A glycyl free radical as the precursor in the synthesis of carbon monoxide and cyanide by the [FeFe]‐hydrogenase maturase HydG

Nicolet, Yvain; Martin, Lydie; Tron, Cécile; Fontecilla‐Camps, J.C.

doi:10.1016/j.febslet.2010.09.008

Cited by 52 publications

(78 citation statements)

References 35 publications

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“…The presence of a serine residue as a naturally occurring ligand for a protein bound [4Fe-4S] cluster has not been previously observed, but a serine for cysteine site directed mutation has often been used to modulate or eliminate cluster activity [46][47][48][49][50][51]. Examination of the electron density near the auxiliary cluster ( Figure 3B) shows clear density for the serine ligand (Ser283) with a short Fe-O bond length (1.8 Å in the MTA complex), which likely reflects deprotonation of the serine ligand.…”

Section: Discussionmentioning

confidence: 99%

“…This cluster is held in place by three conserved cysteine residues [14] and an unanticipated ligand from close to the C-terminus, Ser283. Serine is previously unreported as a natural ligand for [4Fe-4S] clusters, although this residue has been introduced as a cluster ligand by site directed mutagenesis [46][47][48][49][50][51]. In the TeLipA2:SAH complex structure, the Ser283 alkoxy oxygen atom lies in close proximity to an additional metal atom, which because of its abundance in the crystallization medium (initially approximately 160 mM), has been modeled as a sodium ion (Na-O distance 2.5 Å, Figure 3B).…”

Section: Structure Of Telipa2mentioning

confidence: 99%

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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%

Section: Structure Of Telipa2mentioning

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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“…Work from several laboratories has shown that the maturation of the [FeFe] center requires at least three protein maturases: HydF that has GTPase activity and appears to be both a [FeFe] center scaffold and carrier (8,9), HydG that synthesizes CO and CN − from tyrosine (10)(11)(12)(13), and HydE that, by elimination, should be involved in the synthesis of the DTMA bridge (14,15). Both HydE and HydG are members of the large radical S-adenosyl-L-methionine (SAM) protein family (16,17).…”

mentioning

confidence: 99%

CO and CN ⁻ syntheses by [FeFe]-hydrogenase maturase HydG are catalytically differentiated events

Pagnier

Martin

Zeppieri

et al. 2015

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

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The synthesis and assembly of the active site [FeFe] unit of [FeFe]-hydrogenases require at least three maturases. The radical S-adenosyl-L-methionine HydG, the best characterized of these proteins, is responsible for the synthesis of the hydrogenase CO and CN − ligands from tyrosine-derived dehydroglycine (DHG). We speculated that CN − and the CO precursor − :CO 2 H may be generated through an elimination reaction. We tested this hypothesis with both wild type and HydG variants defective in second ironsulfur cluster coordination by measuring the in vitro production of CO, CN − , and − :CO 2 H-derived formate. We indeed observed formate production under these conditions. We conclude that HydG is a multifunctional enzyme that produces DHG, CN − , and CO at three well-differentiated catalytic sites. We also speculate that homocysteine, cysteine, or a related ligand could be involved in Fe(CO) x (CN) y transfer to the HydF carrier/scaffold. (4) showed that the active site is composed of a conventional [4Fe-4S] cubane connected by a cysteine thiolate to a binuclear FeFe unit, in which each iron ion is terminally coordinated by one CN − ligand and one CO ligand and by a third CO molecule that bridges the two metals (5). Unexpectedly, we also found that a small molecule first postulated (6), and now indirectly confirmed (7), to be dithiomethylamine (DTMA) bridges the two Fe ions (Fig. 1).The [4Fe-4S] cubane bridged to the binuclear [FeFe] unit has been collectively called the H-cluster (1). Work from several laboratories has shown that the maturation of the [FeFe] center requires at least three protein maturases: HydF that has GTPase activity and appears to be both a [FeFe] center scaffold and carrier (8, 9), HydG that synthesizes CO and CN − from tyrosine (10-13), and HydE that, by elimination, should be involved in the synthesis of the DTMA bridge (14, 15). Both HydE and HydG are members of the large radical S-adenosyl-L-methionine (SAM) protein family (16,17). With the recent reports of HydG crystal structures from Carboxydothermus hydrogenoformans (Ch) by us (18) and from Thermoanaerobacter italicus (Ti) by Dinis et al. (19), X-ray models are now available for the three maturases (20, 21); however, unambiguous structure-function relationships have been proposed only in the case of HydG. Indeed, site-directed mutational studies have shown that CO and CN − syntheses are affected by either the deletion of the maturase C-terminal region, where a second iron-sulfur cluster binds (22), or Cys-to-Ser mutations in its corresponding CxxCx 22 C binding motif (10, 13). In addition, it has been shown that HydG synthesizes Fe(CO) x (CN) y precursors (x = 1 or 2; y = 1) of the [FeFe] catalytic unit (23). The two HydG crystal structures are very similar at the SAM and [4Fe-4S] cluster-containing (β/α) 8 TIM-like barrel, common to several radical SAM proteins (16) (Fig. 2). Conversely, there are significant differences in the composition of the extra C-terminal second (s) iron-sulfur cluster. In our crystals, ChHydG lacks th...

