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.

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.

MqnA, the only chorismate dehydratase known so far, catalyzes the initial step in the biosynthesis of menaquinone via the futalosine pathway. Details of the MqnA reaction mechanism remain unclear. Here, we present crystal structures of Streptomyces coelicolor MqnA and its active site mutants in complex with chorismate and the product 3-enolpyruvyl-benzoate, produced during heterologous expression in Escherichia coli. Together with activity studies, our data are in line with dehydration proceeding via substrate assisted catalysis, with the enol pyruvyl group of chorismate acting as catalytic base. Surprisingly, structures of the mutant Asn17Asp with copurified ligand suggest that the enzyme converts to a hydrolase by serendipitous positioning of the carboxyl group. All complex structures presented here exhibit a closed Venus flytrap fold, with the enzyme exploiting the characteristic ligand binding properties of the fold for specific substrate binding and catalysis. The conformational rearrangements that facilitate complete burial of substrate/product, with accompanying topological changes to the enzyme surface, could foster substrate channeling within the biosynthetic pathway.

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.

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.

Nonhost resistance of Arabidopsis thaliana against Phytophthora infestans, a filamentous eukaryotic microbe and the causal agent of potato late blight, is based on a multilayered defense system. Arabidopsis thaliana controls pathogen entry through the penetration-resistance genes PEN2 and PEN3, encoding an atypical myrosinase and an ABC transporter, respectively, required for synthesis and export of unknown indole compounds. To identify pathogen-elicited leaf surface metabolites and further unravel nonhost resistance in Arabidopsis, we performed untargeted metabolite profiling by incubating a P. infestans zoospore suspension on leaves of WT or pen3 mutant Arabidopsis plants. Among the plant-secreted metabolites, 4-methoxyindol-3-yl-methanol and S-(4-methoxy-indol-3-yl-methyl) cysteine were detected in spore suspensions recollected from WT plants, but at reduced levels from the pen3 mutant plants. In both whole-cell and microsome-based assays, 4-methoxyindol-3-yl-methanol was transported in a PEN3-dependent manner, suggesting that this compound is a PEN3 substrate. The syntheses of both compounds were dependent on functional PEN2 and phytochelatin synthase 1. None of these compounds inhibited mycelial growth of P. infestans in vitro. Of note, exogenous application of 4-methoxyindol-3-yl methanol slightly elevated cytosolic Ca2+ levels and enhanced callose deposition in hydathodes of seedlings treated with a bacterial pathogen-associated molecular pattern (PAMP), flagellin (flg22). Loss of flg22-induced callose deposition in leaves of pen3 seedlings was partially reverted by the addition of 4-methoxyindol-3-yl methanol. In conclusion, we have identified a specific indole compound that is a substrate for PEN3 and contributes to the plant defense response against microbial pathogens.

Ubiquitination is a prevalent post-translational modification involved in all aspects of cell physiology. It is mediated by an enzymatic cascade and the E2 ubiquitin-conjugating enzymes (UBCs) lie at its heart. Even though E3 ubiquitin ligases determine the specificity of the reaction, E2s catalyse the attachment of ubiquitin and have emerged as key mediators of chain assembly. They are largely responsible for the type of linkage between ubiquitin moieties and thus, the fate endowed onto the modified substrate. However, in vivo E2-E3 pairing remains largely unexplored. We therefore interrogated the interaction selectivity between 37 Arabidopsis E2s and PUB22, a U-box type E3 ubiquitin ligase that is involved in the dampening of immune signalling. We show that while the U-box domain, which mediates E2 docking, is able to interact with 18 out of 37 tested E2s, the substrate interacting armadillo (ARM) repeats impose a second layer of specificity, allowing the interaction with eleven E2s. In vitro activity assayed by autoubiquitination only partially recapitulated the in vivo selectivity. Moreover, in vivo pairing was modulated during the immune response; pairing with group VI UBC30 was inhibited, while interaction with the K63 chain-building UBC35 was increased. Functional analysis of ubc35 ubc36 mutants shows that they partially mimic pub22 pub23 pub24 enhanced activation of immune responses. Together, our work provides a framework to interrogate in vivo E2-E3 pairing and reveals a multi-tiered and dynamic E2-E3 network.

Elastin is an essential vertebrate protein responsible for the elasticity of force-bearing tissues such as those of the lungs, blood vessels, and skin. One of the key features required for the exceptional properties of this durable biopolymer is the extensive covalent cross-linking between domains of its monomer molecule tropoelastin. To date, elastin’s exact molecular assembly and mechanical properties are poorly understood. Here, using bovine elastin, we investigated the different types of cross-links in mature elastin to gain insight into its structure. We purified and proteolytically cleaved elastin from a single tissue sample into soluble cross-linked and non-cross-linked peptides that we studied by high-resolution MS. This analysis enabled the elucidation of cross-links and other elastin modifications. We found that the lysine residues within the tropoelastin sequence were simultaneously unmodified and involved in various types of cross-links with different other domains. The Lys-Pro domains were almost exclusively linked via lysinonorleucine, whereas Lys-Ala domains were found to be cross-linked via lysinonorleucine, allysine aldol, and desmosine. Unexpectedly, we identified a high number of intramolecular cross-links between lysine residues in close proximity. In summary, we show on the molecular level that elastin formation involves random cross-linking of tropoelastin monomers resulting in an unordered network, an unexpected finding compared with previous assumptions of an overall beaded structure.