Active site geometry of oxalate decarboxylase from Flammulina velutipes: Role of histidine-coordinated manganese in substrate recognition

Abstract

Oxalate decarboxylase (OXDC) from the wood-rotting fungus Flammulina velutipes, which catalyzes the conversion of oxalate to formic acid and CO(2) in a single-step reaction, is a duplicated double-domain germin family enzyme. It has agricultural as well as therapeutic importance. We reported earlier the purification and molecular cloning of OXDC. Knowledge-based modeling of the enzyme reveals a beta-barrel core in each of the two domains organized in the hexameric state. A cluster of three histidines suitably juxtaposed to coordinate a divalent metal ion exists in both the domains. Involvement of the two histidine clusters in the catalytic mechanism of the enzyme, possibly through coordination of a metal cofactor, has been hypothesized because all histidine knockout mutants showed total loss of decarboxylase activity. The atomic absorption spectroscopy analysis showed that OXDC contains Mn(2+) at up to 2.5 atoms per subunit. Docking of the oxalate in the active site indicates a similar electrostatic environment around the substrate-binding site in the two domains. We suggest that the histidine coordinated manganese is critical for substrate recognition and is directly involved in the catalysis of the enzyme.

Fig. 1.

Multiple sequence alignment of OXDC with the other double-domain germin family proteins. Alignment of oxalate-decarboxylase (OXDC) with phaseolin (2phl), canavalin (1cau), oxalate-co A-decarboxylate (OXAOXA), hypothetical proteins from Bacillus subtilis (B. sub) and Synechocystis (Syn). Also shown is the alignment with single-domain oxalate-oxidase (OXOX). The germin box region within each of the domains is marked. The residues of OXDC that interact with oxalate and the corresponding residues from the other proteins have been highlighted.

Fig. 2.

Structural model of OXDC. Stereoview of the ribbon drawing showing trimeric protein. Each monomer of the trimer, with two structurally equivalent domains, is shown in a different color. The three histidines in each domain, which appear to juxtapose for metal coordination, are indicated in blue. The possible metal site is shown in black.

Fig. 3.

Site-directed mutations in OXDC delineate functionally critical histidines. (A) Immunoblot showing the presence of 55-kD OXDC expressed in pROD and all the mutants for histidine. The native OXDC showed a 64-kD band, whereas the vector (pREP1) did not show any signal. The soluble protein fraction (equivalent to 25 μg protein) from the wild type and mutants along with 50 ng of native OXDC was subjected to 10% SDS-PAGE followed by immunoblot analysis using OXDC antibody. The molecular mass of the proteins is indicated. (B) The OXDC activity in the wild type and all mutants was determined as nmole CO₂ evolved per mg of protein per min at 37°C. Mean values of triplicate determinations are presented.

Fig. 4.

Oligomeric organization of OXDC. The purified protein from F. velutipes was subjected to 6% nondenaturating PAGE under nonreducing conditions (A and B) and detected by staining with silver stain (A) and immunoblot analysis using anti-OXDC antibody (B). On 10% SDS-PAGE, the protein was analyzed under different conditions as indicated and detected by silver staining (C) or immunoblot (D). The overexpressed and mutant OXDC were analyzed on 10% SDS-PAGE under native conditions as indicated and detected by immunoblot (E). Monomers (M), trimers (T), and hexamers (H) are indicated by arrows. Lanes: C, catalase; F, ferritin; T, thyroglobulin; O, oxalate decarboxylase; N, native dye; NS, native dye with SDS; NM, native dye with βME; NSM, native dye with SDS and βME; FV, F. velutipes.

Fig. 5.

Hexameric assembly of OXDC. Stereoview of space-filling model of the dimer of trimers shown (A) along the threefold axis of the hexamer and (B) along an axis perpendicular to the threefold axis. The three subunits of the trimeric subassembly are shown in red, green, and blue. The top trimer is in light colors, and the bottom trimer is in dark colors.

Fig. 6.

Binding of oxalate to recombinant and mutant OXDC expressed in fission yeast. A typical decarboxylation reaction without cold substrate was initiated with 10 nmoles of ¹⁴C-oxalic acid and 5 μg equivalent protein extract from recombinant or each independent mutant OXDC. The binding of oxalic acid was determined by percent incorporation of radioactivity in end product (CO₂) and remaining reaction mix. The fractions were prepared by trapping CO₂, followed by mechanical separation of bound OXDC and free oxalic acid.

Fig. 7.

Geometric description of oxalic acid interactions in two domains of OXDC. The interaction of oxalic acid with residues of OXDC and manganese ion in the N-terminal domain (A) and the C-terminal domain (B). The backbone is shown as a ribbon (green) and sidechains as rods. Manganese is shown in black. A water molecule completing metal coordination is also shown. The hydrogen bonds are depicted as thin lines.