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“…Several mechanisms can be envisaged for the fragmentation of dehydroglycine and one of the simpler possibilities (Figure 3a) can be accelerated by an active site base (B in Figure 3a) which may be provided by the sidechains of conserved Arg129 or Arg165 (TiHydG numbering), and/or an electrophile (Figure 3a, E + ) which might be a proton from an acidic residue or a Lewis acid such as the labile iron. Using a CaHydG mutant that has two cysteine residues that normally bind the auxiliary cluster mutated to serine, and therefore does not bind an intact auxiliary cluster, the formation of cyanide without CO formation has been measured [24] and this mutant produces formate in place of CO [25]. This implies that under these conditions, cleavage of dehydroglycine to form cyanide occurs prior to the conversion of the formyl moiety into carbon monoxide [25].…”

Section: Structure Of Hydgmentioning

confidence: 99%

“…The structure of HydE (Figure 4a) includes a large cavity (991 Å 3 ) within the TIM barrel that is well suited to bind a second substrate (in addition to SAM). Efforts to identify this substrate include in silico [24] and in vitro screening experiments [32,33]. The rate of SAM turnover to form 5ʹ-deoxyadenosine was recently used to screen a range of putative HydE second substrates, leading to the conclusion that the HydE substrate likely contains a thiol [33].…”

Section: Hyde: 'A Riddle Wrapped In a Mystery Inside An Enigma'mentioning

confidence: 99%

Metallocofactor assembly for [FeFe]-hydrogenases

Dinis

Wieckowski

Roach

2016

Current Opinion in Structural Biology

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Hydrogenases are a potential source of environmentally benign bioenergy, using complex cofactors to catalyze the reversible reduction of protons to form hydrogen. AddressesChemistry and the Institute for Life Sciences, University of Southampton, Highfield Campus, Southampton SO17 1BJ, UK.Corresponding author: Peter L. Roach (plr2@soton.ac.uk). AbstractHydrogenases are a potential source of environmentally benign bioenergy, using complex cofactors to catalyze the reversible reduction of protons to form hydrogen. The most active subclass, the [FeFe]-hydrogenases, is dependent on a metallocofactor, the H cluster, that consists of a two iron subcluster ([2Fe] H ) bridging to a classical cubane cluster ([4Fe-4S] H ). The ligands coordinating to the diiron subcluster include an azadithiolate, three carbon monoxides, and two cyanides. To assemble this complex cofactor, three maturase enzymes, HydG, HydE and HydF are required. The biosynthesis of the diatomic ligands proceeds by an unusual fragmentation mechanism, and structural studies in combination with spectroscopic analysis have started to provide insights into the HydG mediated assembly of a [2Fe] H subcluster precursor.

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A glycyl free radical as the precursor in the synthesis of carbon monoxide and cyanide by the [FeFe]‐hydrogenase maturase HydG

Abstract: a b s t r a c t

Cited by 52 publications

References 35 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

CO and CN ⁻ syntheses by [FeFe]-hydrogenase maturase HydG are catalytically differentiated events

Metallocofactor assembly for [FeFe]-hydrogenases

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A glycyl free radical as the precursor in the synthesis of carbon monoxide and cyanide by the [FeFe]‐hydrogenase maturase HydG

Abstract: a b s t r a c t

Cited by 52 publications

References 35 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

CO and CN − syntheses by [FeFe]-hydrogenase maturase HydG are catalytically differentiated events

Metallocofactor assembly for [FeFe]-hydrogenases

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CO and CN ⁻ syntheses by [FeFe]-hydrogenase maturase HydG are catalytically differentiated events