Publications by authors named "Karl Gruber"

174 Publications

Efficient Entropy-Driven Inhibition of Dipeptidyl Peptidase III by Hydroxyethylene Transition-State Peptidomimetics.

Chemistry 2021 Jul 27. Epub 2021 Jul 27.

Institute of Organic Chemistry, Graz University of Technology, Stremayrgasse 9, 8010, Graz, Austria.

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 from the closing binding cleft. Here, the design, synthesis and biophysical characterization of peptidomimetic inhibitors is reported, using for the first time an hydroxyethylene transition-state mimetic for a metalloprotease. Efficient routes for the synthesis of both stereoisomers of the pseudopeptide core were developed, which allowed the synthesis of peptidomimetic inhibitors mimicking the VVYPW-motif of tynorphin. The best inhibitors inhibit DPP3 in the low μM range. Biophysical characterization by means of ITC measurement and X-ray crystallography confirm the unusual entropy-driven mode of binding. Stability assays demonstrated the desired stability of these inhibitors, which efficiently inhibited DPP3 in mouse brain homogenate.
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http://dx.doi.org/10.1002/chem.202102204DOI Listing
July 2021

Structural basis for inhibition of the AAA-ATPase Drg1 by diazaborine.

Nat Commun 2021 06 9;12(1):3483. Epub 2021 Jun 9.

Institute of Molecular Biosciences, University of Graz, Graz, Austria.

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 hydrolysis in D2. The mode of inhibition was unknown. Here we show the first cryo-EM structure of Drg1 revealing the inhibitory mechanism. Diazaborine forms a covalent bond to the 2'-OH of the nucleotide in D2, explaining its specificity for this site. As a consequence, the D2 domain is locked in a rigid, inactive state, stalling the whole Drg1 hexamer. Resistance mechanisms identified include abolished drug binding and altered positioning of the nucleotide. Our results suggest nucleotide-modifying compounds as potential novel inhibitors for AAA-ATPases.
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http://dx.doi.org/10.1038/s41467-021-23854-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8190095PMC
June 2021

Diversification of the mygalomorph spider genus Aname (Araneae: Anamidae) across the Australian arid zone: Tracing the evolution and biogeography of a continent-wide radiation.

Mol Phylogenet Evol 2021 07 2;160:107127. Epub 2021 Mar 2.

Collections and Research Centre, Western Australian Museum, Welshpool, WA 6106, Australia; School of Biological Sciences, The University of Western Australia, Crawley, WA 6009, Australia.

The assembly of the Australian arid zone biota has long fascinated biogeographers. Covering over two-thirds of the continent, Australia's vast arid zone biome is home to a distinctive fauna and flora, including numerous lineages which have diversified since the Eocene. Tracing the origins and speciation history of these arid zone taxa has been an ongoing endeavour since the advent of molecular phylogenetics, and an increasing number of studies on invertebrate animals are beginning to complement a rich history of research on vertebrate and plant taxa. In this study, we apply continent-wide genetic sampling and one of the largest phylogenetic data matrices yet assembled for a genus of Australian spiders, to reconstruct the phylogeny and biogeographic history of the open-holed trapdoor spider genus Aname L. Koch, 1873. This highly diverse lineage of Australian mygalomorph spiders has a distribution covering the majority of Australia west of the Great Dividing Range, but apparently excluding the high rainfall zones of eastern Australia and Tasmania. Original and legacy sequences were obtained for three mtDNA and four nuDNA markers from 174 taxa in seven genera, including 150 Aname specimen terminals belonging to 102 species-level operational taxonomic units, sampled from 32 bioregions across Australia. Reconstruction of the phylogeny and biogeographic history of Aname revealed three radiations (Tropical, Temperate-Eastern and Continental), which could be further broken into eight major inclusive clades. Ancestral area reconstruction revealed the Pilbara, Monsoon Tropics and Mid-West to be important ancestral areas for the genus Aname and its closest relatives, with the origin of Aname itself inferred in the Pilbara bioregion. From these origins in the arid north-west of Australia, our study found evidence for a series of subsequent biome transitions in separate lineages, with at least eight tertiary incursions back into the arid zone from more mesic tropical, temperate or eastern biomes, and only two major clades which experienced widespread (primary) in situ diversification within the arid zone. Based on our phylogenetic results, and results from independent legacy divergence dating studies, we further reveal the importance of climate-driven biotic change in the Miocene and Pliocene in shaping the distribution and composition of the Australian arid zone biota, and the value of continent-wide studies in revealing potentially complex patterns of arid zone diversification in dispersal-limited invertebrate taxa.
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http://dx.doi.org/10.1016/j.ympev.2021.107127DOI Listing
July 2021

