A Refined Model for the Structure of Acireductone Dioxygenase from Klebsiella ATCC 8724 Incorporating Residual Dipolar Couplings

Abstract

SummaryAcireductone dioxygenase (ARD) from Klebsiella ATCC 8724 is a metalloenzyme that is capable of catalyzing different reactions with the same substrates (acireductone and O 2 ) depending upon the metal bound in the active site. A model for the solution structure of the paramagnetic Ni 2+ -containing ARD has been refined using residual dipolar couplings (RDCs) measured in two media. Additional dihedral restraints based on chemical shift (TALOS) were included in the refinement, and backbone structure in the vicinity of the active site was modeled from a crystallographic structure of the mouse homolog of ARD. The incorporation of residual dipolar couplings into the structural refinement alters the relative orientations of several structural features significantly, and improves local secondary structure determination. Comparisons between the solution structures obtained with and without RDCs are made, and structural similarities and differences between mouse and bacterial enzymes are described. Finally, the biological significance of these differences is considered.

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

“…In our refinement of the ARD structure, we replaced the canavalin coordinates used in the original modeling of the active site with those from the corresponding residues in the 1VR3 structure. We note that this did not result in change in the proposed ligands for metal binding (12). Both of the models agree on the identity of His 96, His 98, His 140 and Glu 102 as the metal binding ligands in the active site of ARD (Figure 2).…”

“…4B) structures at the C-terminus of the polypeptide. In ARD, residues 158 to 164 form a tight turn (Asn 158-Pro-159-Glu 160-Gly 161) and a short antiparallel extended structure stabilized by hydrogen bonds to the backbone C=O and NH of Thr 97 (12). This effectively places the side chain indole of Trp 162 near the entrance to the active site.…”

“…In ARD, residues 39-50 form a largely disordered loop between strand D of the β-sandwich and the beginning of the E helix, with few NOEs between residues in this loop and other parts of the protein (9,12). In ARD′, however, this loop becomes more ordered, particularly those residues near the side chain of Tyr 57 on the E helix.…”

“…RDCs were obtained and analyzed using the methods recently described for ARD (12). Reference 1 H, 15 N HSQC spectra were acquired without 1 H decoupling in the indirect dimension at 25 °C without alignment, followed immediately by acquisition of identical spectra under aligning conditions.…”

“…Cupins are a functionally diverse group of proteins distinguished by the presence of an antiparallel β-helix (sometimes called a jellyroll) that forms the central feature of the cupin fold (10,11). We previously described a structural model of Ni-containing ARD based on NMR-derived restraints and molecular modeling in the vicinity of the metal center (9) (PDB entry 1M4O), and have recently published a refinement of that structure that included the use of residual dipolar couplings as restraints in the calculations (PDB entry 1ZRR) (12). Paramagnetism broadens 1 H resonance lines within ~9 Å of the metal center in both Ni-and Fe-containing forms of ARD, making it impossible to determine local structure in the active site by standard NMR methods.…”