TABLE 1.

A survey of the molecular mechanisms identified in natural and laboratory evolution

Identified molecular mechanism of evolution
Starting enzyme	Evolved function	New catalytic interaction	Active site reshaping	Conformational tinkering	Alteration of dynamics	Substrate repositioning	Repositioning cofactors	Description	Method	Refs
Atrazine chlorohydrolase (AtzA)	Melamine deaminase	+						Emergence of a novel catalytic Cys–Asp dyad enabling Cys331 to donate a proton to the melamine LG, and Asp328 to abstract a proton from Cys331, preventing the creation of a high‐energy thiolate.	N	18
Tyrocidine synthetase 1, A domain (TycA)	l‐alanine/l‐threonine adenylation		+					Mutations decrease the size of the binding pocket, creating steric discrimination. An increase in AS hydrophobicity benefits hydrophobic AAs.	RD	19
	(S)‐β‐Phe/l‐Phe specific adenylation		+					AS remodeling strongly enhanced shape complementarity to the aryl group. W239S enlarged the AS pocket by ~220 Å3, facilitating binding for the propargyl substituent used for screening during substrate walking. A conserved salt‐bridge with D235, mutation A236V, and loop β13–β14 remodeling, caused substrate repositioning.	RD	20
Carbonic anhydrase II (hCAII)	Esterase		+					The removal of steric hindrance in the AS appears to promote substrate binding. No change in TS stabilization (βLG remains unchanged)	DE	21
Cytochrome P450	Propane hydroxylation		+					A dramatic narrowing of the substrate access channel was observed (∆V = −127 Å3), providing better solvent exclusion for the smaller evolved substrate. The new channel now appears partitioned. A hydrophobic pocket near the heme cofactor was enlarged by 29 Å3 to enable substrate binding	RD/DE	22, 23
ancDHCH1	Methyl‐parathion hydrolase		+					Four mutations enlarged the ancestor's AS to eliminate a steric clash with the evolved substrate. One deletion altered the conformation of an AS loop. These mutations appear to enable productive binding modes that did not exist in the ancestor.	ASR	24
ancMALS	Maltase		+					Two mutations enhanced the enzyme's complementarity for maltose, while sterically hindering the binding of isomaltose.	ASR	25
ancIMA1‐4	Isomaltase	+		+				Mutation Q279 is predicted to form a new interaction with isomaltose. A repositioning of AS residues stabilized Q279 and caused it to further protrude into the AS cavity, optimizing the interactions with isomaltose over maltose.
ancM/L‐malate/lactate dehydrogenase	Lactate dehydrogenase	+		+				The deletion of a key electrostatic contact between an AS residue and the original substrate, and a loop insertion, created better hydrophobic packing of the new substrate via W107f	ASR	26
Haloalkane dehalogenase (DhaA)	Enantioselectivity for β‐bromoalkanes	+			+			The AS transplantation between DbjA and DhaA led to very limited activity enhancement despite identical positioning of the catalytic pentad in mutant DhaA12. For enantioselectivity to occur, enzyme dynamics seem to be required. Distinct hydration and amplitude of motions in the vicinity of the access tunnel seem to mediate the enantioselectivity in these nearly identical homologous enzymes.	RD	27
β‐Lactamase (NDM1)	Phosphonate monoester hydrolase		+	+				Mutation W93G removed steric hindrance and caused substantial rearrangement of loops 3 and 10, enhancing E–S complementarity.	DE	28
Arylsulfatase (PAS)	Phosphonate monoester hydrolase		+			+		The tuning of the ES E•S complex eliminated a steric clash with the phenyl substituent of the evolved substrate. This led to substrate repositioning and enhanced TS stabilization (evidenced by large changes in βLG).	DE	29
β subunit of tryptophan synthase (TrpB)	Non‐canonical AA synthesis (standalone Trp)			+	+			Increase in heterogeneity of the COMM domain, makes active states accessible in absence of the allosteric activator, TrpA. The authors observed a modification of the conformational landscape, that is, the distribution of open (O), partially open (PC) and closed (C) states of the COMM domain that mediate the transitions between multiple steps along the catalytic cycle.	DE	30, 31
CypA proline isomerase (+ S99T)	Restoration of pro‐isomerization			+	+			AS rewiring minimized side‐chains clashes. Altered dynamics of a cluster of residues increased F113's motions, restoring the existence of two interconverting catalytic CypA substates that can distinctly bind the cis or trans states of the substrate.	DE	32
Lactonase (PON1)	Paraoxonase				+		+	Mutation H115W causes a 1.8 Å (and up to 2.4 Å in further evolved variants) upward displacement of the catalytic Ca2+. The new metal conformation is more competent for the evolved function. This conformation already existed in the WT, but H115W creates a population shift towards the upward position of Ca2+. Increased disorder in an AS loop enables paraoxon binding. The repositioning of metal‐ligating residues enhances catalysis.	DE	33, 34
