Unspecific peroxygenases (UPOs) are fungal enzymes that attract significant attention for their ability to perform versatile oxyfunctionalization reactions using H2O2. Unlike other oxygenases, UPOs do not require additional reductive equivalents or electron transfer chains that complicate basic and applied research. Nevertheless, UPOs generally exhibit low to no heterologous production levels and only four UPO structures have been determined to date by crystallography limiting their usefulness and obstructing research. To overcome this bottleneck, we implemented a workflow that applies PROSS stability design to AlphaFold2 model structures of 10 unique and diverse UPOs followed by a signal peptide shuffling to enable heterologous production. Nine UPOs were functionally produced in Pichia pastoris, including the recalcitrant CciUPO and three UPOs derived from oomycetes the first nonfungal UPOs to be experimentally characterized. We conclude that the high accuracy and reliability of new modeling and design workflows dramatically expand the pool of enzymes for basic and applied research.

Unspecific peroxygenases (UPOs) perform oxy-functionalizations for a wide range of substrates utilizing H2O2 without the need for further reductive equivalents or electron transfer chains. Tailoring these promising enzymes toward industrial application was intensely pursued in the last decade with engineering campaigns addressing the heterologous expression, activity, stability, and improvements in chemo- and regioselectivity. One hitherto missing integral part was the targeted engineering of enantioselectivity for specific substrates with poor starting enantioselectivity. In this work, we present the engineering of the short-type MthUPO toward the enantiodivergent hydroxylation of the terpene model substrate, β-ionone. Guided by computational modeling, we designed a small smart library and screened it with a GC−MS setup. After two rounds of iterative protein evolution, the activity increased up to 17-fold and reached a regioselectivity of up to 99.6% for the 4-hydroxy-β-ionone. Enantiodivergent variants were identified with enantiomeric ratios of 96.6:3.4 (R) and 0.3:99.7 (S), respectively.

In recent years, the engineering of flexible loops to improve enzyme properties has gained attention in biocatalysis. Herein, we report a loop engineering strategy to improve the stability of the substrate access tunnels, which reveals the molecular mechanism between loops and tunnels. Based on the dynamic tunnel analysis of CYP116B3, five positions (A86, T91, M108, A109, T111) in loops B-B′ and B′-C potentially affecting tunnel frequent occurrence were selected and subjected to simultaneous saturation mutagenesis. The best variant 8G8 (A86T/T91L/M108N/A109M/T111A) for the dealkylation of 7-ethoxycoumarin and the hydroxylation of naphthalene was identified with considerably increased activity (134-fold and 9-fold) through screening. Molecular dynamics simulations showed that the reduced flexibility of loops B-B′ and B′-C was responsible for increasing the stability of the studied tunnel. The redesign of loops B-B′ and B′-C surrounding the tunnel entrance provides loop engineering with a powerful and likely general method to kick on/off the substrate/product transportation.

Engineering proteins and enzymes with the desired functionality has broad applications in molecular biology, biotechnology, biomedical sciences, health, and medicine. The vastness of protein sequence space and all the possible proteins it represents can pose a considerable barrier for enzyme engineering campaigns through directed evolution and rational design. The nonlinear effects of coevolution between amino acids in protein sequences complicate this further. Data-driven models increasingly provide scientists with the computational tools to navigate through the largely undiscovered forest of protein variants and catch a glimpse of the rules and effects underlying the topology of sequence space. In this review, we outline a complete theoretical journey through the processes of protein engineering methods such as directed evolution and rational design and reflect on these strategies and data-driven hybrid strategies in the context of sequence space. We discuss crucial phenomena of residue coevolution, such as epistasis, and review the history of models created over the past decade, aiming to infer rules of protein evolution from data and use this knowledge to improve the prediction of the structure− function relationship of proteins. Data-driven models based on deep learning algorithms are among the most promising methods that can account for the nonlinear phenomena of sequence space to some degree. We also critically discuss the available models to predict evolutionary coupling and epistatic effects (classical and deep learning) in terms of their capabilities and limitations. Finally, we present our perspective on possible future directions for developing data-driven approaches and provide key orientation points and necessities for the future of the fast-evolving field of enzyme engineering.

Enzymatic hydroxylation of activated and nonactivated sp3-carbons attracts keen interest from the chemistry community as it is one of the most challenging tasks in organic synthesis. Nature provides a vast number of enzymes with an enormous catalytic versatility to fulfill this task. Given that those very different enzymes have a distinct specificity in substrate scope, selectivity, activity, stability, and catalytic cycle, it is interesting to outline similarities and differences. In this Review, we intend to delineate which enzymes possess considerable advantages within specific issues. Heterologous production, crystal structure availability, enzyme engineering potential, and substrate promiscuity are essential factors for the applicability of these biocatalysts.

