The enzyme mechanism of a de novo designed and evolved aldolase

The combination of computational enzyme design and laboratory evolution is a successful strategy for the development of biocatalysts with non-natural function, one example being the artificial retroaldolase RA95.1,2 This enzyme utilizes amine catalysis via a reactive lysine residue to cleave the unnatural aldol substrate methodol (Figure 1A). The low initial catalytic activity of the computational design was improved tremendously over many rounds of directed evolution, yielding an efficient biocatalyst for both aldol cleavage as well as synthesis with rate acceleration and stereoselectivity comparable to natural aldolases (Figure 1B).3,4 Key to this success was an ultrahigh-throughput (uHTP) screening technique applied for the late stages of optimization.4 In this work, we analyzed changes in enzyme mechanism along the evolutionary trajectory of RA95 that led to more efficient catalysis. To that end, we determined the rate-limiting step for different enzyme variants by probing individual steps of the aldolase mechanism kinetically. We found a shift towards product release being overall rate-limiting for aldol cleavage catalyzed by highly evolved variants of RA95. Specifically, the conversion between Schiff base and enamine intermediate formed from acetone, a (de-)protonation-dependent process, is the slowest step we probed. Our results indicate that uHTP screening is essential to efficiently evolve a multi-step enzyme mechanism, as it allows the optimization of several mechanistic steps in parallel. By comparing our findings to kinetic and structural studies on natural aldolases, we provide valuable feedback to improve future laboratory evolution approaches as well as the success rate of computational enzyme design. Please click Additional Files below to see the full abstract

Consensus Protein Design without Phylogenetic Bias

Consensus design is an appealing strategy for the stabilization of proteins. It exploits amino acid conservation in sets of homologous proteins to identify likely beneficial mutations. Nevertheless, its success depends on the phylogenetic diversity of the sequence set available. Here, we show that randomization of a single protein represents a reliable alternative source of sequence diversity that is essentially free of phylogenetic bias. A small number of functional protein sequences selected from binary-patterned libraries suffice as input for the consensus design of active enzymes that are easier to produce and substantially more stable than individual members of the starting data set. Although catalytic activity correlates less consistently with sequence conservation in these extensively randomized proteins, less extreme mutagenesis strategies might be adopted in practice to augment stability while maintaining function

Direct NMR observation and DFT calculations of a hydrogen bond at the active site of a 44 kDa enzyme

A hydrogen bond between the amide backbone of Arg7 and the remote imidazole side chain of His106 has been directly observed by improved TROSY-NMR techniques in the 44kDa trimeric enzyme chorismate mutase from Bacillus subtilis. The presence of this hydrogen bond in the free enzyme and its complexes with a transition state analog and the reaction product was demonstrated by measurement of 15N-15N and 1H-15N trans-hydrogen bond scalar couplings, 2h J NN and 1h J HN, and by transfer of nuclear polarization across the hydrogen bond. The conformational dependences of these coupling constants were analyzed using sum-over-states density functional perturbation theory (SOS-DFPT). The observed hydrogen bond might stabilize the scaffold at the active site of BsCM. Because the Arg7-His106 hydrogen bond has not been observed in any of the high resolution crystal structures of BsCM, the measured coupling constants provide unique information about the enzyme and its complexes that should prove useful for structural refinement of atomic model

Iterative approach to computational enzyme design

A general approach for the computational design of enzymes to catalyze arbitrary reactions is a goal at the forefront of the field of protein design. Recently, computationally designed enzymes have been produced for three chemical reactions through the synthesis and screening of a large number of variants. Here, we present an iterative approach that has led to the development of the most catalytically efficient computationally designed enzyme for the Kemp elimination to date. Previously established computational techniques were used to generate an initial design, HG-1, which was catalytically inactive. Analysis of HG-1 with molecular dynamics simulations (MD) and X-ray crystallography indicated that the inactivity might be due to bound waters and high flexibility of residues within the active site. This analysis guided changes to our design procedure, moved the design deeper into the interior of the protein, and resulted in an active Kemp eliminase, HG-2. The cocrystal structure of this enzyme with a transition state analog (TSA) revealed that the TSA was bound in the active site, interacted with the intended catalytic base in a catalytically relevant manner, but was flipped relative to the design model. MD analysis of HG-2 led to an additional point mutation, HG-3, that produced a further threefold improvement in activity. This iterative approach to computational enzyme design, including detailed MD and structural analysis of both active and inactive designs, promises a more complete understanding of the underlying principles of enzymatic catalysis and furthers progress toward reliably producing active enzymes