Kemp Eliminase* (KE07)	Kemp eliminase		+	+				Mutations on the floor of the AS increased the basicity of the catalytic base Glu101, thus its reactivity. Evolution corrected a “default” introduced in the original design: The elimination of a salt‐bridge repositioned the conformation of Glu101. Upon G202R, a conformational shift of an AS loop and several residues may have promoted new interactions with the TS.	RD/DE	35
		+	+	+				Mutations triggered the emergence of 3 distinct, catalytically competent, AS configurations. During evolution, a single, more stable and catalytically productive conformation, was selected. In this conformation, W50 is repositioned, enabling better alignment and orientation with the reactants, and a higher pK a of Glu101.	DE	36
Kemp Eliminase* (KE70)	Kemp eliminase		+	+	+			Evolution created a deeper, tighter pocket by reshaping the AS. A repositioning of AS residues surrounding the catalytic dyad rewired several interactions, which contributed to an increased basicity of the dyad (D45), thus, its reactivity. MDs suggest that the catalytic dyad and other AS residues become less mobile.	RD/DE	37
TEM‐1	Cefotaxime		+	+	+			Mutation G238S alone causes local perturbations in the 238‐loop controlling access to the AS (i.e., the open conformation allows larger substrates to reach the catalytic center). R164S alone increases the conformational freedom of the Ω‐loop, also promoting substrate access. Surprisingly, combining both mutations disturb the AS preorganization; a misalignment between N170 and the deacylating water results in poor k cat and a shift in equilibrium toward nonproductive conformations, over catalytically competent ones.	N	38
Diels‐alderase* (DA_20_00)	Diels‐alderase		+	+	+			DE and loop grafting resulted in high E–S complementarity, constraining the substrate in a productive orientation, promoted by 88 VDW contacts between ligands/peptide backbone, aromatic side chains and a buried water molecule. Increased disorder of an interhelical loop and a supporting helix is observed.	RD/DE	39, 40
Acyltransferase (LovD)	Acyltransferase (simvastatin synthesis)		+	+	+			The active site becomes more buried, and a narrowing of the substrate access channel is observed. Evolved backbone dynamics were necessary to gradually displace the catalytic triad residues from a nonproductive conformation to a precise, competent geometry for acylation. In the WT, this mechanism was achieved by protein–protein interaction‐induced conformational sampling.	DE	41
N‐acyl homoserine lactonase (AiiA)	Paraoxonase		+	+		+		Remote mutations cause a narrowing of the AS upon a shift in loop 3's position, enabling a new π–π stacking interaction between F68 and the LG. MDs suggest a repositioning of the paraoxon with respect to the bimetallic core and the nucleophile.	DE	42
Metallo‐β‐lactamase (BcII)	Cephalexinase			+	+		+	A shift in the position of one AS metal ions shortens the distance between the two Zn2+ by 0.5–1.0 Å. this enables the stabilization of a negatively charged reaction intermediate by Zn2+, altering the rate‐limiting step of the reaction. Sign epistasis between mutation N70S and G262S results in an increase in conformational dynamics along the trajectory (AS loop flexibility), rendering the AS cavity more accessible, which leads to a wider substrate spectrum.	DE	43, 44
Phosphotriesterase (PTE)	Arylesterase	+	+	+	+			AS reshaping enhanced E–S complementarity with 2‐naphthyl hexanoate. Mutations rewired a key AS hydrogen bonding network, stabilizing the “bent” conformation of the mutated H254R, which promotes LG assistance via π‐cation interaction. Fine‐tuning of loops dynamics mediate the stabilization of productive substrate binding modes, and freezes unproductive ones.	DE	45, 46
AA–binding protein (AABP, AncCDT)	Cyclohexadienyl dehydratase	+	+	+	+			Mutation V173E introduced general acid catalysis, whereas other mutations shift W60 and increase E–S complementarity. A new hydrogen‐bonding network interacts with the departing hydroxyl and carboxylate groups, enhancing TS stabilization. The removal of a steric clash and the introduction of new interactions increases E–S complementarity. Stabilization of the closed conformation in efficient extant enzymes vs. largely open conformations in AABP and Anc demonstrate a role of conformational tuning in evolution. Altering the conformational landscape to preferentially sample the closed conformation minimizes nonproductive binding.	ASR/DE	47, 48
Bifunctional PROFAR/PRA isomerase (HisA dup13‐15, D10G)	PRA isomerase (TrpF function)	+	+	+	+			The VVR duplication in loop 1 creates a TrpF‐active conformation that introduces R15[c] in an optimal position to interact with the PRA substrate. Mutations V106L and G102A abolish the second phosphate binding site causing specialization toward TrpF (as PRA is monophosphorylated). Dynamic conformational shifts between loop 1 and 5 mediate the dual substrate specificity in the bifunctional enzyme.	DE	49, 50
	PROFAR isomerase (HisA function)	+	+	+	+			In the HisA‐active conformation, W145 forms a key stacking interaction with the ProFAR carboxamide aminoimidazole moiety. D10G increased dynamics in loop 1, enabling loop 5 to compete for the AS, which favors the HisA function over TrpF.