Unspecific peroxygenases (UPOs) enable oxyfunctionalizations of a broad substrate range with unparalleled activities. Tailoring these enzymes for chemo- and regioselective transformations represents a grand challenge due to the difficulties in their heterologous productions. Herein, we performed protein engineering in Saccharomyces cerevisiae using the MthUPO from Myceliophthora thermophila. More than 5300 transformants were screened. This protein engineering led to a significant reshaping of the active site as elucidated by computational modelling. The reshaping was responsible for the increased oxyfunctionalization activity, with improved kcat/Km values of up to 16.5-fold for the model substrate 5-nitro-1,3-benzodioxole. Moreover, variants were identified with high chemo- and regioselectivities in the oxyfunctionalization of aromatic and benzylic carbons, respectively. The benzylic hydroxylation was demonstrated to perform with enantioselectivities of up to 95% ee. The proposed evolutionary protocol and rationalization of the enhanced activities and selectivities acquired by MthUPO variants represent a step forward toward the use and implementation of UPOs in biocatalytic synthetic pathways of industrial interest.

In the last decade thiotaurine, 2-aminoethane thiosulfonate, has been investigated as an inflammatory modulating agent as a result of its ability to release hydrogen sulfide (H2S) known to play regulatory roles in inflammation. Thiotaurine can be included in the “taurine family” due to structural similarity to taurine and hypotaurine, and is characterized by the presence of a sulfane sulfur moiety. Thiotaurine can be produced by different pathways, such as the spontaneous transsulfuration between thiocysteine – a persulfide analogue of cysteine – and hypotaurine as well as in vivo from cystine. Moreover, the enzymatic oxidation of cysteamine to hypotaurine and thiotaurine in the presence of inorganic sulfur can occur in animal tissues and last but not least thiotaurine can be generated by the transfer of sulfur from mercaptopyruvate to hypotaurine catalyzed by a sulfurtransferase. Thiotaurine is an effective antioxidant agent as demonstrated by its ability to counteract the damage caused by pro-oxidants in the rat. Recently, we observed the influence of thiotaurine on human neutrophils functional responses. In particular, thiotaurine has been found to prevent human neutrophil spontaneous apoptosis suggesting an alternative or additional role to its antioxidant activity. It is likely that the sulfane sulfur of thiotaurine may modulate neutrophil activation via persulfidation of target proteins. In conclusion, thiotaurine can represent a biologically relevant sulfur donor acting as a biological intermediate in the transport, storage and release of sulfide.

Multi-component reactions of building blocks with more than one MCR-reactive group will give rise to oligomeric MCR products. The proper choice of at least two bifunctional building blocks will give either a polymeric or a cyclic product. Apart from polymerization, repetitive or consecutive Ugi reactions have been used to produce linear MCR-heterooligomers with such building blocks.

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Conformational analysis by NMR spectroscopy and molecular modeling revealed a left-handed PPII helix-like structure for Trp2-Tat(1–9) (cis and trans) and an even more flexible structure for TXA2-R(1–9).PPII helices form a well-defined structural class comparable with the other structures defined in proteins and are characterized by exposed, mobile structures with 4–8 residues, mostly found on the protein surface. Polyproline II helices are mainly identified by their torsion angles of φ∼−75° and Ψ∼145−. They do not form regular interchain hydrogen bonds, but are hydrogen bonded with water molecules. PPII helices have a strong preference for the amino acid proline, although it is not necessarily present. These features were also reported for the parent peptide Tat(1–9)4 as well as for the well known DP IV substrates neuropeptide Y and pancreatic polypeptide5 suggesting that PPII-like helical structures represent a favored structural class for the interaction with DP IV.Thus, the considerable enhancement of the inhibition capacity of both Trp2-Tat(1–9) and TXA2-R(1–9) compared to the moderate inhibitor Tat(1–9)2, Ki=2.68±0.01 10−4 M, can only be due to tryptophan in the second position suggesting that its side chain is favored to exhibit attractive hydrophobic interactions with DP IV compared with aspartic acid.On the other hand, we could show recently that Tat(1–9) and its analogues as well as TXA2-R(1–9) inhibit DP IV according to different inhibition mechanisms (Lorey et al., manuscript submitted). One possible explanation for these findings might be enzyme-ligand interactions relying on multiple weak binding sites as described for PPII helices5 rather than specific lock and key binding. Certainly, only an X-ray structure of DP IV would help to understand the interaction of DP IV with inhibitors.