Gene Duplication and Fusion: Strategy for Active Site Control and Starting Point for New Catalysts

Abstract

Catalase–peroxidases (KatGs) have two peroxidase-like domains. The N-terminal domain contains the heme-dependent, bifunctional active site. Though the C-terminal domain lacks the ability to bind heme or directly catalyze any reaction, it has been proposed to serve as a platform to direct the folding of the N-terminal domain. Toward such a purpose, its I′-helix is highly conserved and appears at the interface between the two domains. Single and multiple substitution variants targeting highly conserved residues of the I′-helix were generated for intact KatG as well as the stand-alone C-terminal domain (KatGC). Single variants of intact KatG produced only subtle variations in spectroscopic and catalytic properties of the enzyme. However, the double and quadruple variants showed spectroscopic and catalytic properties similar to that observed for the N-terminal domain on its own (KatGN). The analogous variants of KatGC showed a much more profound loss of function as evaluated by their ability to return KatGN to its active conformation. These results suggest that the I′-helix is central to direct structural adjustments in the adjacent N-terminal domain. In particular, substitution of E695, a strictly conserved residue of the I’helix was especially destructive to KatG active site integrity. Available structures of KatG indicate that E695 is a central component of several hydrogen-bonded networks that also include strictly conserved R126 and W159 from the adjacent N-terminal domain. Both W159 (N-terminus of the D-helix) and R126 (BC helical connecting loop) are part of core structures of the peroxidase-like N-terminal domain with connections to the active site. This points to a potential connection between the I’-helix and its influence on active site conformation and function. To evaluate this hypothesis, we replaced R126, W159 and E695 with alanine, singly and in combination, not only for intact KatG, but also for the stand-alone KatGN or KatGC, as appropriate. Single variants of intact KatG showed a substantial loss of stability, particularly at the active site. The analogous variants of KatGC and KatGN showed a profound loss of function as evaluated by the return KatGN (or its variants) to a functional conformation, suggesting C-terminal domain through these interactions directs active site structural adjustments that are essential for catalytic function in the active site 25 Å away. The C-terminal domain not only directs conformational adjustments in its N-terminal domain partner, but due to its origin from gene duplication, also still retains the helical architecture of a typical peroxidase active site. This site lacks the residues necessary for catalytic activity or even heme binding. As such, the C-terminal domain appears to provide an ideal “blank slate” for engineering new heme-dependent catalysts. Spectroscopic measurements showed that a M616G/R617H variant of KatGC was sufficient to restore heme binding, producing a hexacoordinate low-spin ferric state. Additional modifications produced spectra very similar to those observed for KatGN.