Karl Gruber

• Dr.
• Professor at University of Graz

The Old Yellow Enzyme from Ferrovum sp. JA12 (FOYE) displays an unusual thermal stability for an enzyme isolated from a mesophilic organism. We determined the crystal structure of this enzyme and performed bioinformatic characterization to get insights into its thermal stability. The enzyme displays a tetrameric quaternary structure; however, unlik...

The reduction of oximes was recently identified as a promiscuous activity of Old Yellow Enzymes (OYEs). This reaction involves a two‐step reduction of α‐oxime‐ß‐ketoesters to the corresponding amines, which spontaneously dimerise to yield pyrazine derivatives. This biotransformation is currently limited to substrates with small substituents like me...

Advancing climate change increases the risk of future infectious disease outbreaks, particularly of zoonotic diseases, by affecting the abundance and spread of viral vectors. Concerningly, there are currently no approved drugs for some relevant diseases, such as the arboviral diseases chikungunya, dengue or zika. The development of novel inhibitors...

Human proteins are crucial players in both health and disease. Understanding their molecular landscape is a central topic in biological research. Here, we present an extensive dataset of predicted protein structures for 42,042 distinct human proteins, including splicing variants, derived from the UniProt reference proteome UP000005640. To ensure hi...

According to the World Health Organization, Chagas disease (CD) is the most prevalent poverty-promoting neglected tropical disease. Alarmingly, climate change is accelerating the geographical spreading of CD causative parasite, Trypanosoma cruzi, which additionally increases infection rates. Still, CD treatment remains challenging due to a lack of...

Stereoselective synthesis of quaternary stereocenters represents a significant challenge in organic chemistry. Herein, we describe the use of ene-reductases OPR3 and YqjM for the efficient asymmetric synthesis of chiral 4,4-disubstituted 2-cyclohexenones via desymmetrizing hydrogenation of prochiral 4,4-disubstituted 2,5-cyclohexadienones. This tra...

Proteins are used in various biotechnological applications, often requiring the optimization of protein properties by introducing specific amino‐acid exchanges. Deep mutational scanning (DMS) is an effective high‐throughput method for evaluating the effects of these exchanges on protein function. DMS data can then inform the training of a neural ne...

Enzymatic decarboxylation of biobased hydroxycinnamic acids gives access to phenolic styrenes for adhesive production. Phenolic acid decarboxylases are proficient enzymes that have been applied in aqueous systems, organic solvents, biphasic systems, and deep eutectic solvents, which makes stability a key feature. Stabilization of the enzyme would i...

The reduction of C=X (X = N, O) bonds is a cornerstone in both synthetic organic chemistry and biocatalysis. Conventional reduction mechanisms usually involve a hydride ion targeting the less electronegative carbon atom. In a departure from this paradigm, our investigation into Old Yellow Enzymes (OYEs) reveals a mechanism involving transfer of hyd...

The monkeypox virus (MPX) belongs to the Orthopoxvirus genus of the Poxviridae family, is endemic in parts of Africa and causes a disease in humans similar to smallpox. The most recent outbreak of MPX is already affecting 110 countries, with 86,956 confirmed cases since May 2022 and has consequently become a focus of interest. In particular, a mole...

The COVID‐19 pandemic continues to pose a substantial threat to human lives and is likely to do so for years to come. Despite the availability of vaccines, searching for efficient small‐molecule drugs that are widely available, including in low‐ and middle‐income countries, is an ongoing challenge. In this work, we report the results of an open sci...

Selective covalent labelling of enzymes using small molecule probes has advanced the scopes of protein profiling. The covalent bond formation to a specific target is the key step of activity‐based protein profiling (ABPP), a method which has become an indispensable tool for measuring enzyme activity in complex matrices. With respect to carbohydrate...

In this work, we present DrugSolver CavitomiX, a novel computational pipeline for drug repurposing and identifying ligands and inhibitors of target enzymes. The pipeline is based on cavity point clouds representing physico-chemical properties of the cavity induced solely by the protein. To test the pipeline’s ability to identify inhibitors, we chos...

The COVID-19 pandemic continues to pose a substantial threat to human lives and is likely to do so for years to come. Despite the availability of vaccines, searching for efficient small-molecule drugs that are widely available, including in low- and middle-income countries, is an ongoing challenge. In this work, we report the results of a community...

The combination of gold(I) and enzyme catalysis has provided access to a series of nor(pseudo)ephedrine derivatives in a regio‐ and stereoselective manner. The approach involves developing IPrAuNTf2‐catalyzed hydration of 1‐phenylprop‐2‐yn‐1‐yl acetate or N‐(1‐phenylprop‐2‐yn‐1‐yl)acetamide, followed by (dynamic) asymmetric biotransamination or bio...

