Radical<i>S</i>-Adenosylmethionine Enzymes

Broderick, Joan B.; Duffus, Benjamin R.; Duschene, Kaitlin S.; Shepard, Eric M.

doi:10.1021/cr4004709

Cited by 711 publications

(891 citation statements)

References 684 publications

(1,530 reference statements)

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“…A proposed mechanism for lipoyl synthase, including the highly unusual suggestion that the auxiliary cluster acts as a sacrificial sulfur donor, has been developed in the light of biochemical and spectroscopic studies [29,30,52,54]. Using a N Ɛ -octanoyl lysinyl peptide as a substrate analogue, a monothiolated species was isolated from activity assays and NMR analysis demonstrated that the sulfur insertion had occurred exclusively at C-6 (and not at C-8) [29].…”

Section: Discussionmentioning

confidence: 99%

Structures of lipoyl synthase reveal a compact active site for controlling sequential sulfur insertion reactions

Harmer

Hiscox

Dinis

et al. 2014

Biochemical Journal

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Summary Statement: The biosynthesis of lipoyl cofactors requires two lipoyl synthase mediated sulfur insertions. We report the crystal structures of a lipoyl synthase complexed with S-adenosylhomocysteine or 5'-methylthioadenosine. Models based on these structures identify likely substrate binding sites.Keywords: radical SAM, cofactors, crystal structure, enzyme catalysis, sulfur Abbreviations used: LipA, lipoyl synthase; BioB, biotin synthase; SAM, Sadenosylmethionine; LCD, lipoyl carrier domain; MTA, 5'-methylthioadenosine; RS, radical SAM; ACP, acyl carrier protein; SsLipA, Sulfolobus solfataricus LipA; TeLipA2 Thermosynechococcus elongatus LipA2; Ec, Escherichia coli; 5'-dA, 5'-deoxyadenosine. 2 ABSTRACTLipoyl cofactors are essential for living organisms and are produced by the insertion of two sulfur atoms into the relatively unreactive C-H bonds of an octanoyl substrate. This reaction requires lipoyl synthase, a member of the radical SAM enzyme superfamily. Herein we present crystal structures of lipoyl synthase with two [4Fe-4S] clusters bound at opposite ends of the TIM barrel, the usual fold of the radical SAM superfamily. The cluster required for reductive SAM cleavage conserves the features of the radical SAM superfamily, but the auxiliary cluster is bound by a CX 4 CX 5 C motif unique to lipoyl synthase. The fourth ligand to the auxiliary cluster is an extremely unusual serine residue. Site directed mutants show this conserved serine ligand is essential for the sulfur insertion steps. One crystallized LipA complex contains MTA, a breakdown product of SAM, bound in the likely SAM binding site. Modelling has identified an 18 Å deep channel, well-proportioned to accommodate an octanoyl substrate. These results suggest the auxiliary cluster is the likely sulfur donor, but access to a sulfide ion for the second sulfur insertion reaction requires the loss of an iron atom from the auxiliary cluster, which the serine ligand may enabled.3

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Section: Discussionmentioning

confidence: 99%

Structures of lipoyl synthase reveal a compact active site for controlling sequential sulfur insertion reactions

Harmer

Hiscox

Dinis

et al. 2014

Biochemical Journal

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“…Increases in visible absorption bands as well as iron/sulfur contents observed after reconstitution of the purified complex were similar among the wild-type QhpD and QhpD G161A and QhpD R373A mutants but were considerably greater in the QhpD C116S and QhpD C353S mutants over the wild-type enzyme. These results suggest that in these Cys mutants, additional iron and sulfur atoms are adventitiously bound to surface residues on the protein (21) and/or to the remaining Cys residues in the canonical and auxiliary [4Fe-4S] cluster-binding sites possibly in different chemical structures (12).…”

mentioning

confidence: 90%

The Radical S-Adenosyl-l-methionine Enzyme QhpD Catalyzes Sequential Formation of Intra-protein Sulfur-to-Methylene Carbon Thioether Bonds

Nakai

Ito

Kobayashi

et al. 2015

Journal of Biological Chemistry

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Background:The small subunit of quinohemoprotein amine dehydrogenase contains three Cys-to-Asp/Glu thioether bonds. Results:The radical S-adenosyl-L-methionine (SAM) enzyme QhpD catalyzes the single-turnover reaction of thioether bond formation in the protein substrate. Conclusion:The thioether bond formation by QhpD proceeds sequentially in an N-to C-terminal direction of the polypeptide. Significance: Our findings uncover another challenging reaction of radical SAM superfamily of enzymes.

