One protein, two enzymes revisited: a structural entropy switch interconverts the two isoforms of acireductone dioxygenase

Abstract

Acireductone dioxygenase (ARD) catalyzes different reactions between O2 and 1,2-dihydroxy-3-oxo-5-(methylthio)pent-1-ene (acireductone) depending upon the metal bound in the active site. Ni2+ -ARD cleaves acireductone to formate, CO and methylthiopropionate. If Fe2+ is bound (ARD'), the same substrates yield methylthioketobutyrate and formate. The two forms differ in structure, and are chromatographically separable. Paramagnetism of Fe2+ renders the active site of ARD' inaccessible to standard NMR methods. The structure of ARD' has been determined using Fe2+ binding parameters determined by X-ray absorption spectroscopy and NMR restraints from H98S ARD, a metal-free diamagnetic protein that is isostructural with ARD'. ARD' retains the beta-sandwich fold of ARD, but a structural entropy switch increases order at one end of a two-helix system that bisects the beta-sandwich and decreases order at the other upon interconversion of ARD and ARD', causing loss of the C-terminal helix in ARD' and rearrangements of residues involved in substrate orientation in the active site.

Figure 1

An outline of the methionine salvage pathway in Klebsiella ATCC 8724. Carbon atoms 1, 2 and 3 in acireductone 4 are labeled for reference in the text.

Figure 2

Comparison of the active sites of H98S (left), ARD′ (current work, PDB 2HJI, center), and ARD (PDB 1ZRR, ref. 9) right. The H98S active site structure was determined using experimentally determined NOE and RDC data. The ARD′ active site structure shows Fe⁺² as an umber sphere and the ARD active site shows the Ni⁺² ion in green. The positions of backbone heavy atoms and β-carbons of ligands His 96, His 98, Glu 102 and His 140 in the ARD′ structure are based on NOE data and residual dipolar couplings from H98S ARD, with metal-ligand bond lengths determined by Fe XAS of ARD′. The corresponding atom positions in the ARD structure are based on the crystal structure of mouse ARD (1VR3, see text) () and bond lengths determined from Ni XAS experiments (). Note that the indole sidechain of Trp 162 partially occludes the active site opening in ARD. In H98S and ARD′, the polypeptide including Trp 162 is disordered and does not occlude the active site. All three sites are shown in approximately the same orientation using the backbone atom positions of the structurally conserved β-sandwich (residues shown in yellow, Pro 137-Phe 142) as reference.

Figure 3

a) Ring current-shifted aliphatic amino acid methyl resonances in the 600 MHz ¹H NMR spectra of ARD (green trace), H98S ARD (red trace) and ARD′ (blue trace) with resonance assignments shown. Note the similar patterns for H98S ARD and ARD′, with different shift patterns for ARD. b) Overlay of the ¹H, ¹⁵N HSQC spectra of ARD′ (peaks shown in red) and H98S ARD (peaks shown in black). Note close correspondence of most peaks in the spectra. Peaks that occur in the H98S spectrum but not in the ARD′ spectrum (labeled) are within ~9 Å of the metal center. The indole NɛH of Trp 162 (lower left) is observed in both ARD′ and H98S ARD (see text for discussion). c) Overlay of the ¹H, ¹⁵N HSQC spectra of ARD (peaks shown in red) and H98S ARD (peaks shown in black). Different shift patterns indicate significant differences in the tertiary structure. All spectra were taken at 25 °C, pH 7.4 at 600 MHz ¹H observe frequency.

Figure 3

a) Ring current-shifted aliphatic amino acid methyl resonances in the 600 MHz ¹H NMR spectra of ARD (green trace), H98S ARD (red trace) and ARD′ (blue trace) with resonance assignments shown. Note the similar patterns for H98S ARD and ARD′, with different shift patterns for ARD. b) Overlay of the ¹H, ¹⁵N HSQC spectra of ARD′ (peaks shown in red) and H98S ARD (peaks shown in black). Note close correspondence of most peaks in the spectra. Peaks that occur in the H98S spectrum but not in the ARD′ spectrum (labeled) are within ~9 Å of the metal center. The indole NɛH of Trp 162 (lower left) is observed in both ARD′ and H98S ARD (see text for discussion). c) Overlay of the ¹H, ¹⁵N HSQC spectra of ARD (peaks shown in red) and H98S ARD (peaks shown in black). Different shift patterns indicate significant differences in the tertiary structure. All spectra were taken at 25 °C, pH 7.4 at 600 MHz ¹H observe frequency.