Proteins get used in a wide variety of applications. For some applications, a specific protein property needs to be improved. Deep mutational scanning data can then be used to train a neural network, which can predict the effect of variants that have not been experimentally determined. Many approaches use the protein’s sequence in one form or anoth...

Cold-active enzymes maintain a large part of their optimal activity at low temperatures. Therefore, they can be used to avoid side reactions and preserve heat-sensitive compounds. Baeyer-Villiger monooxygenases (BVMO) utilize molecular oxygen as a co-substrate to catalyze reactions widely employed for steroid, agrochemical, antibiotic, and pheromon...

Oxidative alkene cleavage is a highly interesting reaction to obtain aldehydes and ketones. The Mn‐dependent protein TM1459 from Thermotoga maritima can catalyse alkene cleavage of styrene derivatives in the presence of tert‐butyl hydroperoxide. Despite the high thermal stability of the enzyme, it gets inactivated during the reaction. The data repo...

Erianthus produces substantial biomass, exhibits good Brix value, and shows wider environmental adaptability, rendering it a potential biofuel plant. In contrast to closely related sorghum and sugarcane, Erianthus can grow in degraded soils, thus releasing pressure on agricultural lands used for biofuel production. However, the lack of genomic reso...

The biocatalytic reduction of the oxime moiety to the corresponding amine group has only recently been found to be a promiscuous activity of ene-reductases transforming α-oximo β-keto esters. However, the reaction pathway of this two-step reduction remained elusive. By studying the crystal structures of enzyme oxime complexes, analyzing molecular d...

Treatment of COVID-19 with a soluble version of ACE2 that binds to SARS-CoV-2 virions before they enter host cells is a promising approach, however it needs to be optimized and adapted to emerging viral variants. The computational workflow presented here consists of molecular dynamics simulations for spike RBD-hACE2 binding affinity assessments of...

Introduction The current coronavirus pandemic is being combated worldwide by nontherapeutic measures and massive vaccination programs. Nevertheless, therapeutic options such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main-protease (M pro ) inhibitors are essential due to the ongoing evolution toward escape from natural or induc...

The synthesis of aldehydes from carboxylic acids has long been a challenge in chemistry. In contrast to the harsh chemically driven reduction, enzymes such as carboxylic acid reductases (CARs) are considered appealing biocatalysts for aldehyde production. Although structures of single- and didomains of microbial CARs have been reported, to date no...

To date, more than 263 million people have been infected with SARS-CoV-2 during the COVID-19 pandemic. In many countries, the global spread occurred in multiple pandemic waves characterized by the emergence of new SARS-CoV-2 variants. Here we report a sequence and structural-bioinformatics analysis to estimate the effects of amino acid substitution...

Treatment of COVID-19 with a soluble version of ACE2 that binds to SARS-CoV-2 virions before they enter host cells is a promising approach, but it needs to be optimized and adapted to emerging viral variants. The computational workflow presented here consists of molecular dynamics simulations for RBD-ACE2 binding affinity assessments of ACE2 or RBD...

Catalysis by radical enzymes dependent on coenzyme B12 (AdoCbl) relies on the reactive primary 5′‐deoxy‐5′adenosyl radical, which originates from reversible Co−C bond homolysis of AdoCbl. This bond homolysis is accelerated roughly 10¹²‐fold upon binding the enzyme substrate. The structural basis for this activation is still strikingly enigmatic. As...

The current COVID-19 pandemic poses a challenge to medical professionals and the general public alike. In addition to vaccination programs and nontherapeutic measures being employed worldwide to encounter SARS-CoV-2, great efforts have been made towards drug development and evaluation. In particular, the main protease (M pro ) makes an attractive d...

The current coronavirus pandemic is being combated worldwide by nontherapeutic measures and massive vaccination programs. Nevertheless, therapeutic options such as SARS-CoV-2 main-protease (M pro ) inhibitors are essential due to the ongoing evolution toward escape from natural or induced immunity. While antiviral strategies are vulnerable to the e...

Catalysis by radical enzymes dependent on coenzyme B 12 (AdoCbl) relies on the reactive primary 5’‐deoxy‐5’adenosyl radical, which originates from reversible Co‐C bond homolysis of AdoCbl. This bond homolysis is accelerated roughly 10 12 ‐fold upon binding the enzyme substrate. The structural basis for this activation is still strikingly enigmatic....

