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

Dinis, Pedro; Suess, Daniel L. M.; Fox, Stephen; Harmer, Jenny E.; Driesener, Rebecca C.; Paz, Liliana De La; Swartz, James R.; Essex, Jonathan W.; Britt, R. David; Roach, Peter L.

doi:10.1073/pnas.1417252112

Cited by 96 publications

(175 citation statements)

References 49 publications

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“…Third, the cluster of TtuA is ligated by only three cysteines and thus, has a free coordination site on the fourth iron, which can be occupied by a hydrosulfide ligand. Precedents for exogenous sulfur species bound to an iron site of a [4Fe-4S] cluster have been reported: a comparable [4Fe-5S] cluster has been observed in HydG, an enzyme involved in maturation of hydrogenases (17), as well as 2-hydroxyisocaproyl-CoA dehydratase (18). Moreover, in the structure of the methylthiotransferase RimO, a polysulfide terminal ligand to a [4Fe-4S] cluster has been observed (19).…”

Section: Discussionmentioning

confidence: 95%

Nonredox thiolation in tRNA occurring via sulfur activation by a [4Fe-4S] cluster

Arragain

Bimaï

Legrand

et al. 2017

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

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Sulfur is present in several nucleosides within tRNAs. In particular, thiolation of the universally conserved methyl-uridine at position 54 stabilizes tRNAs from thermophilic bacteria and hyperthermophilic archaea and is required for growth at high temperature. The simple nonredox substitution of the C2-uridine carbonyl oxygen by sulfur is catalyzed by tRNA thiouridine synthetases called TtuA. Spectroscopic, enzymatic, and structural studies indicate that TtuA carries a catalytically essential [4Fe-4S] cluster and requires ATP for activity. A series of crystal structures shows that (i) the cluster is ligated by only three cysteines that are fully conserved, allowing the fourth unique iron to bind a small ligand, such as exogenous sulfide, and (ii) the ATP binding site, localized thanks to a proteinbound AMP molecule, a reaction product, is adjacent to the cluster. A mechanism for tRNA sulfuration is suggested, in which the unique iron of the catalytic cluster serves to bind exogenous sulfide, thus acting as a sulfur carrier.T he cellular translation machinery contains essential components such as tRNAs. To achieve their function, they feature a great variety of well-conserved posttranscriptional chemical modifications. Sulfur is present in several of these modified nucleosides: thiouridine and derivatives (s 4 U8, s 2 U34, and m 5 s 2 U54), 2-thioadenosine derivatives (ms 2 i 6 A37 and ms 2 t 6 A37), and 2-thiocytidine (s 2 C32). However, mechanisms of sulfur insertion into tRNAs are largely unknown, and the enzymes responsible for these reactions are incompletely characterized. Whereas redox conversion of a C-H to a C-S bond (synthesis of ms 2 i 6 A37 and ms 2 t 6 A37) depends on redox enzymes from the Radical-S-adenosyl-L-methionine iron-sulfur enzyme family, simple nonredox conversion of C = O to C = S group (synthesis of s 2 U34 and s 4 U8) is not expected to require such redox clusters. Intriguingly, we recently discovered that the ATPdependent formation of s 2 C32 in some tRNAs is catalyzed by an iron-sulfur enzyme, TtcA (1). However, the role of its cluster has not been defined. In the same superfamily, TtuA enzymes catalyze the C2-thiolation of uridine 54 in the T loop of thermophilic tRNAs (Fig. 1A), allowing stabilization of tRNAs at high temperature in thermophilic microorganisms. Sequences analysis shows that they share conserved cysteines and ATP binding motif (Fig. S1). Here, we report a detailed biochemical and structural characterization of TtuA that shows the presence of a [4Fe-4S] cluster essential for activity. The crystal structures of Pyrococcus horikoshii TtuA (PhTtuA) show that the cluster, chelated by only three cysteines, is adjacent to the ATP binding site. The presence of electron density near the fourth iron, nonbonded to the protein, indicates that the cluster can bind an exogenous substrate. We propose that thiolation occurs via sulfur binding to the cluster and transfer to the tRNA substrate. The fact that the catalytic [4Fe-4S] cluster serves as a sulfur carrier during a nonredox ...