Figure 3

a) Ring current-shifted aliphatic amino acid methyl resonances in the 600 MHz ¹H NMR spectra of ARD (green trace), H98S ARD (red trace) and ARD′ (blue trace) with resonance assignments shown. Note the similar patterns for H98S ARD and ARD′, with different shift patterns for ARD. b) Overlay of the ¹H, ¹⁵N HSQC spectra of ARD′ (peaks shown in red) and H98S ARD (peaks shown in black). Note close correspondence of most peaks in the spectra. Peaks that occur in the H98S spectrum but not in the ARD′ spectrum (labeled) are within ~9 Å of the metal center. The indole NɛH of Trp 162 (lower left) is observed in both ARD′ and H98S ARD (see text for discussion). c) Overlay of the ¹H, ¹⁵N HSQC spectra of ARD (peaks shown in red) and H98S ARD (peaks shown in black). Different shift patterns indicate significant differences in the tertiary structure. All spectra were taken at 25 °C, pH 7.4 at 600 MHz ¹H observe frequency.

Figure 4

Comparison of the structures of ARD′ (A) and ARD (B). Letters reference to the ARD sequence as follows: A (Ala 2-Phe 6), B (Leu 15-Ser 18), C (Glu 23-Lys 31), E (Thr 50-Tyr 57), E′ (Ile 61-Lys 68), F (Ser 72-Leu 78), G (Lys 85-Glu 90), H (Phe 92-Glu 95), I (Arg 104-Val 107), J (Gly 111-Ile 117), K (Glu 120-Leu 125), L (Asn 129-Ile 132), M (His 140-Met 144), N (Phe 150-Phe 156), O (Gly 161-Gly 168), P (Ile 171-Ala 174). The positions of metal ions are indicated by gray (Fe⁺²) and blue (Ni⁺²) spheres. Residues 157-175 (loop O and helix P in ARD) are disordered in ARD′, and so for clarity are not shown in Figure 4A. C) Positions of heavy atoms in structure 10 of the ensemble shown in Fig. 4D. This structure is closest to the average of the ensemble. Residues close to the active site are shown in neon, Phe 92-Arg 104 in magenta and Val 134-Asp 143 in yellow. The position of the iron is indicated by a green sphere. D) Superposition of 14 accepted structures of ARD′. Statistics are shown in Table 1. Residues 39-49 are shown in green, and residues 65-73 are shown in red.

Figure 5

Top) Relative displacement of the E and E′ helices in ARD and ARD′. For this comparison, the backbone coordinates of the conserved β-sandwiches of both proteins were superimposed, but only the helices and leading/following peptides are shown. ARD is shown in red, ARD′ in blue neon. Bottom) Side chain packing differences in ARD and ARD′ using the same superposition as in the top figure. Aromatic residues that give rise to ring current shifts observed in Fig. 3a are shown in light lines, shifted aliphatic side chains are shown in neon. Red corresponds to ARD, blue to ARD′.

Figure 6

Proposed mechanisms for regioselectivity in ARD′ (left) and ARD (right). Bidentate ligation of the metal by the substrate via the O1 and O2 positions (ARD′) versus O1 and O3 positions (ARD) results in Lewis acid activation of the O2 position in ARD′ and O3 position in ARD for intramolecular nucleophilic attack by peroxide anion, giving ring closure to the 4-membered cyclic peroxide in the case of ARD′ and the 5-membered cyclic peroxide in the case of ARD. For more detail concerning these mechanisms, see references and .

Figure 7

Comparison of the active sites of ARD′ (left) and ARD (right) modeled with substrate acireductone 4 bound. Modeled structures were generated using AMBER 8.0 (). Initial structure for ARD′ was from the current work, starting structure for ARD was from PDB entry 1ZRR (). In both cases, the two equatorial water ligands were replaced by oxygens from acireductone (shown in blue in both structures). In ARD′, 4 is ligated to the Fe⁺² via O1 and O2 (ACRC), while in ARD, 4 is ligated to Ni⁺² via O1 and O3 (ACRT). Complexes were subjected to 4000 steps of minimization to remove close contacts. The two structures are not in the same orientations, but are rotated to obtain clear views. Due to paramagnetic broadening, the positions of side chains within the active site of ARD (1ZRR) are not as precisely defined as in the ARD′ structure. Figures were generated using MOLMOL ().