The monkeypox virus (MPX) belongs to the orthopoxvirus genus of the Poxviridae family, is endemic in parts of Africa, and causes a disease in humans similar to smallpox. The most recent outbreak of MPX in 2022 is already affecting 19 countries on different continents and has consequently become a focus of interest. In particular, a molecular unders...

Chronically elevated circulating fatty acid levels promote lipid accumulation in nonadipose tissues and cause lipotoxicity. Adipose triglyceride lipase (ATGL) critically determines the release of fatty acids from white adipose tissue, and accumulating evidence suggests that inactivation of ATGL has beneficial effects on lipotoxicity-driven disorder...

Environmentally friendly functionalization and recycling processes for synthetic polymers have recently gained momentum, and enzymes play a central role in these procedures. However, natural enzymes must be engineered to accept synthetic polymers as substrates. To enhance the activity on synthetic polyesters, the canonical amino acid methionine in...

Efficient amide formation is of high importance for the chemical and pharmaceutical industry. The direct biocatalytic one-pot transformation of acids into amides without substrate activation is a highly desirable but...

To date, more than 263 million people have been infected with SARS-CoV-2 during the COVID-19 pandemic. In many countries, the global spread came in several pandemic waves characterized by the emergence of new SARS-CoV-2 variants. Here, we report on a sequence- and structural-bioinformatics analysis by which we estimate the impact of amino acid exch...

Dipeptidyl peptidase III (DPP3) is a ubiquitously expressed Zn‐dependent protease, which plays an important role in regulating endogenous peptide hormones, such as enkephalins or angiotensins. In previous biophysical studies, it could be shown that substrate binding is driven by a large entropic contribution due to the release of water molecules fr...

The hexameric AAA-ATPase Drg1 is a key factor in eukaryotic ribosome biogenesis and initiates cytoplasmic maturation of the large ribosomal subunit by releasing the shuttling maturation factor Rlp24. Drg1 monomers contain two AAA-domains (D1 and D2) that act in a concerted manner. Rlp24 release is inhibited by the drug diazaborine which blocks ATP...

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes TMPRSS2 receptor to enter target human cells and subsequently causes coronavirus disease 19 (COVID-19). TMPRSS2 belongs to the type II serine proteases of subfamily TMPRSS, which is characterized by the presence of the serine-protease domain. TMPRSS4 is another TMPRSS member,...

Since the worldwide outbreak of the infectious disease COVID-19, several studies have been published to understand the structural mechanism of the novel coronavirus SARS-CoV-2. During the infection process, the SARS-CoV-2 spike (S) protein plays a crucial role in the receptor recognition and cell membrane fusion process by interacting with the huma...

Monolignol oxidoreductases are members of the berberine bridge enzyme–like (BBE-like) protein family (pfam 08031) that oxidize monolignols to the corresponding aldehydes. They are FAD-dependent enzymes that exhibit the para-cresolmethylhydroxylase-topology, also known as vanillyl oxidase-topology. Recently, we have reported the structural and bioch...

Levetiracetam is an active pharmaceutical ingredient widely used to treat epilepsy. We describe a new synthesis of levetiracetam by a dynamic kinetic resolution and a ruthenium-catalysed ex-cell anodic oxidation. For the enzymatic resolution, we tailored a high throughput screening method to identify Comamonas testosteroni nitrile hydratase variant...

Since the worldwide outbreak of the infectious disease COVID-19, several studies have been published to understand the structural mechanism of the novel coronavirus SARS-CoV-2. During the infection process, the SARS-CoV-2 spike (S) protein plays a crucial role in the receptor recognition and cell membrane fusion process by interacting with the huma...

Since the worldwide outbreak of the infectious disease COVID-19, several studies have been published to understand the structural mechanism of the novel coronavirus SARS-CoV-2. During the infection process, the SARS-CoV-2 spike (S) protein plays a crucial role in the receptor recognition and cell membrane fusion process by interacting with the huma...

This work is about synergy of theory and experiment in revealing mechanism of binding of dipeptidyl peptidase III (DPP III) and Kelch-like ECH-associated protein 1 (KEAP1), the main cellular sensor of oxidative stress. The NRF2 ̶ KEAP1 signaling pathway is important for cell protection, but it is also impaired in many cancer cells where NRF2 target...

Ascorbate oxidases are an enzyme group that has not been explored to a large extent. So far, mainly ascorbate oxidases from plants and only a few from fungi have been described. Although ascorbate oxidases belong to the well-studied enzyme family of multi-copper oxidases, their function is still unclear. In this study, Af_AO1, an enzyme from the fu...

