Kinetic and spectroscopic studies of bicupin oxalate oxidase and putative active site mutants

Abstract

Ceriporiopsis subvermispora oxalate oxidase (CsOxOx) is the first bicupin enzyme identified that catalyzes manganese-dependent oxidation of oxalate. In previous work, we have shown that the dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be unprotonated. CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC) and the 241-244DASN region of the N-terminal Mn binding domain of CsOxOx is analogous to the lid region of OxDC that has been shown to determine reaction specificity. We have prepared a series of CsOxOx mutants to probe this region and to identify the carboxylate residue implicated in catalysis. The pH profile of the D241A CsOxOx mutant suggests that the protonation state of aspartic acid 241 is mechanistically significant and that catalysis takes place at the N-terminal Mn binding site. The observation that the D241S CsOxOx mutation eliminates Mn binding to both the N- and C- terminal Mn binding sites suggests that both sites must be intact for Mn incorporation into either site. The introduction of a proton donor into the N-terminal Mn binding site (CsOxOx A242E mutant) does not affect reaction specificity. Mutation of conserved arginine residues further support that catalysis takes place at the N-terminal Mn binding site and that both sites must be intact for Mn incorporation into either site.

Figure 1. The reaction catalyzed by Ceriporiopsis subvermispora oxalate oxidase.

Figure 2. Model of free-radical mechanisms for oxalate oxidase and oxalate decarboxylase.

Modified from .

Figure 3. Manganese binding sites of the oxalate decarboxylase monomer and homology models of the manganese binding sites of CsOxOx.

(A) OxDC (PDB ID 1UW8) with manganese ions (purple), metal coordinating residues (atoms colored as follows: C, cyan; N, blue; O, red), conserved active site arginine residues (dark blue) and the N-terminal lid region (green) highlighted. (B) Homology model of the N-terminal CsOxOx Mn binding site metal coordinating residues and the DASN of the lid region. (C) Homology model of the C-terminal CsOxOx Mn binding site metal coordinating residues. The homology model of CsOxOx was constructed using its amino acid sequence and the experimentally solved structure of Bacillus subtilis OxDC (PDB ID 1UW8) using Swiss-Model (The Swiss Institute of Bioinformatics) , , . Figure generated using Pymol (The PyMOL Molecular Graphics System, Schrödinger, LLC).

Figure 4. The effect of pH on the affinity of recombinant CsOxOx D241A for oxalate.

pH dependence of CsOxOx D241A mutant on kinetic parameters V_max/K_m (•) and V_max (▴) for the CsOxOx catalyzed reaction.

Figure 5. CD spectra of recombinant, wild-type CsOxOx and the putative active site mutants.

All samples were 938 ug/mL in phosphate buffer (pH 7.0): wild-type CsOxOx, black; D241A, dark blue; D241S, grey; A242E, orange; DASN241-244SENS, red; R169K, cyan; R349K, yellow.

Figure 6. EPR spectra of PBN radical adducts obtained from the incubation of 100

mM oxalate, 100 mM KCl, 20 mM PBN, and 50 µM CsOxOx. (A) PBN-oxalate derived radical adduct from the recombinant, wild type CsOxOx catalyzed reaction. (B) PBN-oxalate derived radical adduct from the CsOxOx A242E mutant catalyzed reaction showed no signal over background. (C) PBN-oxalate derived radical adduct from the CsOxOx D241A mutant catalyzed reaction. (D) All reaction components without enzyme.

Figure 7. X-band EPR of the Mn(II) centers of wild type CsOxOx and CsOxOx A242E mutant under several different conditions at 5

K. 7A: wild type CsOxOx in imidazole buffer at pH 7.0, as taken from the original preparation. 7B: CsOxOx A242E mutant in imidazole buffer at pH 7.0, as taken from the original preparation. 7C: Same sample as 7A, thawed and after addition of 100 mM acetate buffer pH 4.0. 7D: Same sample as 7B, thawed and after addition of 100 mM acetate buffer pH 4.0. 7E: Same sample as in 7C thawed and after addition of 50 mM oxalate and allowed to further react for approximately 2 min. 7F: Same sample as in 7D thawed and after addition of 50 mM oxalate and allowed to further react for approximately 2 min.

Figure 8. pH dependence of recombinant, wild-type CsOxOx and A242E CsOxOx mutant at 416.0

GHz. 8A: Recombinant, wild type CsOxOx A: Same sample as C after the addition of 100 mM glycine buffer, pH 3.0. B: Same sample as C after the addition of 100 mM acetate buffer, pH 4.0. C: in 25 mM Imidazole-Cl, pH 7.0. 8B: A242E CsOxOx mutant. A: Same sample as B after the addition of 100 mM acetate buffer, pH 4.0, B: in 25 mM Imidazole-Cl, pH 7.0.