Molecular basis for the substrate specificity and catalytic mechanism of thymine-7-hydroxylase in fungi

Abstract

TET proteins play a vital role in active DNA demethylation in mammals and thus have important functions in many essential cellular processes. The chemistry for the conversion of 5mC to 5hmC, 5fC and 5caC catalysed by TET proteins is similar to that of T to 5hmU, 5fU and 5caU catalysed by thymine-7-hydroxylase (T7H) in the nucleotide anabolism in fungi. Here, we report the crystal structures and biochemical properties of Neurospora crassa T7H. T7H can bind the substrates only in the presence of cosubstrate, and binding of different substrates does not induce notable conformational changes. T7H exhibits comparable binding affinity for T and 5hmU, but 3-fold lower affinity for 5fU. Residues Phe292, Tyr217 and Arg190 play critical roles in substrate binding and catalysis, and the interactions of the C5 modification group of substrates with the cosubstrate and enzyme contribute to the slightly varied binding affinity and activity towards different substrates. After the catalysis, the products are released and new cosubstrate and substrate are reloaded to conduct the next oxidation reaction. Our data reveal the molecular basis for substrate specificity and catalytic mechanism of T7H and provide new insights into the molecular mechanism of substrate recognition and catalysis of TET proteins.

Structural and stereoelectronic insights into oxygenase-catalyzed formation of ethylene from 2-oxoglutarate

“…It is possible that the unusual conformations of 2OG observed with PsEFE are diagnostic of ethylene formation. Interestingly, the PsEFE active-site architecture has similarities with those of the ten-eleven translocation (TET) 2OG oxygenases (for PsEFE and the TET from Neurospora crassa Cα rmsd = 1.2 Å over 185 residues) (42). Arg171 of PsEFE is conserved in the two enzymes; in the NcTET structure, the analogous Arg190 interacts with its nucleosome substrate and with the 2OG C-1 carboxylate (SI Appendix, Fig.…”

Radical Tropolone Biosynthesis

“…Overlaying TropC with T7H revealed several shared residues which define the substrate binding site in the T7H structure (Supplementary Figure S31). 33 Specifically, residues F284 and F213 align well with two conserved aromatic residues in T7H, F292 and Y217, respectively. 33 F292 and Y217 have been demonstrated as critical for substrate binding and alignment in T7H, with F292 providing π-π stacking interactions that align thymine in the enzyme active site for productive catalysis.…”

“…33 F292 and Y217 have been demonstrated as critical for substrate binding and alignment in T7H, with F292 providing π-π stacking interactions that align thymine in the enzyme active site for productive catalysis. 33 The conservation of these aromatic residues in homologs of T7H and their alignment with F284 and F213 in TropC suggests that these residues may play an analogous role in substrate alignment. 33 In addition, bound metal ion was observed in the structure of TropC, which was refined to be Fe(III).…”

“…This proposal is likewise analogous to the reported role of F292 and Y217 in T7H, suggesting a similar function in aligning the TropC substrate to achieve catalysis. 33 To further investigate this hypothesis, we generated the isosteric variant TropC F284Y which reconstituted the ring expansion activity of the enzyme, providing further evidence that F284 is a critical residue for determining the product mixtures generated by TropC. We aimed to further analyze this reaction by generating a TropC F213A variant but were unable to produce soluble protein with this construct.…”

Az első eukarióta nikotinsav hasznosítási útvonal felderítése, szabályozása és enzimeinek vizsgálata Aspergillus nidulans modellszervezetben

“…• HxnY is an -ketoglutarate dependent dioxygenase. It is probable that the substrate of HxnY is a nicotinate derivative, with a closed pyridine ring (the nearest structural analogue of HxnY is thymine-7-hydroxylase of N. crassa (Li et al, 2015)).…”