Dipeptidyl peptidase 3 (DPP3) is a zinc-dependent hydrolase involved in degrading oligopeptides with 4–12 amino acid residues. It has been associated with several pathophysiological processes, including blood pressure regulation, pain signaling, and cancer cell defense against oxidative stress. However, the physiological substrates and the cellular...

The biocatalytic Friedel-Crafts acylation has been identified recently for the acetylation of resorcinol using activated acetic acid esters for the synthesis of acetophenone derivatives catalyzed by an acyltransferase. Because the wild-type enzyme is limited to acetic and propionic derivatives as the substrate, variants were designed to extend the...

Regioselective hydroxylation on inactivated C−H bonds is among the dream reactions of organic chemists. Cytochrome P450 enzymes (CYPs) perform this reaction in general with high regio‐ and stereoselectivity (e. g. for steroids as substrates). Furthermore, enzyme engineering may allow to tune the properties of the enzyme. Regioselective hydroxylatio...

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a human FAD‐dependent enzyme that plays a crucial role in the antioxidant defense system. A naturally occurring single nucleotide polymorphism (NQO1*2) in the NQO1 gene leads to an amino acid substitution (P187S), which severely compromises the activity and stability of the enzyme. The NQO1*2 genotype has...

Replacing the central cobalt ion of vitamin B12 by other metals has been a long‐held aspiration within the B12‐field. Herein, we describe the synthesis from hydrogenobyric acid of zincobyric acid (Znby) and zincobalamin (Znbl), the Zn‐analogues of the natural cobalt‐corrins cobyric acid and vitamin B12, respectively. The solution structures of Znby...

Zincobyrsäure und Zincobalamin, die Zn‐Analoge von Cobyrsäure und Vitamin B12, wurden aus dem metallfreien Corrin Hydrogenobyrsäure hergestellt. Die Kristallstruktur des Zincobyrats, die erste eines Zn‐Corrins, zeigt eine deutliche Fehlpassung des geschlossenschaligen ZnII‐Ions und des Corrin‐Liganden. Die lumineszenten Zn‐Mimetika von Vitamin B12...

The B12 cofactors instill a natural curiosity regarding the primordial selection and evolution of their corrin ligand. Surprisingly, this important natural macrocycle has evaded molecular scrutiny, and its specific role in predisposing the incarcerated cobalt ion for organometallic catalysis has remained obscure. Herein, we report the biosynthesis...

Hydrogenobyrsäure, der metallfreie Corrin‐Ligand von natürlichen B12‐Derivaten, wurde durch Biosynthese hergestellt. Seine Struktur zeigt einen ringkontrahierten, stark helikalen Koordinationsraum, der Cobalt‐Ionen in einem entatisch gespannten Zustand bei koordinativer Fehlanordnung fest bindet. Die B12‐Cofaktoren werden dadurch für die Spaltung i...

Podophyllotoxin is probably the most prominent representative of lignan natural products. Deoxy‐, epi‐, and podophyllotoxin, which are all precursors to frequently used chemotherapeutic agents, were prepared by a stereodivergent biotransformation and a biocatalytic kinetic resolution of the corresponding dibenzylbutyrolactones with the same 2‐oxogl...

The addition of water to non‐activated carbon–carbon double bonds catalyzed by fatty acid hydratases (FAHYs) allows for highly regio‐ and stereoselective oxyfunctionalization of renewable oil feedstock. So far, the applicability of FAHYs has been limited to free fatty acids, mainly owing to the requirement of a carboxylate function for substrate re...

Gebändigtes Eisen: Podophyllotoxin und Derivate wurden durch chemoenzymatische Totalsynthese unter Verwendung der Eisen‐abhängigen Dioxygenase aus Podophyllum hexandrum hergestellt. Mittels Substrat‐Engineering konnte der biologische Schritt von einem enantiodivergenten zu einem racematspaltenden Schlüsselschritt umgelenkt werden. Derivate des natü...

The addition of water to non‐activated carbon‐carbon double bonds catalyzed by fatty acid hydratases (FAHYs) allows for highly regio‐ and stereoselective oxyfunctionalization of renewable oil feedstock. So far, the applicability of FAHYs has been limited to free fatty acids, mainly owing to the requirement of a carboxylate function for substrate re...

The enzyme 4-oxalocrotonate tautomerase shows remarkable catalytic versatility due to the secondary amine of its N-terminal proline moiety. In this work, we incorporated a range of proline analogues into the enzyme and examined the effects on structure and activity. While the structure of the enzyme remained unperturbed, its promiscuous Michael-typ...