An artificial metalloenzyme for a bimolecular Diels–Alder reaction

The Diels–Alder reaction, one of the most important in organic chemistry, forms functionalized six-membered cycloadducts in a single step. While widely used to construct complex biologically active molecules in the laboratory, [4+2] cycloadditions are rarely employed for natural product biosynthesis in cells owing to the lack of appropriate enzymes. Creating artificial metalloenzymes able to exploit Lewis acid catalysis for substrate activation could change this situation. Embedding a metal ion in a chiral protein binding pocket potentially combines the best aspects of two worlds – transition metal and enzymatic catalysis – to achieve both high activity and selectivity. Here we report the transformation of a zinc-binding helical bundle into an artificial metalloenzyme that efficiently catalyzes a hetero-Diels–Alder reaction between 3-vinyl indole and an azachalcone derivative by a process of design and laboratory evolution. The best enzyme, DA7, performed \u3e15,000 turnovers per active site and produced only a single product stereoisomer (\u3e99% ee). Detailed kinetic analysis showed that this catalyst is more than two orders of magnitude more proficient than other known Diels–Alderases, including many designed catalysts and natural enzymes involved in polyketide natural products biosynthesis. The remarkable activity of DA7 can be ascribed to the Zn(II) ion, which activates the heterodiene for reaction, and a shape complementary binding pocket that preorganized the reactants for efficient reaction and exacting control over chemo-, diastereo-, and enantioselectivity. These results establish the feasibility of combining design and evolution to harness the structural and functional properties of metal ions to produce remarkably active enzymes for an important abiological reaction. Extending this approach to metal ions other than zinc, and to scaffolds beyond helical bundles, can be expected to produce proficient custom-metalloenzymes for a wide spectrum of unnatural chemical transformations

Whi3 mnemon association with endoplasmic reticulum membranes confines the memory of deceptive courtship to the yeast mother cell

Prion-like proteins are involved in many aspects of cellular physiology, including cellular memory. In response to deceptive courtship, budding yeast escapes pheromone-induced cell-cycle arrest through the coalescence of the G1/S inhibitor Whi3 into a dominant, inactive super-assembly. Whi3 is a mnemon (Whi3(mnem)), a protein that conformational change maintains as a trait in the mother cell but is not inherited by the daughter cells. How the maintenance and asymmetric inheritance of Whi3(mnem) are achieved is unknown. Here, we report that Whi3(mnem) is closely associated with endoplasmic reticulum (ER) membranes and is retained in the mother cell by the lateral diffusion barriers present at the bud neck. Strikingly, barrier defects made Whi3(mnem) propagate in a mitotically stable, prion-like manner. The amyloid-forming glutamine-rich domain of Whi3 was required for both mnemon and prion-like behaviors. Thus, we propose that Whi3(mnem) is in a self-templating state, lending temporal maintenance of memory, whereas its association with the compartmentalized membranes of the ER prevents infectious propagation to the daughter cells. These results suggest that confined self-templating super-assembly is a powerful mechanism for the long-term encoding of information in a spatially defined manner. Yeast courtship may provide insights on how individual synapses become potentiated in neuronal memory.Peer reviewe

Design and optimization of enzymatic activity in a de novo β-barrel scaffold

While native scaffolds offer a large diversity of shapes and topologies for enzyme engineering, their often unpredictable behavior in response to sequence modification makes de novo generated scaffolds an exciting alternative. Here we explore the customization of the backbone and sequence of a de novo designed eight stranded β-barrel protein to create catalysts for a retro-aldolase model reaction. We show that active and specific catalysts can be designed in this fold and use directed evolution to further optimize activity and stereoselectivity. Our results support previous suggestions that different folds have different inherent amenability to evolution and this property could account, in part, for the distribution of natural enzymes among different folds

Upregulation of an Artificial Zymogen by Proteolysis

Regulation of enzymatic activity is vital to living organisms. Here, we report the development and the genetic optimization of an artificial zymogen requiring the action of a natural protease to upregulate its latent asymmetric transfer hydrogenase activity