Directed evolution of the final enzyme in the lovastatin biosynthetic pathway yields a variant with 29 mutations that does not require a carrier protein and displays altered dynamics of the catalytic residues, spending more time in the catalytically active conformation. Natural enzymes have evolved to perform their cellular functions under complex selective pressures, which often require their catalytic activities to be regulated by other proteins. We contrasted a natural enzyme, LovD, which acts on a protein-bound (LovF) acyl substrate, with a laboratory-generated variant that was transformed by directed evolution to accept instead a small free acyl thioester and no longer requires the acyl carrier protein. The resulting 29-mutant variant is 1,000-fold more efficient in the synthesis of the drug simvastatin than the wild-type LovD. This is to our knowledge the first nonpatent report of the enzyme currently used for the manufacture of simvastatin as well as the intermediate evolved variants. Crystal structures and microsecond-scale molecular dynamics simulations revealed the mechanism by which the laboratory-generated mutations free LovD from dependence on protein-protein interactions. Mutations markedly altered conformational dynamics of the catalytic residues, obviating the need for allosteric modulation by the acyl carrier LovF.

Since fullerenes are available in macroscopic quantities from fullerene soot, large efforts have been geared toward designing efficient strategies to obtain highly pure fullerenes, which can be subsequently applied in multiple research fields. Here we present a supramolecular nanocage synthesized by metal-directed self-assembly, which encapsulates fullerenes of different sizes. Direct experimental evidence is provided for the 1:1 encapsulation of C₆₀, C₇₀, C₇₆, C₇₈ and C₈₄, and solid state structures for the host–guest adducts with C₆₀ and C₇₀ have been obtained using X-ray synchrotron radiation. Furthermore, we design a washing-based strategy to exclusively extract pure C₆₀ from a solid sample of cage charged with a mixture of fullerenes. These results showcase an attractive methodology to selectively extract C₆₀ from fullerene mixtures, providing a platform to design tuned cages for selective extraction of higher fullerenes. The solid-phase fullerene encapsulation and liberation represent a twist in host–guest chemistry for molecular nanocage structures.

Enzymes exist as a dynamic ensemble of conformations, each potentially playing a key role in substrate binding, the chemical transformation, or product release. We discuss recent advances in the evaluation of the enzyme conformational dynamics and its evolution towards new functions or substrate preferences.

Enzymes exist as ensembles of conformations that are important for function. Tuning these populations of conformational states through mutation enables evolution toward additional activities. Here we computationally evaluate the population shifts induced by distal and active site mutations in a family of computationally designed and experimentally optimized retro-aldolases. The conformational landscape of these enzymes was significantly altered during evolution, as pre-existing catalytically active conformational substates became major states in the most evolved variants. We further demonstrate that key residues responsible for these substate conversions can be predicted computationally. Significantly, the identified residues coincide with those positions mutated in the laboratory evolution experiments. This study establishes that distal mutations that affect enzyme catalytic activity can be predicted computationally and thus provides the enzyme (re)design field with a rational strategy to determine promising sites for enhancing activity through mutation.

Bio-catalytic micro- and nanomotors self-propel by the enzymatic conversion of substrates into products. Despite the advances in the field, the fundamental aspects underlying enzyme-powered self-propulsion have rarely been studied. In this work, we select four enzymes (urease, acetylcholinesterase, glucose oxidase, and aldolase) to be attached on silica microcapsules and study how their turnover number and conformational dynamics affect the self-propulsion, combining both an experimental and molecular dynamics simulations approach. Urease and acetylcholinesterase, the enzymes with higher catalytic rates, are the only enzymes capable of producing active motion. Molecular dynamics simulations reveal that urease and acetylcholinesterase display the highest degree of flexibility near the active site, which could play a role on the catalytic process. We experimentally assess this hypothesis for urease micromotors through competitive inhibition (acetohydroxamic acid) and increasing enzyme rigidity (β-mercaptoethanol). We conclude that the conformational changes are a precondition of urease catalysis, which is essential to generate self-propulsion.