The three‐dimensional structure of the enzymes provides very relevant information on the arrangement of the catalytic machinery and structural elements gating the active site pocket. The recent success of the neural network Alphafold2 in predicting the folded structure of proteins from the primary sequence with high levels of accuracy has revolutionized the protein design field. However, the application of Alphafold2 for understanding and engineering function directly from the obtained single static picture is not straightforward. Indeed, understanding enzymatic function requires the exploration of the ensemble of thermally accessible conformations that enzymes adopt in solution. In the present study, we evaluate the potential of Alphafold2 in assessing the effect of the mutations on the conformational landscape of the beta subunit of tryptophan synthase (TrpB). Specifically, we develop a template‐based Alphafold2 approach for estimating the conformational heterogeneity of several TrpB enzymes, which is needed for enhanced stand‐alone activity. Our results show the potential of Alphafold2, especially if combined with molecular dynamics simulations, for elucidating the changes induced by mutation in the conformational landscapes at a rather reduced computational cost, thus revealing its plausible application in computational enzyme design.

Halohydrin dehalogenases (HHDH) are industrially relevant biocatalysts exhibiting a promiscuous epoxide-ring opening reactivity in the presence of small nucleophiles, thus giving access to novel carbon–carbon, carbon–oxygen, carbon–nitrogen, and carbon–sulfur bonds. Recently, the repertoire of HHDH has been expanded, providing access to some novel HHDH subclasses exhibiting a broader epoxide substrate scope. In this work, we develop a computational approach based on the application of linear and non-linear dimensionality reduction techniques to long time-scale Molecular Dynamics (MD) simulations to study the HHDH conformational landscapes. We couple the analysis of the conformational landscapes to CAVER calculations to assess their impact on the active site tunnels and potential ability towards bulky epoxide ring opening reaction. Our study indicates that the analyzed HHDHs subclasses share a common breathing motion of the halide binding pocket, but present large deviations in the loops adjacent to the active site pocket and N-terminal regions. Such conformational differences affect the available tunnels for epoxide binding to the active site. The superior activity of the HHDH G subclass towards bulkier substrates is explained by the additional structural elements delimiting the active site region, its rich conformational heterogeneity, and the substantially wider and frequently observed active site tunnels. This study therefore provides key information for HHDH promiscuity and engineering.

DNA-encoded chemical library (DEL) technology is a powerful tool in early-stage drug discovery. Although widely applied in industry and academia, challenges persist in generating DELs with high quality and chemical diversity. Low yields in building-block incorporation, limited selectivity and, most importantly, DNA damage from harsh reaction conditions compromise library quality, reduce signal-to-noise in affinity selections and ultimately hinder drug discovery. Here we show that tailored enzymes can be harnessed for the effective construction of molecular diversity on DNA under mild conditions. Targeting amide bond formation, we designed a cascade of complementary coenzyme A ligases and rationally tailored N -acyltransferases to access a broad amide scope on-DNA (>120 examples), identifying structural elements that optimize the biocatalysts’ DNA compatibility in the process. Integrating the enzymatic cascade with chemical synthesis led to the construction of a diverse DEL without damage to the DNA barcode, highlighting the biocatalysts’ applicability for early scaffold construction and late-stage functionalization.

Halohydrin dehalogenases (HHDHs) are powerful enzymes for the asymmetric diversification of oxyfunctionalized synthons. They feature two characteristic sequence motifs that distinguish them from homologous short-chain dehydrogenases and reductases. Sequence motif 1, carrying a conserved threonine, glycine and a central aromatic residue, lines the nucleophile binding pocket of HHDHs. It could therefore impact nucleophile binding and presumably also activity of the enzymes. However, experimental evidence supporting this theory is largely missing. Herein, we systematically studied the mutability of the three conserved motif 1 residues as well as their resulting impact on enzyme activity, stability and selectivity in two model HHDHs: HheC from Agrobacterium radiobacter AD1 and HheG from Ilumatobacter coccineus. In both HheC and HheG, the conserved threonine and glycine only tolerated mutations to structurally similar amino acids. In contrast, the central aromatic (i.e., phenylalanine or tyrosine) residue of motif 1 demonstrated much higher variability in HheC. Remarkably, some of these variants featured drastically altered activity, stability and selectivity characteristics. For instance, variant HheC F12Y displayed up to 5-fold increased specific activity in various epoxide ring opening and dehalogenation reactions as well as enhanced enantioselectivity (e.g., an E-value of 74 in the azidolysis of epichlorohydrin compared to E = 22 for HheC wild type), while exhibiting also a 10 K higher apparent melting temperature. QM and MD simulations support the experimentally observed activity increase of HheC F12Y and revealed alterations in the hydrogen bonding network within the active site. As such, our results demonstrate that multiple enzyme properties of HHDHs can be altered through targeted mutagenesis of conserved motif 1 residues. In addition, this work illustrates that motif 1 plays vital roles beyond nucleophile binding by impacting solubility and stability properties. These insights advance our understanding of HHDH active sites and will facilitate their future engineering.

Our study successfully explores strategies to effectively improve the photocontrol efficiency of light‐sensitive enzymes, dubbed photoxenases, with photoswitchable unnatural amino acids (UAAs). The engineering of photoxenases is a versatile method for the reversible photocontrol in various applications. To boost the photocontrol of an established allosteric and heterodimeric photoxenase based on imidazole glycerol phosphate synthase, we turned from an ineffective tuning of the UAA photochemistry to a semi‐rational enzyme design. Remarkably, mutations at the catalytically important heterodimer interface increased the light‐regulation factor (LRF) for the k _cat up to ∼100 with near‐quantitative reversibility. Steady‐state kinetic investigations combined with computationally determined correlation‐based Shortest‐Path‐Map (SPM) analysis and conformational landscapes revealed how photocontrol was altered in the two best hits. The LRF( k _cat ) correlated with a shift of a conformational equilibrium between an active and inactive population at the targeted active site and a tuned population productivity upon irradiation. While the overall reduced k _cat values originated from a rewiring of the allosteric signal transmission, the increased LRF( k _cat ) resulted from a change in i) the size of the conformational shift, ii) the population productivity, and iii) the conformational heterogeneity. With this, our findings provide initial guidelines to boost photocontrol and underscore the power of photoxenase engineering.