Chorismate mutase randomization

It is in the realm of very large combinatorial libraries that selection rather than screening gains crucial importance. As the focus shifts from randomizing an eight-residue peptide to a 100 amino acid protein (the typical size of a small functional domain, for example a chorismate mutase domain), the number of sequence permutations rises to an astronomical 20100. The ability to assay even a tiny fraction of this sequence space in directed molecular evolution experiments demands selection, even though initial development of an appropriate system may be considerably more involved than the setup of a screening procedure. 

To test these possibilities, random segments of four to seven residues were inserted into the middle of the EcCM HI helix [95]. The individual libraries (designated L4, L5, L6 and L7) have a maximum theoretical diversity of 160,000 (204), 3.2 x 106 (205), 6.4 x 107 (206), and 1.28 x 109 (207) distinct members, respectively. In each case, transformation of chorismate mutase-deficient bacteria yielded roughly 107 clones, giving fully diverse and redundant coverage of the L4 and L5 libraries, 10 % sequence coverage of the L6 library, and 1 % coverage of the L7 library. 

To explore the feasibility of such an approach for the design of active catalysts, we have systematically replaced the secondary structural elements in the homodimeric helical bundle chorismate mutase (Fig. 3.18) with binary-patterned units of random sequence. Genetic selection was then used to assess the catalytic capabilities of the proteins in the resulting libraries, providing quantitative information about the robustness of this particular protein scaffold and insight into the subtle interactions needed to form a functional active site [119]. 

In the first stage of our experiment, modules that correspond to the HI and H2/H3 helices were combined individually with the appropriate complementary wild-type helical segments of the thermostable MjCM , a protease-resistant version of the dimeric M.jannaschii chorismate mutase (Fig. 3.18) [37]. This amounted to randomizing 37 % and 42 % of the entire protein for the HI and H2/H3 replacement modules,... 

Fig. 3.18. Generation of large binary-patterned libraries ofAroQ chorismate mutase genes. (A) A two-stage strategy was adopted involving separate randomization and selection of functional variants ofthe HI (light gray) and H2/H3 helices (dark gray), followed by combination of functional binary-patterned segments from the initial libraries and reselection. In the final constructs, 80 % of the protein was randomized. (B) The...

In another study, the promiscuous chorismate mutase activity of isochorismate pyruvate-lyase (PchB) was used to derive mechanistic insights into its native activity (isochorismate pyruvate lyase). Presumed key active-site residues were randomized, and the resulting variants of PchB were selected for the promiscuous chorismate mutase activity. Consequently, a common mechanism was proposed for both functions of PchB, with the rare [l,5]-sigmatropic rearrangement for the lyase activity, being distinct from other pyruvate lyases. 

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