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Section: Discussionmentioning

confidence: 95%

Nonredox thiolation in tRNA occurring via sulfur activation by a [4Fe-4S] cluster

Arragain

Bimaï

Legrand

et al. 2017

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

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“…The SAM sulfonium sulfur atom is poised 3.4 Å from the proximal iron, an arrangement that closely resembles SAM binding in other radical SAM enzymes [23]. The structure of co-crystals of HydG with tyrosine have not yet been reported, but this complex has been modelled [20] with the tyrosine binding pocket adjacent to the 5ʹ-C of SAM (Figure 2b). …”

Section: Structure Of Hydgmentioning

confidence: 81%

“…Two HydG crystal structures have been described [19,20] but the structure of the enzyme from Thermoanaerobacter italicus (TiHydG) is the first reported with an intact auxiliary structure ( Figure 2). The asymmetric unit contains two HydG monomers and the core structure of HydG is a complete triose phosphate isomerase (TIM) barrel, typical of RS enzymes the bind a small second substrate (such as PylB [21], BioB [22] and NosL [11]) (Figure 2a).…”

Section: Structure Of Hydgmentioning

confidence: 99%

“…This fifth iron has an occupancy of 0.73 and approximately octahedral geometry. It is held in place by a single bonding interaction to the protein, coordination from imidazole of His265, which is highly conserved in HydG sequences [20]. The four remaining ligands to the fifth iron include two water molecules and a nonproteinacous amino acid whose side chain was not sufficiently well ordered to permit unequivocal identification (although it is suspected to be methionine arising from SAM degradation during crystallization).…”

Section: Structure Of Hydgmentioning

confidence: 99%

“…The partial occupancy of the fifth iron in monomer A and complete absence in monomer B suggested it might be loosely bound. To capture this concept, we termed it the 'labile iron' and suggested it as a potential site for the assembly of the Fe(CO) 2 CN synthon which might be readily released to a downstream acceptor such as HydF for further elaboration into the [2Fe] H subcluster [20].…”

Section: Structure Of Hydgmentioning

confidence: 99%

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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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Characterization of auxiliary iron–sulfur clusters in a radical S‐adenosylmethionine enzyme PqqE from Methylobacterium extorquens AM1

et al. 2017

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PqqE is a radical S ‐adenosyl‐ l ‐methionine ( SAM ) enzyme that catalyzes the initial reaction of pyrroloquinoline quinone ( PQQ ) biosynthesis. PqqE belongs to the SPASM (subtilosin/ PQQ /anaerobic sulfatase/mycofactocin maturating enzymes) subfamily of the radical SAM superfamily and contains multiple Fe – S clusters. To characterize the Fe – S clusters in PqqE from Methylobacterium extorquens AM 1, Cys residues conserved in the N‐terminal signature motif ( CX 3 CX 2 C) and the C‐terminal seven‐cysteine motif ( CX 9–15 GX 4 CX n CX 2 CX 5 CX 3 CX n C; n = an unspecified number) were individually or simultaneously mutated into Ser. Biochemical and Mössbauer spectral analyses of as‐purified and reconstituted mutant enzymes confirmed the presence of three Fe – S clusters in PqqE: one [4Fe – 4S] 2+ cluster at the N‐terminal region that is essential for the reductive homolytic cleavage of SAM into methionine and 5′‐deoxyadenosyl radical, and one each [4Fe – 4S] 2+ and [2Fe – 2S] 2+ auxiliary clusters in the C‐terminal SPASM domain, which are assumed to serve for electron transfer between the buried active site and the protein surface. The presence of [2Fe – 2S] 2+ cluster is a novel finding for radical SAM enzyme belonging to the SPASM subfamily. Moreover, we found uncommon ligation of the auxiliary [4Fe – 4S] 2+ cluster with sulfur atoms of three Cys residues and a carboxyl oxygen atom of a conserved Asp residue.

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

Cited by 96 publications

References 49 publications

Nonredox thiolation in tRNA occurring via sulfur activation by a [4Fe-4S] cluster

Nonredox thiolation in tRNA occurring via sulfur activation by a [4Fe-4S] cluster

Metallocofactor assembly for [FeFe]-hydrogenases

Characterization of auxiliary iron–sulfur clusters in a radical S‐adenosylmethionine enzyme PqqE from Methylobacterium extorquens AM1

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