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“…Radical SAM proteins use the reductive cleavage of S-adenosyl methionine to initiate free radical chemistry, and can accomplish a wide variety of reactions, including carbon-carbon bond formation (8,9). PqqE is a founding member of the SPASM domain-containing radical SAM proteins, which contain one or more auxiliary clusters in their C-terminal regions, and of which several are known to modify small peptides or proteins (10,11).…”

Section: Pyrroloquinoline Quinone (Pqq)mentioning

confidence: 99%

Demonstration That the Radical S-Adenosylmethionine (SAM) Enzyme PqqE Catalyzes de Novo Carbon-Carbon Cross-linking within a Peptide Substrate PqqA in the Presence of the Peptide Chaperone PqqD

Barr

Latham

Iavarone

et al. 2016

Journal of Biological Chemistry

110

181

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The radical S-adenosylmethionine (SAM) protein PqqE is predicted to function in the pyrroloquinoline quinone (PQQ) biosynthetic pathway via catalysis of carbon-carbon bond formation between a glutamate and tyrosine side chain within the small peptide substrate PqqA. We report here that PqqE activity is dependent on the accessory protein PqqD, which was recently shown to bind PqqA tightly. In addition, PqqE activity in vitro requires the presence of a flavodoxin-and flavodoxin reductasebased reduction system, with other reductants leading to an uncoupled cleavage of the co-substrate SAM. These results indicate that PqqE, in conjunction with PqqD, carries out the first step in PQQ biosynthesis: a radical-mediated formation of a new carbon-carbon bond between two amino acid side chains on PqqA.Pyrroloquinoline quinone (PQQ) 2 is employed by a wide variety of bacteria, where it functions as a redox cofactor in substrate oxidation via an alternate (non-glycolytic) pathway for production of cellular ATP (1). Hundreds of bacterial species are thought to produce PQQ, based on the presence in their genomes of the strongly conserved pqq operon (2) (Fig. 1A). Although the existence of this important redox cofactor has been known for decades, knowledge of the biosynthetic pathway for PQQ has remained scant (3, 4). The PQQ molecule derives from the evolutionarily conserved glutamate and tyrosine side chains within a ribosomally produced peptide substrate PqqA (Fig. 1, B and C). To produce PQQ, a large number of chemical modifications are required: (i) the formation of a new carbon-carbon bond between the ␥-carbon of glutamate and the 3-position of tyrosine (labeled in green in Fig. 1C); (ii) the addition of two additional hydroxyl groups to the tyrosine ring; (iii) a condensation between the 4-OH of tyrosine and the backbone amine of glutamate to produce a heterocyclic ring; and (iv) an 8-electron oxidation catalyzed by PqqC, a cofactorless enzyme that converts 3a-(2-amino-2-carboxyethyl)-4,5-dioxo-4,5,6,7,8,9-hexahydroquinoline-7,9-dicarboxylic acid (AHQQ) to PQQ in the final step of the pathway (5-7).The radical SAM enzyme PqqE has been the primary candidate as the catalyst for the formation of the new carbon-carbon bond between glutamate and tyrosine. Radical SAM proteins use the reductive cleavage of S-adenosyl methionine to initiate free radical chemistry, and can accomplish a wide variety of reactions, including carbon-carbon bond formation (8, 9). PqqE is a founding member of the SPASM domain-containing radical SAM proteins, which contain one or more auxiliary clusters in their C-terminal regions, and of which several are known to modify small peptides or proteins (10, 11). Recently, a small (ϳ10 kDa) protein, PqqD, has been shown to form a strong, sub-micromolar K D complex with the peptide substrate PqqA. This complex was further demonstrated to associate with PqqE (12). These findings suggested that PqqD would serve as a chaperone to deliver PqqA to PqqE, functioning as a necessary and heretofore missing componen...

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RadicalS-Adenosylmethionine Enzymes

Cited by 711 publications

References 684 publications

Structures of lipoyl synthase reveal a compact active site for controlling sequential sulfur insertion reactions

Structures of lipoyl synthase reveal a compact active site for controlling sequential sulfur insertion reactions

The Radical S-Adenosyl-l-methionine Enzyme QhpD Catalyzes Sequential Formation of Intra-protein Sulfur-to-Methylene Carbon Thioether Bonds

Demonstration That the Radical S-Adenosylmethionine (SAM) Enzyme PqqE Catalyzes de Novo Carbon-Carbon Cross-linking within a Peptide Substrate PqqA in the Presence of the Peptide Chaperone PqqD

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