Metal-Dependent Function of a Mammalian Acireductone Dioxygenase

Abstract

The two acireductone dioxygenase (ARD) isozymes from the methionine salvage pathway of Klebsiella oxytoca are the only known pair of naturally occurring metalloenzymes with distinct chemical and physical properties determined solely by the identity of the divalent transition metal ion (Fe(2+) or Ni(2+)) in the active site. We now show that this dual chemistry can also occur in mammals. ARD from Mus musculus (MmARD) was studied to relate the metal ion identity and three-dimensional structure to enzyme function. The iron-containing isozyme catalyzes the cleavage of 1,2-dihydroxy-3-keto-5-(thiomethyl)pent-1-ene (acireductone) by O2 to formate and the ketoacid precursor of methionine, which is the penultimate step in methionine salvage. The nickel-bound form of ARD catalyzes an off-pathway reaction resulting in formate, carbon monoxide (CO), and 3-(thiomethyl) propionate. Recombinant MmARD was expressed and purified to obtain a homogeneous enzyme with a single transition metal ion bound. The Fe(2+)-bound protein, which shows about 10-fold higher activity than that of others, catalyzes on-pathway chemistry, whereas the Ni(2+), Co(2+), or Mn(2+) forms exhibit off-pathway chemistry, as has been seen with ARD from Klebsiella. Thermal stability of the isozymes is strongly affected by the metal ion identity, with Ni(2+)-bound MmARD being the most stable, followed by Co(2+) and Fe(2+), and Mn(2+)-bound ARD being the least stable. Ni(2+)- and Co(2+)-bound MmARD were crystallized, and the structures of the two proteins found to be similar. Enzyme-ligand complexes provide insight into substrate binding, metal coordination, and the catalytic mechanism.

Figure 1. Thermal stability of MmARD as a function of the bound metal ion

The data indicate that Ni²⁺-bound MmARD has the highest melting temperature (58 °C) and Mn2+-bound MmARD has the lowest melting temperature (43 °C).

Figure 2. Quantitation of on-pathway and off-pathway products by MmARD bound to different metal ions

The quantitation of ARD catalyzed oxidation of desthio-acireductone was done for a reaction mixture volume of 1mL. The error bars are represented as standard deviations.

Figure 3. X-ray crystal structure of Ni-MmARD

a) The X-ray crystal structure of Ni-MmARD with the Ni atom shown as a green sphere, b) The active site with the Ni atom as a green sphere and its protein ligands H88, H90, H133, E94 represented as sticks and two water molecules shown as red spheres forming an octahedral coordination geometry. The metal co-ordination distances are shown as red dotted lines with distances measured in Å.

Figure 4. Metal identity established by anomalous scattering experiments

a) Anomalous electron density map of Ni-MmARD at 8.4328 keV showing the anomalous signal (green) of Ni (green sphere) in the active site; b) anomalous electron density map of Co-MmARD at 7.8089 keV showing the anomalous signal (magenta) of Co (magenta sphere) in the active site.

Figure 5. The active site of Ni-MmARD showing the on-pathway product KMTB

The Ni atom is shown as a green sphere. KMTB and active site residues are shown in stick representation. Waters are shown as small red spheres. The residues F84, F105, F135, A145, V143 and I98 which interact with the alkyl group of KMTB are shown in yellow. KMTB is within hydrogen bonding distance of R96 and with the two water molecules bound to Ni. The hydrogen bonding distances are shown as red dotted lines with distances measured in Å.

Figure 6. The active site of Ni-MmARD showing the off-pathway product analog valeric acid (VA)

The Ni atom is shown as a green sphere. VA and active site residues are shown in stick representation. Waters are shown as small red spheres. The residues F84, F105, F135, A145, V143 and I98 which interact with the alkyl group of VA are shown in yellow. VA is within hydrogen bonding distance of R96 and with one of the two water molecules bound to Ni. The hydrogen bonding distances are shown as red dotted lines with distances measured in Å.

Figure 7. The active site of Ni-MmARD showing D-Lactic acid (DLA)

The Ni atom is shown as a green sphere. DLA and active site residues are shown in stick representation. DLA is coordinating with Ni²⁺ and is within hydrogen bonding distance of R96. Residues F84, F105 and F135 which interact with the alkyl group of DLA are shown in yellow. The hydrogen bonding distances are shown as red dotted lines with distances measured in Å.

Scheme 1. Methionine Salvage Pathway

Scheme 2. ARD reactions using model E1 substrate reaction

Scheme 3. Proposed mechanisms of Ni-ARD versus Fe-ARD enzymatic catalysis explained by Chelate hypothesis

The results of incorporation of ¹⁸O and ¹⁴C labeling studies are indicated by the blue O atoms and the asterisk. (Figure adapted from ref 39).