Maturation of the [FeFe]-hydrogenase active site depends on at least the expression of three gene products called HydE, HydF, and HydG. Although the exact role of HydE in the formation of the cofactor in [FeFe]-hydrogenase is still unknown, it is thought to be involved in the formation and incorporation of the -C#N and C#O ligands.
Nicolet Y, Rubach JK, Posewitz MC, Amara P, Mathevon C, Atta M, Fontecave M, Fontecilla-Camps JC.
X-ray structure of the [FeFe]-hydrogenase maturase HydE from Thermotoga maritima
▸ Abstract
Maturation of the [FeFe]-hydrogenase active site depends on at least the expression of three gene products called HydE, HydF, and HydG. We have solved the high resolution structure of recombinant, reconstituted S-adenosine-L-methionine-dependent HydE from Thermotoga maritima. Besides the conserved [Fe(4)S(4)] cluster involved in the radical-based reaction, this HydE was reported to have a second [Fe(4)S(4)] cluster coordinated by three Cys residues. However, in our crystals, depending on the reconstitution and soaking conditions, this second cluster is either a [Fe(2)S(2)] center, with water occupying the fourth ligand site or is absent. We have carried out site-directed mutagenesis studies on the related HydE from Clostridium acetobutylicum, along with in silico docking and crystal soaking experiments, to define the active site region and three anion-binding sites inside a large, positive cavity, one of which binds SCN(-) with high affinity. Although the overall triose-phosphate isomerase-barrel structure of HydE is very similar to that of biotin synthase, the residues that line the internal cavity are significantly different in the two enzymes.
Rohac R, Amara P, Benjdia A, Martin L, Ruffié P, Favier A, Berteau O, Mouesca JM, Fontecilla-Camps JC, Nicolet Y
Carbon-sulfur bond-forming reaction catalysed by the radical SAM enzyme HydE
▸ Abstract
Carbon-sulfur bond formation at aliphatic positions is a challenging reaction that is performed efficiently by radical S-adenosyl-L-methionine (SAM) enzymes. Here we report that 1,3-thiazolidines can act as ligands and substrates for the radical SAM enzyme HydE, which is involved in the assembly of the active site of [FeFe]-hydrogenase. Using X-ray crystallography, in vitro assays and NMR spectroscopy we identified a radical-based reaction mechanism that is best described as the formation of a C-centred radical that concomitantly attacks the sulfur atom of a thioether. To the best of our knowledge, this is the first example of a radical SAM enzyme that reacts directly on a sulfur atom instead of abstracting a hydrogen atom. Using theoretical calculations based on our high-resolution structures we followed the evolution of the electronic structure from SAM through to the formation of S-adenosyl-L-cysteine. Our results suggest that, at least in this case, the widely proposed and highly reactive 5'-deoxyadenosyl radical species that triggers the reaction in radical SAM enzymes is not an isolable intermediate.