Kemp Eliminase* (HG3)	Kemp eliminase	+	+	+		+		Catalysis was solely enhanced by an increase in turnover number, rather than affinity. Evolution tailored a striking shape complementarity with the ligand, eliminating a dual binding mode in the designed scaffold that formed a nonproductive complex in HG2. The introduction of an oxyanion binder (K50Q) promotes TS stabilization, while a better alignment between the catalytic base (D127) and the ligand enhances proton transfer.	RD/DE	51
Anc chalcone isomerase (AncCHI)	Michael addition of chalconaringenin		+	+	+	+		Enlargement of the AS to enable substrate binding and repositioning. A key catalytic residue R34, present in the noncatalytic ancestor, saw an increase in conformational sampling resulting in better positioning with the TS for catalysis. Both mechanisms seem to stabilize the productive substrate binding mode over the nonproductive one.	ASR/DE	52
Kemp Eliminase* (KE59)	Kemp eliminase		+	+	+	+		A change in AS environment enhanced TS stabilization due to better buffering of the negative charge developing at the TS (evidenced by changes in βLG). Enhanced desolvation seems to improve the reactivity of the designed catalytic base (E230). Evolution also created a wider AS entrance. There is a possible emergence of two catalytic binding modes of the substrate. MDs suggest a gain in mobility of the AS residues.	RD/DE	53
PTE arylesterase (AE)	Paraoxonase		+	+	+		+	This evolutionary trajectory demonstrates that an original function can be “reversed” by selecting mutations that “restore” the AS conformation seen in the WT. The AS was reshaped by increasing its volume to reaccommodate the bulky ethyl substituents of paraoxon, steric clashes were removed and the hydrophobicity of the cavity was enhanced to match that of the phosphotriester. The AS residues were repositioned; the distance between the two catalytic Zn2+ was restored to 3.8 Å as seen in wtPTE. Loop 7 motions, essential to paraoxon catalysis, were restored. The characteristic “broken” pattern (nonlinear pKa dependency) observed by LFER for paraoxon in wtPTE, was also reproduced in the evolved paraoxonase.	DE	54
Retro‐aldolase* (RA95 and RA95.5)	(±)‐Methodol cleavage	+	+	+	+	+		During the DE of RA95.0, the function of Lys210 was taken over by mutation T83K, inserted across the AS in an environment that better promotes catalysis. K83 becomes gradually stabilized in the evolved variant. A drastic AS reshaping is seen, leading to a promiscuous intermediate accommodating two substrate binding orientations. L1, L6, and L7 loops of RA95.5 exhibit increased dynamics (enhanced B‐factor).	RD/DE	55, 56
								Further evolution to RA95.5‐8F led to substrate repositioning, once again: The naphthol moiety of the substrate “migrated” back to the original position computed in RA95. F180Y introduces a new catalytic residue flanking the triad that reinforces the catalytic core, forming a highly efficient tetrad. A change in rate‐limiting step, from C─C bond cleavage to product release, was observed upon the emergence of the tetrad, which accelerates Schiff‐base formation and hydrolysis.	RD/DE	57, 58

Note: The symbol + indicates that the mechanism has been specifically identified in the study. A blank indicates that the mechanism has not been identified in this model; note that it does not rule out the existence of other mechanisms at play. Asterisk symbol (*) denotes starting enzymes that were computationally designed.

Abbreviations: AA, amino acids; Anc, ancestor; AS, active site; ASR, ancestral sequence reconstruction; DE, directed evolution; E–S, enzyme−substrate complex; LFER, linear free‐energy relationships; LG, leaving group; MDs, molecular dynamics simulations; N, natural trajectory; TS, transition state; VDW, Van der Waals; RD, rational design.