Characterization of metal binding in the active sites of acireductone dioxygenase isoforms from Klebsiella ATCC 8724

Abstract

The two acireductone dioxygenase (ARD) isozymes from the methionine salvage pathway of Klebsiella ATCC 8724 present an unusual case in which two enzymes with different structures and distinct activities toward their common substrates (1,2-dihydroxy-3-oxo-5-(methylthio)pent-1-ene and dioxygen) are derived from the same polypeptide chain. Structural and functional differences between the two isozymes are determined by the type of M2+ metal ion bound in the active site. The Ni2+-bound NiARD catalyzes an off-pathway shunt from the methionine salvage pathway leading to the production of formate, methylthiopropionate, and carbon monoxide, while the Fe2+-bound FeARD' catalyzes the on-pathway formation of methionine precursor 2-keto-4-methylthiobutyrate and formate. Four potential protein-based metal ligands were identified by sequence homology and structural considerations. Based on the results of site-directed mutagenesis experiments, X-ray absorption spectroscopy (XAS), and isothermal calorimetry measurements, it is concluded that the same four residues, His96, His98, Glu102 and His140, provide the protein-based ligands for the metal in both the Ni- and Fe-containing forms of the enzyme, and subtle differences in the local backbone conformations trigger the observed structural and functional differences between the FeARD' and NiARD isozymes. Furthermore, both forms of the enzyme bind their respective metals with pseudo-octahedral geometry, and both may lose a histidine ligand upon binding of substrate under anaerobic conditions. However, mutations at two conserved nonligand acidic residues, Glu95 and Glu100, result in low metal contents for the mutant proteins as isolated, suggesting that some of the conserved charged residues may aid in transfer of metal from in vivo sources or prevent the loss of metal to stronger chelators. The Glu100 mutant reconstitutes readily but has low activity. Mutation of Asp101 results in an active enzyme that incorporates metal in vivo but shows evidence of mixed forms.

Figure 1

Solution structures of NiARD (left, PDB entry 1ZRR) and FeARD' (center, PDB entry 2HJI) from Klebsiella and crystallographic structure of MmARD from Mus musculus (right, PDB entry 1VR3). Letters reference to the Klebsiella ARD sequence as follows: A (Ala 2-Phe 6), B (Leu15-Ser18), C (Glu23-Lys31), D (Val33-Glu36), E (Thr52-Tyr57), E' (Ile61-Lys68), F (Ser72-Leu78), G (Lys85-Glu90), H (Phe92-Glu95), I (Arg104-Val107), J (Gly111-Ile117), K (Glu120-Leu125), L (Asn129-Ile132), M (His140-Met144), N (Phe150-Phe156), O (Trp162-Phe166), P (Ile171-Ala174). Features in the MmARD structure are lettered to show correspondence with the Klebsiella ARD structures. Blue spheres show position of Ni⁺² in NiARD and unknown metal in MmARD, grey sphere shows position of Fe⁺² in FeARD'. Figures were generated using Molscript (35).

Figure 2

Details of the metal binding site in NiARD (PDB entry 1ZRR) emphasizing residues mutated in this study and discussed in the text. The nickel is shown as a green sphere. For clarity, the two equatorial water ligands that complete the Ni coordination are not shown. Backbone CO-NH hydrogen bonds between Thr 97 and Ile 163 and ligand-metal bonds are shown as thin blue lines. View is from the vantage point of the C-terminal end of the G helix (see Figure 1).

Figure 3

Comparison of the upfield region of the ¹H NMR spectra of various ARD mutants and isoforms. Spectra are arranged so that similarities are emphasized. From top to bottom, E95A as isolated (purple), E102A (carmine), apo-WT ARD (green), FeARD' (red), H98S (carmine), E100A as isolated (green), E100A reconstituted with Ni⁺² (red) and NiARD (purple). All resonances in this region are due to methyl groups in close-packing arrangements with aromatic side chains (15). Note that both E95A and E102A show fewer resonances in this region, indicating a lower degree of order in their structures. ApoARD and FeARD' have very similar folds, as does H98S (15). Apo-E100A shows some similarity to H98S, but after reconstitution with Ni⁺² shows a high degree of similarity with NiARD. Detailed resonance assignments of H98S and NiARD are available from the BMRB database (www.bmrb.wisc.edu), accession numbers 7103 and 4313 respectively.

Figure 4

Fe K-edge XANES of the resting state (black) and ES complex (red, offset) of FeARD'. The region around 7114 eV corresponds to the 1s → 3d transition. Insert: First derivative of the XANES in the edge region.

Figure 5

Fe K-edge (k = 2−12.5 Å⁻¹) EXAFS spectrum for resting FeARD'. Top: k³-weighted spectrum (red line) fit (black line) and difference (data-fit; blue line). Bottom: Fourier-transformed data, fit and difference. The fit shown was calculated for 2 O @ 2.15 Å + 4 N @ 1.98 Å, including 3 histidine ligands, and corresponds to fit R13 in the supplementary table.

Figure 6

Fe K-edge (k = 2−12.5 Å⁻¹) EXAFS spectrum for FeARD' ES complex. Top: k³-weighted spectrum (red line) fit (black line) and difference (data-fit; blue line). Bottom: Fourier-transformed data, fit and difference. The fit shown was calculated for 3 O @ 2.15 Å + 3 N @ 1.98 Å including 1 histidine ligand and corresponds to fit ES08 in the supplementary table.

Figure 7

Comparison of the Fe K-edge Fourier-transformed EXAFS spectra (k − 2 −12.5 Å⁻¹) of FeARD' (solid) and Ni K-edge EXAFS spectra of NiARD (dashed) of the resting state (top) and ES complex (bottom) from Klebsiella ATCC 8724.