Serine 477 plays a crucial role in the interaction of the SARS-CoV-2 spike protein with the human receptor ACE2.

Sci Rep 2021 02 22;11(1):4320. Epub 2021 Feb 22.

Institute of Molecular Bioscience, University of Graz, 8010, Graz, Austria.

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 human angiotensin-converting enzyme 2 (hACE2) receptor. However, new variants of these spike proteins emerge as the virus passes through the disease reservoir. This poses a major challenge for designing a potent antigen for an effective immune response against the spike protein. Through a normal mode analysis (NMA) we identified the highly flexible region in the receptor binding domain (RBD) of SARS-CoV-2, starting from residue 475 up to residue 485. Structurally, the position S477 shows the highest flexibility among them. At the same time, S477 is hitherto the most frequently exchanged amino acid residue in the RBDs of SARS-CoV-2 mutants. Therefore, using MD simulations, we have investigated the role of S477 and its two frequent mutations (S477G and S477N) at the RBD during the binding to hACE2. We found that the amino acid exchanges S477G and S477N strengthen the binding of the SARS-COV-2 spike with the hACE2 receptor.
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http://dx.doi.org/10.1038/s41598-021-83761-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900180PMC
February 2021

Rediscovering Asia's forgotten crops to fight chronic and hidden hunger.

Nat Plants 2021 02 15;7(2):116-122. Epub 2021 Feb 15.

The UWA Institute of Agriculture, The University of Western Australia (UWA), Perth, Western Australia, Australia.

Asia has a rich variety of nutritious 'neglected crops', domesticated since ancient times but mostly forgotten or underutilized today. These crops, including cereals, roots, nuts, pulses, fruits and vegetables, are adapted to their land, resilient to environmental challenges and rich in micronutrients. Changing current agricultural practices from a near monoculture to a diverse cropping portfolio that uses these forgotten crops is a viable and promising approach to closing the current gaps in production and nutrition in Asia. Such an approach was proposed by the Food and Agriculture Organization's Zero Hunger initiative in Asia, which aims to end hunger by 2030. The Zero Hunger initiative is a promising approach to help increase access to nutritious food; however, it faces substantial challenges, such as the lack of farmer willingness to switch crops and adequate governmental support for implementation. Countries such as Nepal have started using these neglected crops, implementing various approaches to overcome challenges and start a new agricultural pathway.
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http://dx.doi.org/10.1038/s41477-021-00850-zDOI Listing
February 2021

The catalytic machinery of the FAD-dependent AtBBE-like protein 15 for alcohol oxidation: Y193 and Y479 form a catalytic base, Q438 and R292 an alkoxide binding site.

Arch Biochem Biophys 2021 03 22;700:108766. Epub 2021 Jan 22.

