Design, selection, and characterization of a split chorismate mutase

Abstract

Split proteins are versatile tools for detecting protein-protein interactions and studying protein folding. Here, we report a new, particularly small split enzyme, engineered from a thermostable chorismate mutase (CM). Upon dissecting the helical-bundle CM from Methanococcus jannaschii into a short N-terminal helix and a 3-helix segment and attaching an antiparallel leucine zipper dimerization domain to the individual fragments, we obtained a weakly active heterodimeric mutase. Using combinatorial mutagenesis and in vivo selection, we optimized the short linker sequences connecting the leucine zipper to the enzyme domain. One of the selected CMs was characterized in detail. It spontaneously assembles from the separately inactive fragments and exhibits wild-type like CM activity. Owing to the availability of a well characterized selection system, the simple 4-helix bundle topology, and the small size of the N-terminal helix, the heterodimeric CM could be a valuable scaffold for enzyme engineering efforts and as a split sensor for specifically oriented protein-protein interactions.

Building Proficient Enzymes with Foldamer Prostheses

“…The 115 amino acid long three-helix fragment Z-C3 was produced recombinantly in a chorismate mutase deficient E. coli strain, thereby excluding the possibility of contamination with endogenous enzyme. [12] Kinetic assay: Kinetic assays were performed by measuring the consumption of chorismate spectroscopically at either 274 nm (e 274 = 2630 m À1 cm…”

“…An engineered heterodimeric chorismate mutase (CM), [12] which catalyzes the sigmatropic rearrangement of chorismate to prephenate (hdCM; Figure 1 a), served as the protein scaffold for the assembly of hybrid enzymes. hdCM was originally constructed by dissecting the helical-bundle CM from Methanocaldococcus jannaschii into a short N- Figure 1.…”

“…[12] The average initial velocities obtained from at least three independent measurements are plotted in Figure S4.…”

“…[12] K d values for variants 5-7, which display minimal CM activity, were determined by monitoring inhibition of the formation of an active complex between Z-C3 and 2. IC 50 values were obtained by plotting the resulting initial velocities at fixed concentrations of 2 and Z-C3 (both 1 mm) as a function of increasing amounts of the N1-Z fragment.…”

Mesophilic and Hyperthermophilic Adenylate Kinases Differ in Their Tolerance to Random Fragmentation

“…This suggests that the use of our novel transposase-based library design strategy with thermophilic proteins may represent an effective way to accelerate the discovery of split regulatory proteins that cooperatively function for synthetic biology applications in mesophiles, 17 without the need for further optimization. 33,54 Proteins with enhanced thermostability may also be poised to evolve new structures and functions through fission. A recent study showed that mesophilic protein fragmentation can result in polypeptide fragments that form functional oligomers with a new quaternary structure, 55 reinforcing a long-standing hypothesis that proteins can adapt new structures through the exchange or shuffling of their polypeptide domain.…”