Gruber K

References (39)

Title : Identification and validation of fusidic acid and flufenamic acid as inhibitors of SARS-CoV-2 replication using DrugSolver CavitomiX - Hetmann_2023_Sci.Rep_13_11783
Author(s) : Hetmann M , Langner C , Durmaz V , Cespugli M , Kochl K , Krassnigg A , Blaschitz K , Groiss S , Loibner M , Ruau D , Zatloukal K , Gruber K , Steinkellner G , Gruber CC
Ref : Sci Rep , 13 :11783 , 2023
Abstract : 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 chose enzymes essential for SARS-CoV-2 replication as a test system. The active-site cavities of the viral enzymes main protease (M(pro)) and papain-like protease (Pl(pro)), as well as of the human transmembrane serine protease 2 (TMPRSS2), were selected as target cavities. Using active-site point-cloud comparisons, it was possible to identify two compounds-flufenamic acid and fusidic acid-which show strong inhibition of viral replication. The complexes from which fusidic acid and flufenamic acid were derived would not have been identified using classical sequence- and structure-based methods as they show very little structural (TM-score: 0.1 and 0.09, respectively) and very low sequence (~5%) identity to M(pro) and TMPRSS2, respectively. Furthermore, a cavity-based off-target screening was performed using acetylcholinesterase (AChE) as an example. Using cavity comparisons, the human carboxylesterase was successfully identified, which is a described off-target for AChE inhibitors.
ESTHER : Hetmann_2023_Sci.Rep_13_11783
PubMedSearch : Hetmann_2023_Sci.Rep_13_11783
PubMedID: 37479788

Title : Residue-Specific Incorporation of the Non-Canonical Amino Acid Norleucine Improves Lipase Activity on Synthetic Polyesters - Haernvall_2022_Front.Bioeng.Biotechnol_10_769830
Author(s) : Haernvall K , Fladischer P , Schoeffmann H , Zitzenbacher S , Pavkov-Keller T , Gruber K , Schick M , Yamamoto M , Kuenkel A , Ribitsch D , Guebitz GM , Wiltschi B
Ref : Front Bioeng Biotechnol , 10 :769830 , 2022
Abstract : 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 Thermoanaerobacter thermohydrosulfuricus lipase (TTL) was exchanged by the residue-specific incorporation method for the more hydrophobic non-canonical norleucine (Nle). Strutural modelling of TTL revealed that residues Met-114 and Met-142 are in close vicinity of the active site and their replacement by the norleucine could modulate the catalytic activity of the enzyme. Indeed, hydrolysis of the polyethylene terephthalate model substrate by the Nle variant resulted in significantly higher amounts of release products than the Met variant. A similar trend was observed for an ionic phthalic polyester containing a short alkyl diol (C5). Interestingly, a 50% increased activity was found for TTL [Nle] towards ionic phthalic polyesters containing different ether diols compared to the parent enzyme TTL [Met]. These findings clearly demonstrate the high potential of non-canonical amino acids for enzyme engineering.
ESTHER : Haernvall_2022_Front.Bioeng.Biotechnol_10_769830
PubMedSearch : Haernvall_2022_Front.Bioeng.Biotechnol_10_769830
PubMedID: 35155387
Gene_locus related to this paper: theet-q3ch51

Title : Small-Molecule Inhibitors Targeting Lipolysis in Human Adipocytes - Grabner_2022_J.Am.Chem.Soc__
Author(s) : Grabner GF , Guttenberger N , Mayer N , Migglautsch-Sulzer AK , Lembacher-Fadum C , Fawzy N , Bulfon D , Hofer P , Zullig T , Hartig L , Kulminskaya N , Chalhoub G , Schratter M , Radner FPW , Preiss-Landl K , Masser S , Lass A , Zechner R , Gruber K , Oberer M , Breinbauer R , Zimmermann R
Ref : Journal of the American Chemical Society , : , 2022
Abstract : 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 disorders including insulin resistance, steatohepatitis, and heart disease, classifying ATGL as a promising drug target. Here, we report on the development and biological characterization of the first small-molecule inhibitor of human ATGL. This inhibitor, designated NG-497, selectively inactivates human and nonhuman primate ATGL but not structurally and functionally related lipid hydrolases. We demonstrate that NG-497 abolishes lipolysis in human adipocytes in a dose-dependent and reversible manner. The combined analysis of mouse- and human-selective inhibitors, chimeric ATGL proteins, and homology models revealed detailed insights into enzyme-inhibitor interactions. NG-497 binds ATGL within a hydrophobic cavity near the active site. Therein, three amino acid residues determine inhibitor efficacy and species selectivity and thus provide the molecular scaffold for selective inhibition.
ESTHER : Grabner_2022_J.Am.Chem.Soc__
PubMedSearch : Grabner_2022_J.Am.Chem.Soc__
PubMedID: 35362954