Graz University of Technology, NAWI Graz, Institute of Biochemistry, Graz, Austria; University of Graz, NAWI Graz, Institute of Molecular Biosciences, Graz, Austria; Acib-Austrian Centre of Industrial Biotechnology, Krenngasse 37, Graz, Austria. Electronic address:

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 biochemical characterization of two monolignol oxidoreductases from Arabidopsis thaliana, AtBBE13 and AtBBE15. Now, we have conducted a comprehensive site directed mutagenesis study for AtBBE15, to expand our understanding of the catalytic mechanism of this enzyme class. Based on the kinetic properties of active site variants and molecular dynamics simulations, we propose a refined, structure-guided reaction mechanism for the family of monolignol oxidoreductases. Here, we propose that this reaction is facilitated stepwise by the deprotonation of the allylic alcohol and a subsequent hydride transfer from the Cα-atom of the alkoxide to the flavin. We describe an excessive hydrogen bond network that enables the catalytic mechanism of the enzyme. Within this network Tyr479 and Tyr193 act concertedly as active catalytic bases to facilitate the proton abstraction. Lys436 is indirectly involved in the deprotonation as this residue determines the position of Tyr193 via a cation-π interaction. The enzyme forms a hydrophilic cavity to accommodate the alkoxide intermediate and to stabilize the transition state from the alkoxide to the aldehyde. By means of molecular dynamics simulations, we have identified two different and distinct binding modes for the substrate in the alcohol and alkoxide state. The alcohol interacts with Tyr193 and Tyr479 while Arg292, Gln438 and Tyr193 form an alkoxide binding site to accommodate this intermediate. The pH-dependency of the activity of the active site variants revealed that the integrity of the alkoxide binding site is also crucial for the fine tuning of the pK of Tyr193 and Tyr479. Sequence alignments showed that key residues for the mechanism are highly conserved, indicating that our proposed mechanism is not only relevant for AtBBE15 but for the majority of BBE-like proteins.
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http://dx.doi.org/10.1016/j.abb.2021.108766DOI Listing
March 2021

Binding of dipeptidyl peptidase III to the oxidative stress cell sensor Kelch-like ECH-associated protein 1 is a two-step process.

J Biomol Struct Dyn 2020 Aug 18:1-12. Epub 2020 Aug 18.

Divison of Organic Chemistry and Biochemistry, Ruđer Bošković Institute, Zagreb, Croatia.

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 gene expression leads to resistance to chemotherapeutic drugs. DPP III competitively binds to KEAP1 in the conditions of oxidative stress and induces release of NRF2 and its translocation into nucleus. The binding is established mainly through the ETGE motif of DPP III and the Kelch domain of KEAP1. However, although part of a flexible loop, ETGE itself is firmly attached to the DPP III surface by strong hydrogen bonds. Using combined computational and experimental study, we found that DPP III ̶ Kelch binding is a two-step process comprising the endergonic loop detachment and exergonic DPP III ̶ Kelch interaction. Substitution of arginines, which keep the ETGE motif attached, decreases the work needed for its release and increases DPP III ̶ Kelch binding affinity. Interestingly, mutations of one of these arginine residues have been reported in cBioPortal for cancer genomics, implicating its possible involvement in cancer development. Communicated by Ramaswamy H. Sarma.
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http://dx.doi.org/10.1080/07391102.2020.1804455DOI Listing
August 2020

A Fungal Ascorbate Oxidase with Unexpected Laccase Activity.

Int J Mol Sci 2020 Aug 11;21(16). Epub 2020 Aug 11.

Institute of Environmental Biotechnology, Department of Agrobiotechnology, University of Natural Resources and Life Sciences (BOKU), 3430 Tulln an der Donau, Austria.

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, _AO1, an enzyme from the fungus , was characterized. Sequence analyses and copper content determination demonstrated _AO1 to belong to the multi-copper oxidase family. Biochemical characterization and 3D-modeling revealed a similarity to ascorbate oxidases, but also to laccases. _AO1 had a 10-fold higher affinity to ascorbic acid ( = 0.16 ± 0.03 mM) than to ABTS ( = 1.89 ± 0.12 mM). Furthermore, the best fitting 3D-model was based on the ascorbate oxidase from var. . The laccase-like activity of _AO1 on ABTS ( = 11.56 ± 0.15 µM/min/mg) was, however, not negligible. On the other hand, other typical laccase substrates, such as syringaldezine and guaiacol, were not oxidized by _AO1. According to the biochemical and structural characterization, _AO1 was classified as ascorbate oxidase with unusual, laccase-like activity.
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http://dx.doi.org/10.3390/ijms21165754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7460845PMC
August 2020

Dipeptidyl peptidase 3 modulates the renin-angiotensin system in mice.

