Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 285 (52), 40515-24

Identification and Characterization of Oxalate Oxidoreductase, a Novel Thiamine Pyrophosphate-Dependent 2-oxoacid Oxidoreductase That Enables Anaerobic Growth on Oxalate

Affiliations

Identification and Characterization of Oxalate Oxidoreductase, a Novel Thiamine Pyrophosphate-Dependent 2-oxoacid Oxidoreductase That Enables Anaerobic Growth on Oxalate

Elizabeth Pierce et al. J Biol Chem.

Abstract

Moorella thermoacetica is an anaerobic acetogen, a class of bacteria that is found in the soil, the animal gastrointestinal tract, and the rumen. This organism engages the Wood-Ljungdahl pathway of anaerobic CO(2) fixation for heterotrophic or autotrophic growth. This paper describes a novel enzyme, oxalate oxidoreductase (OOR), that enables M. thermoacetica to grow on oxalate, which is produced in soil and is a common component of kidney stones. Exposure to oxalate leads to the induction of three proteins that are subunits of OOR, which oxidizes oxalate coupled to the production of two electrons and CO(2) or bicarbonate. Like other members of the 2-oxoacid:ferredoxin oxidoreductase family, OOR contains thiamine pyrophosphate and three [Fe(4)S(4)] clusters. However, unlike previously characterized members of this family, OOR does not use coenzyme A as a substrate. Oxalate is oxidized with a k(cat) of 0.09 s(-1) and a K(m) of 58 μM at pH 8. OOR also oxidizes a few other 2-oxoacids (which do not induce OOR) also without any requirement for CoA. The enzyme transfers its reducing equivalents to a broad range of electron acceptors, including ferredoxin and the nickel-dependent carbon monoxide dehydrogenase. In conjunction with the well characterized Wood-Ljungdahl pathway, OOR should be sufficient for oxalate metabolism by M. thermoacetica, and it constitutes a novel pathway for oxalate metabolism.

Figures

FIGURE 1.
FIGURE 1.
SDS-PAGE of purified OOR. Left lane, molecular mass marker (sizes shown in kDa); right lane, 6 μg of purified OOR.
FIGURE 2.
FIGURE 2.
Sedimentation equilibrium analysis of OOR. The top panel shows a global fit of analytical ultracentrifugation data for three different concentrations of OOR collected at 6000 rpm. Absorbance data were recorded at 430, 460, and 470 nm for 8.3 μm (triangles), 20.7 μm (squares), and 32.3 μm (circles) OOR concentrations, respectively. The solid line through the points is the weighted best least squares fit to an ideal single-species model. Residuals for each fit are shown in the bottom panel. A vertical offset was applied to the residuals from the 8.3 μm (triangles) and 32.3 μm (circles) concentrations for clarity.
FIGURE 3.
FIGURE 3.
Schematic of OOR peptides. Top, arrangement of the three peptide sequences as they align with pyruvate:ferredoxin oxidoreductases from D. africanus and M. thermoacetica. Each rectangle represents a separate gene product. Locations of conserved residues that may be involved in iron-sulfur cluster, TPP, Mg2+, and substrate binding are shown. All residues proposed to ligate the [Fe4S4] clusters are cysteines. Bottom, expanded view of the β subunit of OOR showing both conserved and non-conserved residues that align with the [Fe4S4] cluster and TPP-binding residues of D. africanus PFOR.
FIGURE 4.
FIGURE 4.
Oxalate and pH dependence of OOR activity. A, activity was measured at 25 °C in 50 mm Tris-HCl, pH 7.9, with 10 mm methyl viologen and with varying oxalate concentrations (closed circles) or 1 mm oxalate and varying coenzyme A concentrations (open circles). B, activity was measured at 25 °C in MES (open circles), sodium phosphate (closed circles), borate (open squares), and N-cyclohexyl-3-amino-propanesulfonic acid (closed squares).
FIGURE 5.
FIGURE 5.
UV-visible spectra of OOR. Solid line, as-isolated protein; dashes and dots, oxidized protein; short dashes, oxalate-reduced protein; dots, dithionite-reduced protein. Spectra were measured anaerobically. 4.1 μm protein was prepared in 50 mm sodium phosphate, pH 7.0. Reduced protein was prepared by adding 10 μm sodium oxalate or 15 μm sodium dithionite to the as-isolated protein sample, and the spectra shown were recorded after 20 min. Oxidized protein was prepared by incubation in 6.8 mm CO2 with 80 nm CODH/ACS from M. thermoacetica.
FIGURE 6.
FIGURE 6.
EPR spectra of OOR. 39 μm OOR was reduced with oxalate and then titrated with oxidized cytochrome c. A, as-isolated OOR (1.2 spins/monomer); B, oxalate-reduced OOR (3.4 spins/monomer); C, dithionite-reduced OOR (3.2 spins/monomer); D–F, oxalate reduced OOR, reoxidized by the addition of oxidized cytochrome c (D, 1.3 spins/monomer, E, 0.7 spins/monomer, F, 0.07 spins/monomer). The parameters were as follows: receiver gain, 2 × 102; modulation frequency, 100 kHz; modulation amplitude, 10 G; center field, 3450 G; sweep width, 700 G; microwave power, 0.129 milliwatt at 9 K.
FIGURE 7.
FIGURE 7.
Proposed OOR mechanism. Oxalate binds to OOR (step 1) and undergoes nucleophilic attack by the TPP ylide (step 2), generating oxalyl-TPP, which could be stabilized by protonation from a general base (step 3). Decarboxylation (step 4) leaves an anionic intermediate that would release two electrons to the [Fe4S4] clusters (step 5). The carboxyl-TPP that remains could be decarboxylated to release a second CO2 molecule (step 6) or hydrolyzed to release bicarbonate (not shown), regenerating the starting form of the enzyme.

Similar articles

See all similar articles

Cited by 10 articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback