Stabilization of a Metabolic Enzyme by Library Selection in Thermus thermophilus

Abstract

The anthranilate phosphoribosyl transferase from the hyperthermophilic archaeon Sulfolobus solfataricus (sAnPRT, encoded by strpD), which catalyzes the third step in tryptophan biosynthesis, is a thermostable homodimer with low enzymatic activity at room temperature. We have combined two mutations leading to the monomerization and two mutations leading to the activation of sAnPRT. The resulting "activated monomer" sAnPRT-I36E-M47D+D83G-F149S, which is much more labile than wild-type sAnPRT, was stabilized by a combination of random mutagenesis and metabolic library selection using the extremely thermophilic bacterium Thermus thermophilus as host. This approach led to the identification of five mutations that individually increased the thermal stability of sAnPRT-I36E-M47D+D83G-F149S by 1 to 8 °C, and by 13 °C when combined. The beneficial exchanges were located in different parts of the protein structure, but none of them led to the "re-dimerization" of the enzyme. We observed a negative correlation between thermal stability and catalytic activity of the mutants; this suggests that conformational flexibility is required for catalysis by sAnPRT.

Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently

“…1070,1071 Of course if one uses a thermophile such as T. thermophilus then in vivo selection is possible, too. 1072 …”

“…1070,1071 Of course if one uses a thermophile such as T. thermophilus then in vivo selection is possible, too. 1072 As rehearsed above, protein flexibility (a somewhat ill-defined concept 87,1073 ) is related to k cat , and most residues involved in improving k cat are away from the active site, at the protein surface (where they are bombarded by solvent thermal fluctuations). The connection between flexibility and thermostability is not well understood, and it does not always follow that less flexibility provides greater stability.…”

Activation of a Chimeric Rpb5/RpoH Subunit Using Library Selection

“…The rp5H gene library was generated using error-prone PCR as described previously [29], [30], with the following modifications: The PCR cycling parameters were 95°C for 5 min; 35 cycles at 95°C for 45 s, 55°C for 55 s, 72°C for 40 s, followed by 72°C for 10 min. The PCR mixture contained 1.0 mM MgCl 2 , 1.0 mM MnCl 2 , 0.35 mM dATP, 1.35 mM dTTP, 0.4 mM dCTP, 0.2 mM dGTP, 1 µM of each primer (5′rpb5_EcoRI and 3′rpoH_NotI), 5 U DNA polymerase (GoTag, Promega, Mannheim, Germany), and 25 ng of plasmid pRS423_ rp5H as template.…”

High‐efficiency transformation of archaea by direct PCR products with its application to directed evolution of a thermostable enzyme

“…Besides natural treasure troves of thermostable enzymes (Atomi et al ., 2011; Rollin et al ., 2015; Honda et al ., 2017; You et al ., 2017; Kim et al ., 2018), hyperthermophilic microorganisms could also be an ideal host organism for evolving mesophilic or moderately thermophilic enzymes into their thermostable counterparts. Directed evolution is a powerful tool for protein engineering without the detailed understanding of catalytic mechanism and structure information (Giver et al ., 1998; Schwab and Sterner, 2011; Zhou et al ., 2018). To our limited knowledge, the only hyperthermophilic microorganism S. solfataricus has been demonstrated for directed evolution of mesophilic enzymes to more thermostable ones (Cannio et al ., 2001).…”