J Biol Chem 2020 10 16;295(40):13711-13723. Epub 2020 Jun 16.

Institute of Biochemistry, Graz University of Technology, NAWI Graz, Graz, Austria. Electronic address:

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 pathways that are potentially targeted by DPP3 to mediate these effects remain unknown. Here, we show that global DPP3 deficiency in mice (DPP3) affects the renin-angiotensin system (RAS). LC-MS-based profiling of circulating angiotensin peptides revealed elevated levels of angiotensin II, III, IV, and 1-5 in DPP3 mice, whereas blood pressure, renin activity, and aldosterone levels remained unchanged. Activity assays using the purified enzyme confirmed that angiotensin peptides are substrates for DPP3. Aberrant angiotensin signaling was associated with substantially higher water intake and increased renal reactive oxygen species formation in the kidneys of DPP3 mice. The metabolic changes and altered angiotensin levels observed in male DPP3 mice were either absent or attenuated in female DPP3 mice, indicating sex-specific differences. Taken together, our observations suggest that DPP3 regulates the RAS pathway and water homeostasis by degrading circulating angiotensin peptides.
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http://dx.doi.org/10.1074/jbc.RA120.014183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7535908PMC
October 2020

Rational Engineered C-Acyltransferase Transforms Sterically Demanding Acyl Donors.

ACS Catal 2020 Jan 27;10(2):1094-1101. Epub 2019 Dec 27.

Institute of Chemistry, University of Graz, NAWI Graz, BioTechMed Graz, Heinrichstrasse 28, 8010 Graz, Austria.

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 substrate scope of this enzyme. By rational protein engineering, the key residue in the active site was identified which can be replaced to allow binding of bulkier acyl moieties. The single-point variant F148V enabled the transformation of previously inaccessible medium chain length alkyl and alkoxyalkyl carboxylic esters as donor substrates with up to 99% conversion and up to >99% isolated yield.
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http://dx.doi.org/10.1021/acscatal.9b04617DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6996649PMC
January 2020

Cities as evolutionary experiments: As cities and other man-made environments encroach on natural settings, species evolve to cope.

Authors:
Karl F Gruber

EMBO Rep 2019 11 28;20(11):e49401. Epub 2019 Oct 28.

Perth, WA, Australia.

Cities exert strong selective pressures on plants and animals to adapt to urban life. They provide a unique testing ground for studying evolution in action.
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http://dx.doi.org/10.15252/embr.201949401DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6831994PMC
November 2019

A small molecule chaperone rescues the stability and activity of a cancer-associated variant of NAD(P)H:quinone oxidoreductase 1 in vitro.

FEBS Lett 2020 02 30;594(3):424-438. Epub 2019 Oct 30.

Institute of Biochemistry, Graz University of Technology, Austria.

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 been linked to a higher risk for several types of cancer and poor survival rate after anthracycline-based chemotherapy. In this study, we show that a small molecular chaperone (N-(2-bromophenyl)pyrrolidine-1-sulfonamide) repopulates the native wild-type conformation. As a consequence of the stabilizing effect, the enzymatic activity of the P187S variant protein is strongly improved in the presence of the molecular chaperone in vitro.
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http://dx.doi.org/10.1002/1873-3468.13636DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7027498PMC
February 2020

Zinc Substitution of Cobalt in Vitamin B : Zincobyric acid and Zincobalamin as Luminescent Structural B -Mimics.

Angew Chem Int Ed Engl 2019 10 4;58(41):14568-14572. Epub 2019 Sep 4.

Institute of Organic Chemistry and Center for Molecular Biosciences (CMBI), University of Innsbruck, 6020, Innsbruck, Austria.

Replacing the central cobalt ion of vitamin B by other metals has been a long-held aspiration within the B -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 B , respectively. The solution structures of Znby and Znbl were studied by NMR-spectroscopy. Single crystals of Znby were produced, providing the first X-ray crystallographic structure of a zinc corrin. The structures of Znby and of computationally generated Znbl were found to resemble the corresponding Co -corrins, making such Zn-corrins potentially useful for investigations of B -dependent processes. The singlet excited state of Znby had a short life-time, limited by rapid intersystem crossing to the triplet state. Znby allowed the unprecedented observation of a corrin triplet (E =190 kJ mol ) and was found to be an excellent photo-sensitizer for O (Φ =0.70).
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http://dx.doi.org/10.1002/anie.201908428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6790578PMC
October 2019

The Hydrogenobyric Acid Structure Reveals the Corrin Ligand as an Entatic State Module Empowering B Cofactors for Catalysis.

Angew Chem Int Ed Engl 2019 07 26;58(31):10756-10760. Epub 2019 Jun 26.

Institute of Organic Chemistry and Center for Molecular Biosciences, University of Innsbruck, 6020, Innsbruck, Austria.

The B 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 of the cobalt-free B corrin moiety, hydrogenobyric acid (Hby), a compound crafted through pathway redesign. Detailed insights from single-crystal X-ray and solution structures of Hby have revealed a distorted helical cavity, redefining the pattern for binding cobalt ions. Consequently, the corrin ligand coordinates cobalt ions in desymmetrized "entatic" states, thereby promoting the activation of B -cofactors for their challenging chemical transitions. The availability of Hby also provides a route to the synthesis of transition metal analogues of B .
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http://dx.doi.org/10.1002/anie.201904713DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6771967PMC
July 2019

Chemoenzymatic Total Synthesis of Deoxy-, epi-, and Podophyllotoxin and a Biocatalytic Kinetic Resolution of Dibenzylbutyrolactones.

Angew Chem Int Ed Engl 2019 06 8;58(24):8226-8230. Epub 2019 May 8.

Institute of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Heinrichstrasse 28/II, 8010, Graz, Austria.

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-oxoglutarate-dependent dioxygenase. The reaction can be conducted on 2 g scale, and the enzyme allows tailoring of the initial, "natural" structure and thus transforms various non-natural derivatives. Depending on the substitution pattern, the enzyme performs an oxidative C-C bond formation by C-H activation or hydroxylation at the benzylic position prone to ring closure.
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http://dx.doi.org/10.1002/anie.201900926DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6563474PMC
June 2019

Evolving the Promiscuity of Elizabethkingia meningoseptica Oleate Hydratase for the Regio- and Stereoselective Hydration of Oleic Acid Derivatives.

Angew Chem Int Ed Engl 2019 05 17;58(22):7480-7484. Epub 2019 Apr 17.

Institute of Molecular Biotechnology, Graz University of Technology, NAWI Graz, BioTechMed Graz, Petersgasse 14, 8010, Graz, Austria.

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 recognition and binding. Herein, we describe for the first time the hydration of oleic acid (OA) derivatives lacking this free carboxylate by the oleate hydratase from Elizabethkingia meningoseptica (OhyA). Molecular docking of OA to the OhyA 3D-structure and a sequence alignment uncovered conserved amino acid residues at the entrance of the substrate channel as target positions for enzyme engineering. Exchange of selected amino acids gave rise to OhyA variants which showed up to an 18-fold improved conversion of OA derivatives, while retaining the excellent regio- and stereoselectivity in the olefin hydration reaction.
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http://dx.doi.org/10.1002/anie.201901462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6563698PMC
May 2019

Substituting the catalytic proline of 4-oxalocrotonate tautomerase with non-canonical analogues reveals a finely tuned catalytic system.

Sci Rep 2019 02 25;9(1):2697. Epub 2019 Feb 25.

Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010, Graz, Austria.

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-type activity was severely affected. This finding demonstrates how atomic changes in a biocatalytic system can abolish its activity. Our work provides a toolbox for successful generation of enzyme variants with non-canonical catalytic proline analogues.
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http://dx.doi.org/10.1038/s41598-019-39484-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6389900PMC
February 2019

Structure and Catalytic Mechanism of a Bacterial Friedel-Crafts Acylase.

Chembiochem 2019 01 26;20(1):88-95. Epub 2018 Nov 26.

Austrian Centre of Industrial Biotechnology (ACIB), Petersgasse 14, 8010, Graz, Austria.

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 (PpATase) catalyzes such Friedel-Crafts C-acylation of phenolic substrates in aqueous solution, reaching up to >99 % conversion without the need for CoA-activated reagents. We determined X-ray crystal structures of the native and ligand-bound complexes. This multimeric enzyme consists of three subunits: PhlA, PhlB, and PhlC, arranged in a Phl(A C ) B composition. The structure of a reaction intermediate obtained from crystals soaked with the natural substrate 1-(2,4,6-trihydroxyphenyl)ethanone together with site-directed mutagenesis studies revealed that only residues from the PhlC subunits are involved in the acyl transfer reaction, with Cys88 very likely playing a significant role during catalysis. These structural and mechanistic insights form the basis of further enzyme engineering efforts directed towards enhancing the substrate scope of this enzyme.
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http://dx.doi.org/10.1002/cbic.201800462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6392133PMC
January 2019

Australia tackles HTLV-1.

Authors:
Karl Gruber

Lancet Infect Dis 2018 10;18(10):1073-1074

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http://dx.doi.org/10.1016/S1473-3099(18)30561-9DOI Listing
October 2018

Oxidative cyclization of -methyl-dopa by a fungal flavoenzyme of the amine oxidase family.

J Biol Chem 2018 11 7;293(44):17021-17032. Epub 2018 Sep 7.

From the Institutes of Biochemistry, Graz University of Technology, Petersgasse 12/II, 8010 Graz,

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 harbors a gene that encodes a flavoprotein of the amine oxidase family, termed (fumisoquin biosynthesis gene B). This enzyme catalyzes an oxidative ring closure reaction that leads to the formation of isoquinoline products. This reaction is reminiscent of the oxidative cyclization reported for berberine bridge enzyme and tetrahydrocannabinol synthase. Despite these similarities, amine oxidases and berberine bridge enzyme-like enzymes possess distinct structural properties, prompting us to investigate the structure-function relationships of FsqB. Here, we report the recombinant production and purification of FsqB, elucidation of its crystal structure, and kinetic analysis employing five putative substrates. The crystal structure at 2.6 Å resolution revealed that FsqB is a member of the amine oxidase family with a covalently bound FAD cofactor. -methyl-dopa was the best substrate for FsqB and was completely converted to the cyclic isoquinoline product. The absence of the -hydroxyl group, as in l--methyl-tyrosine, resulted in a 25-fold lower rate of reduction and the formation of the demethylated product l-tyrosine, instead of a cyclic product. Surprisingly, FsqB did not accept the d-stereoisomer of -methyltyrosine, in contrast to -methyl-dopa, for which both stereoisomers were oxidized with similar rates. On the basis of the crystal structure and docking calculations, we postulate a substrate-dependent population of distinct binding modes that rationalizes stereospecific oxidation in the FsqB active site.
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http://dx.doi.org/10.1074/jbc.RA118.004227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222107PMC
November 2018

Asymmetric Reductive Carbocyclization Using Engineered Ene Reductases.

Angew Chem Int Ed Engl 2018 06 14;57(24):7240-7244. Epub 2018 May 14.

Institute of Organic Chemistry, Graz University of Technology, Stremayrgasse 9, 8010, Graz, Austria.

Ene reductases from the Old Yellow Enzyme (OYE) family reduce the C=C double bond in α,β-unsaturated compounds bearing an electron-withdrawing group, for example, a carbonyl group. This asymmetric reduction has been exploited for biocatalysis. Going beyond its canonical function, we show that members of this enzyme family can also catalyze the formation of C-C bonds. α,β-Unsaturated aldehydes and ketones containing an additional electrophilic group undergo reductive cyclization. Mechanistically, the two-electron-reduced enzyme cofactor FMN delivers a hydride to generate an enolate intermediate, which reacts with the internal electrophile. Single-site replacement of a crucial Tyr residue with a non-protic Phe or Trp favored the cyclization over the natural reduction reaction. The new transformation enabled the enantioselective synthesis of chiral cyclopropanes in up to >99 % ee.
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http://dx.doi.org/10.1002/anie.201802962DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6033016PMC
June 2018

Cleaning up pollutants to protect future health.

Authors:
Karl Gruber

Nature 2018 03;555(7695):S20-S22

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http://dx.doi.org/10.1038/d41586-018-02481-5DOI Listing
March 2018

Identification of Key Residues for Enzymatic Carboxylate Reduction.

Front Microbiol 2018 19;9:250. Epub 2018 Feb 19.

Institute of Molecular Biotechnology, Graz University of Technology, NAWI Graz, Graz, Austria.

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 enzymatic carboxylate reduction. A multiple sequence alignment of CARs with confirmed activity recently identified in our lab and from the literature revealed a fingerprint of conserved amino acids. We studied the function of conserved residues by multiple sequence alignments and mutational replacements of these residues. In this study, single-site alanine variants of CAR were investigated to determine the contribution of conserved residues to the function, expressability or stability of the enzyme. The effect of amino acid replacements was investigated by analyzing enzymatic activity of the variants and . Supported by molecular modeling, we interpreted that five of these residues are essential for catalytic activity, or substrate and co-substrate binding. We identified amino acid residues having significant impact on CAR activity. Replacement of His 237, Glu 433, Ser 595, Tyr 844, and Lys 848 by Ala abolish CAR activity, indicating their key role in acid reduction. These results may assist in the functional annotation of CAR coding genes in genomic databases. While some other conserved residues decreased activity or had no significant impact, four residues increased the specific activity of CAR variants when replaced by alanine. Finally, we showed that CAR wild-type and mutants efficiently reduce aliphatic acids.
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http://dx.doi.org/10.3389/fmicb.2018.00250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5826065PMC
February 2018

Cleaning up pollutants to protect future health.

Authors:
Karl Gruber

Nature 2018 Mar;555(7695):S20-S22

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http://dx.doi.org/10.1038/d41586-018-02481-5DOI Listing
March 2018

A novel Porphyromonas gingivalis enzyme: An atypical dipeptidyl peptidase III with an ARM repeat domain.

PLoS One 2017 30;12(11):e0188915. Epub 2017 Nov 30.

Division of Organic Chemistry and Biochemistry, Ruđer Bošković Institute, Zagreb, Croatia.

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 enzyme for its growth. Until now DPP IV, DPP5, 7 and 11 have been extensively investigated. Here, we report the characterization of DPP III using molecular biology, biochemical, biophysical and computational chemistry methods. In addition to the expected evolutionarily conserved regions of all DPP III family members, PgDPP III possesses a C-terminal extension containing an Armadillo (ARM) type fold similar to the AlkD family of bacterial DNA glycosylases, implicating it in alkylation repair functions. However, complementation assays in a DNA repair-deficient Escherichia coli strain indicated the absence of alkylation repair function for PgDPP III. Biochemical analyses of recombinant PgDPP III revealed activity similar to that of DPP III from Bacteroides thetaiotaomicron, and in the range between activities of human and yeast counterparts. However, the catalytic efficiency of the separately expressed DPP III domain is ~1000-fold weaker. The structure and dynamics of the ligand-free enzyme and its complex with two different diarginyl arylamide substrates was investigated using small angle X-ray scattering, homology modeling, MD simulations and hydrogen/deuterium exchange (HDX). The correlation between the experimental HDX and MD data improved with simulation time, suggesting that the DPP III domain adopts a semi-closed or closed form in solution, similar to that reported for human DPP III. The obtained results reveal an atypical DPP III with increased structural complexity: its superhelical C-terminal domain contributes to peptidase activity and influences DPP III interdomain dynamics. Overall, this research reveals multifunctionality of PgDPP III and opens direction for future research of DPP III family proteins.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0188915PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5708649PMC
January 2018

