Substitution of a conserved catalytic dyad into 2‐KPCC causes loss of carboxylation activity

Abstract

The characteristic His-Glu catalytic dyad of the disulfide oxidoreductase (DSOR) family of enzymes is replaced in 2-ketopropyl coenzyme M oxidoreductase/carboxylase (2-KPCC) by the residues Phe-His. 2-KPCC is the only known carboxylating member of the DSOR family and has replaced this dyad potentially to eliminate proton-donating groups at a key position in the active site. Substitution of the Phe-His by the canonical residues results in production of higher relative concentrations of acetone versus the natural product acetoacetate. The results indicate that these differences in 2-KPCC are key in discriminating between carbon dioxide and protons as attacking electrophiles.

The reactive form of a C–S bond–cleaving, CO2-fixing flavoenzyme

“…The enolacetone preferentially reacts with CO 2 when it is saturating to form acetoacetate (8), as shown in prior work. The parameter k cat /K m under CO 2 -saturated conditions has the same bell shape for its pH-rate profile and the same pK a values as in the absence of CO 2 .…”

“…The presence of an available proton so close to 2-KPCC's enolacetone intermediate ( Fig. 4), however, appears to favor formation of the protonated rather than the carboxylated product, according to prior work with the F501H mutant (8).…”

“…Enclosing the 2-KPCC active site in a hydrophobic compartment that lacks an active site histidine is essential for promoting carboxylation instead of protonation of the enolacetone intermediate (8). By invoking a shared proton between Cys CT and Cys INT in reduced 2-KPCC, we can now propose a mechanism for the oxidative half-reaction in which the acid-base functions of the active site histidine are performed instead by Cys CT (Fig.…”

“…The reactions were conducted as described above with the pH varied from 6.5 to 9.5 (200 mM glycine-Tris-phosphate buffer with 200 mM NaCl) in 0.5 pH unit increments. All reactions were carried out on a 1-ml scale with addition of NADPH from a stock of 10 mM to achieve a final concentration of 100 M. Stock solutions of 2-KPC (10 and 100 mM) were made by dissolving solid powder in the working buffer pH and were then added to the reaction cuvette prior to enzyme addition to achieve final concentrations ranging from 100 to 4000 M. For reactions carried out in the presence of CO 2 , 60 mM KHCO 3 was added to each buffer immediately prior to use, consistent with prior work (8). As a control, a plot of specific activity as a function of pH was measured at 10, 20, and 50 mM KHCO 3 , confirming that 60 mM KHCO 3 indeed supplies a saturating concentration of CO 2 across the pH range used (pH 6.5-9.5).…”

“…We previously showed that substitution of a histidine at this position (F501H) completely shifted the product outcome from acetoacetate (carboxylation product) to acetone (protonation product) (8). The hydrophobic residue at this position in 2-KPCC seemingly steers the reactive enolacetone intermedi-ate toward carboxylation and away from protonation.…”