture of the newly defined SPASM/Twitch subclass and explores the roles of auxiliary clusters in radical SAM chemistry with particular focus on the auxiliary clusters of this subclass.…”

Section: Members Of the S-adenosylmethionine (Sam)mentioning

confidence: 99%

“…BtrN contains a Twitch domain (13), which houses one auxiliary [4Fe-4S] cluster. Using SAM radical chemistry, BtrN catalyzes the oxidation of the C3 hydroxyl group of 2-deoxyscyllo-inosamine by a hydrogen atom abstraction, deprotonation, and one-electron oxidation to produce the ketone group in 3-amino-2,3-dideoxy-scyllo-inosose (amino-DOI) (Fig.…”

Section: Function Of Spasm/twitch Subfamilymentioning

confidence: 99%

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SPASM and Twitch Domains in S-Adenosylmethionine (SAM) Radical Enzymes

Grell

Goldman

Drennan

2015

Journal of Biological Chemistry

Self Cite

135

179

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S-Adenosylmethionine (SAM, also known as AdoMet) radical enzymes use SAM and a [4Fe-4S] cluster to catalyze a diverse array of reactions. They adopt a partial triose-phosphate isomerase (TIM) barrel fold with N-and C-terminal extensions that tailor the structure of the enzyme to its specific function. One extension, termed a SPASM domain, binds two auxiliary [4Fe-4S] clusters and is present within peptide-modifying enzymes. The first structure of a SPASM-containing enzyme, anaerobic sulfatase-maturating enzyme (anSME), revealed unexpected similarities to two non-SPASM proteins, butirosin biosynthetic enzyme 2-deoxy-scyllo-inosamine dehydrogenase (BtrN) and molybdenum cofactor biosynthetic enzyme (MoaA). The latter two enzymes bind one auxiliary cluster and exhibit a partial SPASM motif, coined a Twitch domain. Here we review the structure and function of auxiliary cluster domains within the SAM radical enzyme superfamily. Members of the S-adenosylmethionine (SAM)2 radical superfamily catalyze a wide variety of radical-mediated reactions, including complex chemical transformations and rearrangements; modifications of peptides, DNA, and RNA; dehydrogenations; and sulfur insertions (1). Despite this diversity, there are unifying structural and mechanistic themes. For instance, SAM radical enzymes typically bind a [4Fe-4S] cluster using a conserved CX 3 CXC motif (where is an aromatic residue). This motif provides three cysteine ligands to the iron atoms of the cluster, with the fourth ligand coming from the bidentate coordination of SAM to the unique iron (2, 3). Direct ligation of SAM to the cluster facilitates reductive cleavage of the C-S bond through an inner sphere electron transfer event, forming methionine and a 5Ј-deoxyadenosyl radical (5Ј-dAdo ⅐ ) (Fig. 1A) (4). The abstraction of a hydrogen atom from the substrate by 5Ј-dAdo ⅐ , producing a substrate radical, ends the mechanistic similarity between enzymes of this superfamily; each enzyme utilizes a different mechanism to generate product. Structures of the first seven members of the SAM radical superfamily were used to define a core fold for binding SAM and for the generation of 5Ј-dAdo ⅐ species. This core consists of a partial (␤/␣) 6 triose-phosphate isomerase (TIM) barrel (5). Outside of the core fold, the structure can vary greatly, with Nand C-terminal extensions that are functionalized for binding other cofactors or substrates. The SPASM subfamily is an example of a functionalized C-terminal extension for the binding of two auxiliary clusters.Haft and Basu (6, 7) recognized that enzymes with this C-terminal extension appear to be involved in the modification of ribosomally translated peptides. This subclass is referred to as SPASM after the biochemically characterized members, AlbA, PqqE, anSMEs, and MftC, which are involved in subtilosin A, pyrroloquinoline quinone, anaerobic sulfatase, and mycofactocin maturation, respectively. The SPASM subfamily, accession TIGR04085, is composed of 281 sequences. However, recent similarity network analysis by ...

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X-ray analysis of butirosin biosynthetic enzyme BtrN redefines structural motifs for AdoMet radical chemistry

Cited by 68 publications

References 45 publications

Structural studies of viperin, an antiviral radical SAM enzyme

Structural studies of viperin, an antiviral radical SAM enzyme

X-ray crystallographic and EPR spectroscopic analysis of HydG, a maturase in [FeFe]-hydrogenase H-cluster assembly

SPASM and Twitch Domains in S-Adenosylmethionine (SAM) Radical Enzymes

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