Crystal structure of dipeptidyl peptidase III from the human gut symbiont Bacteroides thetaiotaomicron.

PLoS One 2017 2;12(11):e0187295. Epub 2017 Nov 2.

Division of Organic Chemistry and Biochemistry, Ruđer Bošković Institute, Zagreb, Croatia.

Bacteroides thetaiotaomicron is a dominant member of the human intestinal microbiome. The genome of this anaerobe encodes more than 100 proteolytic enzymes, the majority of which have not been characterized. In the present study, we have produced and purified recombinant dipeptidyl peptidase III (DPP III) from B. thetaiotaomicron for the purposes of biochemical and structural investigations. DPP III is a cytosolic zinc-metallopeptidase of the M49 family, involved in protein metabolism. The biochemical results for B. thetaiotaomicron DPP III from our research showed both some similarities to, as well as certain differences from, previously characterised yeast and human DPP III. The 3D-structure of B. thetaiotaomicron DPP III was determined by X-ray crystallography and revealed a two-domain protein. The ligand-free structure (refined to 2.4 Å) was in the open conformation, while in the presence of the hydroxamate inhibitor Tyr-Phe-NHOH, the closed form (refined to 3.3 Å) was observed. Compared to the closed form, the two domains of the open form are rotated away from each other by about 28 degrees. A comparison of the crystal structure of B. thetaiotaomicron DPP III with that of the human and yeast enzymes revealed a similar overall fold. However, a significant difference with functional implications was discovered in the upper domain, farther away from the catalytic centre. In addition, our data indicate that large protein flexibility might be conserved in the M49 family.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0187295PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5667867PMC
December 2017

Regioselective para-Carboxylation of Catechols with a Prenylated Flavin Dependent Decarboxylase.

Angew Chem Int Ed Engl 2017 10 2;56(44):13893-13897. Epub 2017 Oct 2.

Department of Chemistry, Organic & Bioorganic Chemistry, University of Graz, NAWI Graz, BioTechMed Graz, Heinrichstrasse 28/2, 8010, Graz, Austria.

The utilization of CO as a carbon source for organic synthesis meets the urgent demand for more sustainability in the production of chemicals. Herein, we report on the enzyme-catalyzed para-carboxylation of catechols, employing 3,4-dihydroxybenzoic acid decarboxylases (AroY) that belong to the UbiD enzyme family. Crystal structures and accompanying solution data confirmed that AroY utilizes the recently discovered prenylated FMN (prFMN) cofactor, and requires oxidative maturation to form the catalytically competent prFMN species. This study reports on the in vitro reconstitution and activation of a prFMN-dependent enzyme that is capable of directly carboxylating aromatic catechol substrates under ambient conditions. A reaction mechanism for the reversible decarboxylation involving an intermediate with a single covalent bond between a quinoid adduct and cofactor is proposed, which is distinct from the mechanism of prFMN-associated 1,3-dipolar cycloadditions in related enzymes.
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http://dx.doi.org/10.1002/anie.201708091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5656893PMC
October 2017

Genetic basis for response to cancer immunotherapy.

Authors:
Karl Gruber

Lancet Oncol 2017 09 17;18(9):e521. Epub 2017 Aug 17.

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http://dx.doi.org/10.1016/S1470-2045(17)30627-7DOI Listing
September 2017
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