Simultaneous optimization of enzyme activity and quaternary structure by directed evolution

Abstract

Natural evolution has produced efficient enzymes of enormous structural diversity. We imitated this natural process in the laboratory to augment the efficiency of an engineered chorismate mutase with low activity and an unusual hexameric topology. By applying two rounds of DNA shuffling and genetic selection, we obtained a 400-fold more efficient enzyme, containing three non-active-site mutations. Detailed biophysical characterization of the evolved variant suggests that it exists predominantly as a trimer in solution, but is otherwise similarly stable as the parent hexamer. The dramatic structural and functional effects achieved by a small number of seemingly innocuous substitutions highlights the utility of directed evolution for modifying protein-protein interactions to produce novel quaternary states with optimized activities.Keywords: chorismate mutase; directed evolution; genetic selection; DNA shuffling; quaternary structure A large fraction of proteins in living cells exist as symmetric assemblies of multiple subunits (Goodsell and Olson 2000). Oligomerization provides advantageous properties often not available to the monomer, such as increased stability, new intersubunit binding sites, cooperativity, and allosteric regulation. To understand the chemical and genetic basis for the evolutionary transition between monomers and oligomers, several studies have focused on altering the quaternary structure of existing proteins by either reducing or increasing the number of their subunits (Mossing and Sauer 1990;Green et al. 1995;Dickason and Huston 1996; MacBeath et al. 1998b,c).In nature, infrequent but beneficial mutations are identified and propagated by genetic selection. This tool can also be exploited effectively in the laboratory to obtain novel protein oligomers starting from a diverse population of variants (Taylor et al. 2001a;Woycechowsky and Hilvert 2004). It is especially valuable in the case of enzymes, where structural changes are often poorly tolerated, and mutations that alter quaternary structure without causing a significant loss in catalytic activity are relatively rare.We have successfully used genetic selection to alter the topology of homodimeric AroQ chorismate mutases (CMs)-domain-swapped enzymes that catalyze a key step in the biosynthesis of the aromatic amino acids tyrosine and phenylalanine. For example, by inserting a random hinge-loop of eight residues into the H1 helix of the thermostable mutase from Methanococcus jannaschii (MjCM) and selecting for functional variants by genetic complementation, we obtained a monomeric catalyst with near native activity (MacBeath et al. 1998c). The topologically redesigned monomer unexpectedly exhibits the properties of a molten globule (Vamvaca et al. 2004), but structural ordering seen upon ligand binding indicates that protein folding and catalysis can be efficiently coupled.Reprint requests to: Donald Hilvert, Laboratorium fu¨r Organische Chemie, ETH Ho¨nggerberg/HCI F 339, CH-8093 Zu¨rich/Switzerland; e-mail: hilvert@org.chem.ethz...

Relative tolerance of mesostable and thermostable protein homologs to extensive mutation

“…Although the preferences observed at positions 30, 40, and 42 are modest and not readily rationalized, the strong selection for aspartate at positions 15 and 18 in both libraries, and the absolute requirement for lysine at position 29 in the EcCM library, can be explained in structural terms. For example, the aspartates at positions 15 and 18 are second shell residues that can form salt bridges with the essential active site residues Arg51 and Arg28, and thereby help preorganize the binding pocket for catalysis 25, 29, 34. Such interactions are evidently much more important for the less stable protein, since 100% of active EcCM variants have an aspartate at position 18, and 81% have this residue at position 15, compared to only 85% and 75% aspartate at the respective positions in active MjCM mutants.…”

Design, selection, and characterization of a split chorismate mutase

“…18,[22][23][24][36][37][38] Here we show for the first time the construction of a functional heterodimeric CM. Simply cleaving the dimer-spanning N-terminal helix of MjCM to yield a one-helix and a three-helix fragment abolishes enzyme function in vitro and in vivo.…”

“…18,[21][22][23][24] Insertion of short peptide sequences into the middle of the dimer-spanning N-terminal helix converts the natural homodimer into monomers (mMjCM), 18 trimers, 22 and hexamers. 22 The tolerance of the N-terminal helix to disruption between residues 21 and 22 also makes this an attractive cleavage site for the generation of a heterodimeric mutase [ Fig. 1(A)].…”

Dependence of the Enzymatic Velocity on the Substrate Dissociation Rate

“…In fact, natural enzymes that are chemically modified, can catalyze the same biochemical reactions much faster. 7 Recently, Reuveni et al 8,9 proposed a new way of optimizing the rates of enzymatic reactions. They showed that these reactions can be accelerated by increasing the rate of substrate unbinding from the enzyme–substrate complex when 8 “(i) substrate concentrations are high; (ii) the unbinding rate is low; and (iii) the coefficient of variation associated with the distribution of catalytic times is larger than unity.”…”