Title : Small cause, large effect: Structural characterization of cutinases from Thermobifida cellulosilytica - Ribitsch_2017_Biotechnol.Bioeng_114_2481
Author(s) : Ribitsch D , Hromic A , Zitzenbacher S , Zartl B , Gamerith C , Pellis A , Jungbauer A , Lyskowski A , Steinkellner G , Gruber K , Tscheliessnig R , Herrero Acero E , Guebitz GM
Ref : Biotechnol Bioeng , 114 :2481 , 2017
Abstract : We have investigated the structures of two native cutinases from Thermobifida cellulosilytica, namely Thc_Cut1 and Thc_Cut2 as well as of two variants, Thc_Cut2_DM (Thc_Cut2_ Arg29Asn_Ala30Val) and Thc_Cut2_TM (Thc_Cut2_Arg19Ser_Arg29Asn_Ala30Val). The four enzymes showed different activities towards the aliphatic polyester poly(lactic acid) (PLLA). The crystal structures of the four enzymes were successfully solved and in combination with Small Angle X-Ray Scattering (SAXS) the structural features responsible for the selectivity difference were elucidated. Analysis of the crystal structures did not indicate significant conformational differences among the different cutinases. However, the distinctive SAXS scattering data collected from the enzymes in solution indicated a remarkable surface charge difference. The difference in the electrostatic and hydrophobic surface properties could explain potential alternative binding modes of the four cutinases on PLLA explaining their distinct activities. Biotechnol. Bioeng. 2017;114: 2481-2488. (c) 2017 Wiley Periodicals, Inc.
ESTHER : Ribitsch_2017_Biotechnol.Bioeng_114_2481
PubMedSearch : Ribitsch_2017_Biotechnol.Bioeng_114_2481
PubMedID: 28671263
Gene_locus related to this paper: thefu-q6a0i4 , thefu-q6a0i3

Title : PpEst is a novel PBAT degrading polyesterase identified by proteomic screening of Pseudomonas pseudoalcaligenes - Wallace_2017_Appl.Microbiol.Biotechnol_101_2291
Author(s) : Wallace PW , Haernvall K , Ribitsch D , Zitzenbacher S , Schittmayer M , Steinkellner G , Gruber K , Guebitz GM , Birner-Gruenberger R
Ref : Applied Microbiology & Biotechnology , 101 :2291 , 2017
Abstract : A novel esterase, PpEst, that hydrolyses the co-aromatic-aliphatic polyester poly(1,4-butylene adipate-co-terephthalate) (PBAT) was identified by proteomic screening of the Pseudomonas pseudoalcaligenes secretome. PpEst was induced by the presence of PBAT in the growth media and had predicted arylesterase (EC 3.1.1.2) activity. PpEst showed polyesterase activity on both whole and milled PBAT film releasing terephthalic acid and 4-(4-hydroxybutoxycarbonyl)benzoic acid while end product inhibition by 4-(4-hydroxybutoxycarbonyl)benzoic acid was observed. Modelling of an aromatic polyester mimicking oligomer into the PpEst active site indicated that the binding pocket could be big enough to accommodate large polymers. This is the first report of a PBAT degrading enzyme being identified by proteomic screening and shows that this approach can contribute to the discovery of new polymer hydrolysing enzymes. Moreover, these results indicate that arylesterases could be an interesting enzyme class for identifications of polyesterases.
ESTHER : Wallace_2017_Appl.Microbiol.Biotechnol_101_2291
PubMedSearch : Wallace_2017_Appl.Microbiol.Biotechnol_101_2291
PubMedID: 27872998

Title : Engineering of the zinc-binding domain of an esterase from Clostridium botulinum towards increased activity on polyesters - Biundo_2017_Catal.Sci.Technol_7_1440
Author(s) : Biundo A , Steinkellner G , Gruber K , Spreitzhofer T , Ribitsch D , Guebitz GM
Ref : Catal Sci Technol , 7 :1440 , 2017
Abstract : The carboxylesterase from Clostridium botulinum (Cbotu_EstA) has been shown to hydrolyze the surface of the polyester poly.butylene adipate-co-terephthalate) (PBAT) releasing the monomeric building blocks. Cbotu_EstA contains a zinc ion, tetrahedrally coordinated by two histidine and two aspartic acid residues, which is buried inside an extra domain typical for members of the I. 5 lipase family. To elucidate the role of this extra domain with regard to polyester hydrolysis, variants of the zinc-binding domain were constructed and expressed in E. coli BL21-Gold.DE3). These enzyme variants were characterized with respect to their specific activity, kinetic parameters and thermostability on soluble substrates as well as on PBAT. All the variants exhibited a similar affinity towards the small substrate para-nitrophenyl butyrate (pNPB), with KM values between 0.4 and 1.2 mM, while the catalytic efficiency decreased approximately 1000-fold for the zinc-binding variants and 5-fold for the zinc cavity variants. Moreover, all four variants of the zinccoordination site (D130L, H150F, H156F, and D302L) showed a loss of thermostability. However, H156F and D302L revealed a drastic loss of thermostability compared to D130L and H150F. Nevertheless, compared to Cbotu_EstA, variants carrying substitutions of amino acids in the zinc-binding domain were able to release up to 10 times more soluble products from the polymeric substrate PBAT. The thermostability at 50 degrees C was increased in the case of F154Y and W274H, carrying more hydrophilic residues. These data clearly demonstrate the importance of different regions of the zinc-binding domain for the hydrolysis of polyesters like PBAT.
ESTHER : Biundo_2017_Catal.Sci.Technol_7_1440
PubMedSearch : Biundo_2017_Catal.Sci.Technol_7_1440
PubMedID:
Gene_locus related to this paper: clobh-A51055

