Structural basis for glycyl radical formation by pyruvate formate-lyase activating enzyme

Vey, Jessica L.; Yang, Jian; Li, Meng; Broderick, William E.; Broderick, Joan B.; Drennan, Catherine L.

doi:10.1073/pnas.0806640105

Cited by 179 publications

(310 citation statements)

References 43 publications

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“…S1B). This site is the proposed binding location of the physiological reductant, commonly flavodoxin (30,32).…”

Section: Resultsmentioning

confidence: 99%

“…This reliance by SMEs on primarily peptide backbone-based hydrogen bonding interactions is in agreement with the much higher degree of promiscuity that exists in the anSME system in relation to the FGE system. Interestingly, pyruvate formate lyase-activating enzyme (PFL-AE), the only other structurally characterized AdoMet radical enzyme involved in protein modification, also uses primarily peptide backbone-activase interactions (32). As more structures become available, it will be interesting to see if this binding mode will be common to AdoMet radical enzymes that act on protein substrates.…”

Section: Discussionmentioning

confidence: 99%

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X-ray structure of an AdoMet radical activase reveals an anaerobic solution for formylglycine posttranslational modification

Goldman¹,

Grove²,

Sites³

et al. 2013

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

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112

197

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Arylsulfatases require a maturating enzyme to perform a co-or posttranslational modification to form a catalytically essential formylglycine (FGly) residue. In organisms that live aerobically, molecular oxygen is used enzymatically to oxidize cysteine to FGly. Under anaerobic conditions, S-adenosylmethionine (AdoMet) radical chemistry is used. Here we present the structures of an anaerobic sulfatase maturating enzyme (anSME), both with and without peptidyl-substrates, at 1.6-1.8 Å resolution. We find that anSMEs differ from their aerobic counterparts in using backbone-based hydrogen-bonding patterns to interact with their peptidylsubstrates, leading to decreased sequence specificity. These anSME structures from Clostridium perfringens are also the first of an AdoMet radical enzyme that performs dehydrogenase chemistry. Together with accompanying mutagenesis data, a mechanistic proposal is put forth for how AdoMet radical chemistry is coopted to perform a dehydrogenation reaction. In the oxidation of cysteine or serine to FGly by anSME, we identify D277 and an auxiliary [4Fe-4S] cluster as the likely acceptor of the final proton and electron, respectively. D277 and both auxiliary clusters are housed in a cysteinerich C-terminal domain, termed SPASM domain, that contains homology to ∼1,400 other unique AdoMet radical enzymes proposed to use [4Fe-4S] clusters to ligate peptidyl-substrates for subsequent modification. In contrast to this proposal, we find that neither auxiliary cluster in anSME bind substrate, and both are fully ligated by cysteine residues. Instead, our structural data suggest that the placement of these auxiliary clusters creates a conduit for electrons to travel from the buried substrate to the protein surface.iron-sulfur cluster fold | radical SAM dehydrogenase

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“…S1B). This site is the proposed binding location of the physiological reductant, commonly flavodoxin (30,32).…”

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

X-ray structure of an AdoMet radical activase reveals an anaerobic solution for formylglycine posttranslational modification

Goldman¹,

Grove²,

Sites³

et al. 2013

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

Self Cite

112

197

View full text Add to dashboard Cite

show abstract

“…An overlay of TeLipA2, BioB [19], HydE [42] and Pfl-Ae [43] shows the MTA adenine base and ribose ring approximately overlay the positions expected for adenosyl moiety of SAM (Supplementary Figure S3). DTT is bound to directly to the unique iron of the [4Fe-4S] RS through one of the thiols (Figure 2A) and therefore occupies the ligand site typically occupied by the methionine moiety of SAM.…”

Section: Structure Of Telipa2mentioning

confidence: 89%

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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“…1B) (12). A structure of an AE bound to a peptide mimic of the GRE Gly radical domain (13) shows that the Gly radical domain must be extended away from the GRE to fit into the AE active site. However, once activated, the Gly radical domain must return to the inside of the barrel where the Gly radical can undergo hydrogen atom transfer with the catalytic Cys during each round of turnover, and where it is sequestered away from oxidants and reductants that could quench the radical.…”

mentioning

confidence: 99%

Structures of benzylsuccinate synthase elucidate roles of accessory subunits in glycyl radical enzyme activation and activity

Funk¹,

Judd

Marsh

et al. 2014

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

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Significance Glycyl radical enzymes perform many chemical transformations that form the bedrock of microbial anaerobic metabolism. The structure of benzylsuccinate synthase reveals the architecture of an enzyme capable of removing aromatic hydrocarbons from polluted environments. These structures also illustrate a strategy for controlling the generation and utilization of radicals by glycyl radical enzymes through the use of accessory subunits.

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Structural basis for glycyl radical formation by pyruvate formate-lyase activating enzyme

Cited by 179 publications

References 43 publications

X-ray structure of an AdoMet radical activase reveals an anaerobic solution for formylglycine posttranslational modification

X-ray structure of an AdoMet radical activase reveals an anaerobic solution for formylglycine posttranslational modification

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

Structures of benzylsuccinate synthase elucidate roles of accessory subunits in glycyl radical enzyme activation and activity

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