A Structural Element That Facilitates Proton-Coupled Electron Transfer in Oxalate Decarboxylase

Abstract

The conformational properties of an active-site loop segment, defined by residues Ser161-Glu162-Asn163-Ser164, have been shown to be important for modulating the intrinsic reactivity of Mn(II) in the active site of Bacillus subtilis oxalate decarboxylase. We now detail the functional and structural consequences of removing a conserved Arg/Thr hydrogen bonding interaction by site-specific mutagenesis. Hence, substitution of Thr-165 by a valine residue gives an OxDC variant (T165V) that exhibits impaired catalytic activity. Heavy-atom isotope effect measurements, in combination with the X-ray crystal structure of the T165V OxDC variant, demonstrate that the conserved Arg/Thr hydrogen bond is important for correctly locating the side chain of Glu-162, which mediates a proton-coupled electron transfer (PCET) step prior to decarboxylation in the catalytically competent form of OxDC. In addition, we show that the T165V OxDC variant exhibits a lower level of oxalate consumption per dioxygen molecule, consistent with the predictions of recent spin-trapping experiments (Imaram et al., 2011, Free Rad. Biol. Med. 50, 1009–1015). This finding implies that dioxygen might participate as a reversible electron sink in two putative PCET steps and is not merely used to generate a protein-based radical or oxidized metal center.

Observation of superoxide production during catalysis of Bacillus subtilis oxalate decarboxylase at pH 4

“…the k cat / K M for decarboxylase activity in the T165V mutant is approximately ten times lower than in WT [64]. Saylor et al ascribe this to a hindered motion of the flexible SENS161–164 lid, giving it a preference for the open conformation which allows the intermediate CO 2 •− radical to escape into the surrounding solution at a higher rate [64]. Figure 4 shows that the rate of CO 2 •− radical escape is approximately ten times higher in T165V than in WT.…”

“…The trapped radical can therefore be considered as a signature of ‘true’ oxidase activity taking place within the protein. (B) Extra-protein superoxide production through the reaction of the CO 2 •− radical with dissolved dioxygen will also happen (see Saylor et al [64]). In fact it is expected to be enhanced in T165V because of the large amount of released CO 2 •− radical.…”

“…Expression and purification of recombinant His 6 -tagged Bacillus subtilis wild-type and T165V mutant OxDC was carried out following previously published procedures [27, 28, 33, 60, 63, 64]. Final preparation of the enzyme required a series of dialyses to transfer the protein into storage buffer (50 mM Tris-Cl, 0.5 M NaCl pH8.5), as well as to remove excess imidazole.…”

The Structure of Oxalate Decarboxylase at its Active pH

“…Based on kinetic isotope effect studies, a catalytic mechanism was proposed that involves radical intermediates with the surprising conjecture that dioxygen bound to the active-site Mn ion serves as a transient storage container for the electron removed from oxalate in an initial proton-coupled electron transfer (PCET) step (17). Evidence for the intermediate carbon dioxide radical anion, derived from oxalate was obtained by EPR spin trapping (10,11,15,18). A superoxide radical adduct was also observed under turnover conditions (19).…”

“…Yet, the presence of dioxygen is obligatory for catalysis and O2 is generally considered to act as a co-catalyst (6). Several crystal structures of the enzyme have appeared in the literature over the past 16 years all of which refer to enzyme poised at a pH of 7.5 or higher (7)(8)(9)(10)(11)(12)(13)(14). Yet, OxDC is only active at low pH with peak activity at or below pH4 and negligible activity above pH7 (15).…”