C−C bond‐forming reactions are key transformations for setting up the carbon frameworks of organic compounds. In this context, Friedel–Crafts acylation is commonly used for the synthesis of aryl ketones, which are common motifs in many fine chemicals and natural products. A bacterial multicomponent acyltransferase from Pseudomonas protegens (PpATas...

Flavin-dependent enzymes catalyze many oxidations, including formation of ring structures in natural products. The gene cluster for biosynthesis of fumisoquins, secondary metabolites structurally related to isoquinolines, in the filamentous fungus Aspergillus fumigatus harbors a gene that encodes a flavoprotein of the amine oxidase family, termed f...

Ene reductases from the Old Yellow Enzyme (OYE) family reduce the carbon‐carbon double bond in alpha,beta‐unsaturated compounds bearing an electron withdrawing group, e.g. a carbonyl group. This asymmetric reduction has been exploited for biocatalysis. Going beyond its canonical function, we could show that members of this enzyme family can also ca...

Ene reductases from the Old Yellow Enzyme (OYE) family reduce the carbon‐carbon double bond in alpha,beta‐unsaturated compounds bearing an electron withdrawing group, e.g. a carbonyl group. This asymmetric reduction has been exploited for biocatalysis. Going beyond its canonical function, we could show that members of this enzyme family can also ca...

Carboxylate reductases (CARs, E.C. 1.2.1.30) generate aldehydes from their corresponding carboxylic acid with high selectivity. Little is known about the structure of CARs and their catalytically important amino acid residues. The identification of key residues for carboxylate reduction provides a starting point to gain deeper understanding of enzy...

Over the last decades biocatalysis has emerged as an indispensable and versatile tool for the asymmetric synthesis of active pharmaceutical ingredients (APIs). In this context, especially transaminases (TAs) have been successfully used for the preparation of numerous α‐chiral, optically pure amines, serving as important building blocks for APIs. He...

Porphyromonas gingivalis, an asaccharolytic Gram-negative oral anaerobe, is a major pathogen associated with adult periodontitis, a chronic infective disease that a significant percentage of the human population suffers from. It preferentially utilizes dipeptides as its carbon source, suggesting the importance of dipeptidyl peptidase (DPP) types of...

MMS complementation assay. An aliquot of 1 μL serially diluted mid-log phase cultures of BK2118 strain transformed with pUC18PgDPP3, pUC18AlkD_like domain PgDPP3 (C-terminus), pUC18AlkD B. cereus and pUC18AlkD P. gingivalis, and with pET21a empty plasmid, was spotted on LBA plates with 2 mM MMS and incubated for 2 days at 37°C. (DOCX)

Rgyr (Å) profile of PgDPP III. Rgyr (Å) profile of PgDPP III (black), its DPP IIIpart (red) and AlkD like C-terminal domain (green) determined during 200 ns long MD simulation of the PgDPP III—Arg2-2NA complex (replica 1). (DOCX)

The amino acid residues from the enzyme PgDPP III binding pocket that Arg2-2NA molecule interacts with during 200 ns MD simulation of one replica. (TIF)

Stabilization of Arg2-AMC in the PgDPP III active site. Structure obtained after 150 ns of MD simulations. Zn2+ is represented as a gray sphere. (DOCX)

HPLC-MS results. HPLC-MS results of 24 hours incubation of wild type PgDPP III enzyme with ligands angiotensin II, tynorphin and IVYPW, respectively. In all three reactions there is main product detectable and small amount of side products confirming sequential cleavage of dipeptides from N-termini of substrates. (DOCX)

Correlation of the HDX results with the theoretical results based on the MD simulations of the inital (homology modelled) PgDPP III structure. The structure is obtained during 200 ns of MD simulation (50 ns equilibration + 150 productive MD). (DOCX)

Populations (% of the frames sampled during MD simulations) of the selected strong intermolecular hydrogen bonds. (DOCX)

Multiple sequence alignment. Multiple sequence alignment of a selection of DPP III showing conservation of active site motif (M49 family zinc-binding motifs are highlighted). Multiple sequence alignment was obtained using CLUSTAL Omega with DPP III sequences for: Homo sapiens (Q9NY33), Mus musculus (Q99KK7), Danio rerio (Q6DI20), Drosophila melanog...

3D models of P.gingivalis DPP III. Homology modeling of the three-dimensional structure of P. gingivalis DPP III was performed using Phyre2 program. On the right AlkD and AlkF structures from B. cereus. Active site in DPP III is coloured in blue. (DOCX)