XAS Investigation of the Structure and Function of Ni in Acireductone Dioxygenase

Abstract

Acireductone dioxygenases (ARDs) are enzymes involved in the methionine recycle pathway, which regulates aspects of the cell cycle. Klebsiella pneumoniae produces two enzymes that share a common polypeptide sequence and differ only in the metal ion present. Reaction of acireductone (1,2-dihydroxy-3-keto-5-methylthiopentene) with Fe-ARD and dioxygen produces formate and 2-keto-4-methylthiobutanoic acid, the alpha-ketoacid precursor of methionine. Ni-ARD reacts with acireductone and dioxygen to produce methylthiopropionate, CO, and formate and does not lie on the methionine recycle pathway. An X-ray absorption spectroscopy (XAS) study of the structure of the catalytic Ni center in resting Ni-ARD enzyme and the enzyme-substrate complex is reported. This study establishes the structure of the Ni site in resting Ni-ARD as containing a six coordinate Ni site composed of O/N-donor ligands including 3-4 histidine residues, demonstrates that the substrate binds to the Ni center in a bidentate fashion by displacing two ligands, at least one of which is a histidine ligand, and provides insight into the mechanism of catalysis employed by a Ni-containing dioxygenase. Efficiently relaxed and hyperfine-shifted resonances are observed in the (1)H nuclear magnetic resonance spectrum of Ni-ARD that can be attributed to the His imidazoles ligating the paramagnetic Ni ion and are consistent with the XAS results regarding His ligation. These resonances show significant perturbation in the presence of substrate, confirming that the metal ion interacts directly with the substrate.

One Protein, Two Enzymes Revisited: A Structural Entropy Switch Interconverts the Two Isoforms of Acireductone Dioxygenase

“…Overlap of the 1 H, 15 N HSQC spectra of H98S and ARD′ also show very similar fingerprints for the two proteins, while the overlap between H98S and ARD is minimal. The HSQC fingerprint is diagnostic for a particular fold, and it is clear from the comparison in Figure 3B that there is close structural correspondence between H98S and ARD′.…”

“…It is possible that nickel prefers Nδ ligation at His 98. Although the crystal structure of mouse ARD shows all-Nɛ ligation of the metal, patterns of H/D exchange in hyperfine shifted imidazole 1 H resonances in ARD are consistent with ligation of Ni +2 via two His Nɛ and one His Nδ (15). Current progress in our laboratory towards a crystallographic structure of ARD may clarify this issue.…”

“…Furthermore, two sets of resonances can be assigned to residues extending from Gly 161 to Ala 172 in H98S ARD, suggesting that both cis-and trans-isomers of the Asn 158-Pro 159 peptide bond are occupied. The NɛH resonance of the Trp 162 indole ring, which is not detected in ARD due to proximity to the active site metal, can be identified in 1 H, 15 N HSQC spectra of ARD′, indicating that it is no longer adjacent to the active site ( Figure 3B). The disordering of the C-terminal peptide in ARD′ effectively opens the active site, rendering it more accessible to substrate.…”

“…With modifications (e.g., carrier frequencies optimized for aliphatic versus aromatic carbons) and parameter optimization, the implementations of all experiments are those found in the standard Varian BioPack release. Quadrature detection in the 15 N indirect dimension for all NH-detected experiments was obtained using the Rance-Kay method (25), with pulsed field gradient coherence selection for magnetization transfer through 15 N. Quadrature detection in indirectly detected 13 C dimensions was obtained using States-TPPI and coherence selection was obtained by phase cycling. All NMR datasets were initially processed using XWinNMR (Bruker Biospin Inc.) and data analysis performed using XEASY (26).…”

“…Furthermore, one of these mutations, His 98 to serine (H98S), proved to be interesting for other reasons. The H98S mutation resulted in the formation of a stable soluble protein that while structurally different from ARD shows a high degree of similarity to the ARD′ enzyme as determined by a comparison of 1D 1 H NMR and 2D 1 H, 15 N HSQC spectra ( Figure 3). The H98S mutant thus provides a structural model for the Fe-containing form of ARD.…”