Metalloenzyme design and engineering through strategic modifications of native protein scaffolds

Abstract

Metalloenzymes are among the major targets of protein design and engineering efforts aimed at attaining novel and efficient catalysis for biochemical transformation and biomedical applications, due to the diversity of functions imparted by the metallo-cofactors along with the versatility of the protein environment. Naturally evolved protein scaffolds can often serve as robust foundations for sustaining artificial active sites constructed by rational design, directed evolution, or a combination of the two strategies. Accumulated knowledge of structure-function relationship and advancement of tools such as computational algorithms and unnatural amino acids incorporation all contribute to the design of better metalloenzymes with catalytic properties approaching the needs of practical applications.

Figure 1

Recent examples of rational metalloenzyme design. (a) Fe_BMb, designed prior to structure elucidation of NOR, is structurally similar to NOR and displays NOR activity [16]. Incorporation of I107E mutation doubles nitric oxide reduction activity [17]. (b) Biotin conjugated RhCp*^biotinCl₂ catalyst anchored in streptavidin, with additional secondary coordination sphere interactions catalyzes C-H bond activation (From reference [29]. Reprinted with permission from AAAS) and asymmetric transfer hydrogenation (Reprinted with permission from reference [30]. Copyright (2012) American Chemical Society). (c) Design strategy for RosettaMatch design of BpyAla-based metal binding site. Crystal structure of Co (pink) bound form of the designed protein, compared with the predicted model (gray). Reprinted with permission from reference [26], Copyright (2013) American Chemical Society.

Figure 2

(a) Reactions catalyzed by cytochrome P450 variants. (b) 3D structure of hRXRα (left, PDB 1RXR) and ligase 10C (right, PDB 2LZE, flexible termini omitted for clarity).

Figure 3

(a) Flowchart of cofactor switch guide for the KARI enzyme family [50]. (b) Top: the designed organophosphate hydrolysis reaction; Bottom: transition state of the reaction and designed complementary hydrogen bonding interactions by RosettaMatch algorithm or shape complementarity by RosettaDesign algorithm. Reprinted by permission from Macmillan Publishers Ltd: [Nature Chemical Biology] (reference [53]), copyright (2012).