Title : Characterization of a poly(butylene adipate-co-terephthalate)-hydrolyzing lipase from Pelosinus fermentans - Biundo_2016_Appl.Microbiol.Biotechnol_100_1753
Author(s) : Biundo A , Hromic A , Pavkov-Keller T , Gruber K , Quartinello F , Haernvall K , Perz V , Arrell MS , Zinn M , Ribitsch D , Guebitz GM
Ref : Applied Microbiology & Biotechnology , 100 :1753 , 2016
Abstract : Certain alpha/beta hydrolases have the ability to hydrolyze synthetic polyesters. While their partial hydrolysis has a potential for surface functionalization, complete hydrolysis allows recycling of valuable building blocks. Although knowledge about biodegradation of these materials is important regarding their fate in the environment, it is currently limited to aerobic organisms. A lipase from the anaerobic groundwater organism Pelosinus fermentans DSM 17108 (PfL1) was cloned and expressed in Escherichia coli BL21-Gold(DE3) and purified from the cell extract. Biochemical characterization with small substrates showed thermoalkalophilic properties (T opt = 50 degrees C, pHopt = 7.5) and higher activity towards para-nitrophenyl octanoate (12.7 U mg-1) compared to longer and shorter chain lengths (C14 0.7 U mg-1 and C2 4.3 U mg-1, respectively). Crystallization and determination of the 3-D structure displayed the presence of a lid structure and a zinc ion surrounded by an extra domain. These properties classify the enzyme into the I.5 lipase family. PfL1 is able to hydrolyze poly(1,4-butylene adipate-co-terephthalate) (PBAT) polymeric substrates. The hydrolysis of PBAT showed the release of small building blocks as detected by liquid chromatography-mass spectrometry (LC-MS). Protein dynamics seem to be involved with lid opening for the hydrolysis of PBAT by PfL1.
ESTHER : Biundo_2016_Appl.Microbiol.Biotechnol_100_1753
PubMedSearch : Biundo_2016_Appl.Microbiol.Biotechnol_100_1753
PubMedID: 26490551
Gene_locus related to this paper: 9firm-a0a0a0ymq9

Title : An Esterase from Anaerobic Clostridium hathewayi Can Hydrolyze Aliphatic-Aromatic Polyesters - Perz_2016_Environ.Sci.Technol_50_2899
Author(s) : Perz V , Hromic A , Baumschlager A , Steinkellner G , Pavkov-Keller T , Gruber K , Bleymaier K , Zitzenbacher S , Zankel A , Mayrhofer C , Sinkel C , Kueper U , Schlegel K , Ribitsch D , Guebitz GM
Ref : Environ Sci Technol , 50 :2899 , 2016
Abstract : Recently, a variety of biodegradable polymers have been developed as alternatives to recalcitrant materials. Although many studies on polyester biodegradability have focused on aerobic environments, there is much less known on biodegradation of polyesters in natural and artificial anaerobic habitats. Consequently, the potential of anaerobic biogas sludge to hydrolyze the synthetic compostable polyester PBAT (poly(butylene adipate-co-butylene terephthalate) was evaluated in this study. On the basis of reverse-phase high-performance liquid chromatography (RP-HPLC) analysis, accumulation of terephthalic acid (Ta) was observed in all anaerobic batches within the first 14 days. Thereafter, a decline of Ta was observed, which occurred presumably due to consumption by the microbial population. The esterase Chath_Est1 from the anaerobic risk 1 strain Clostridium hathewayi DSM-13479 was found to hydrolyze PBAT. Detailed characterization of this esterase including elucidation of the crystal structure was performed. The crystal structure indicates that Chath_Est1 belongs to the alpha/beta-hydrolases family. This study gives a clear hint that also micro-organisms in anaerobic habitats can degrade manmade PBAT.
ESTHER : Perz_2016_Environ.Sci.Technol_50_2899
PubMedSearch : Perz_2016_Environ.Sci.Technol_50_2899
PubMedID: 26878094
Gene_locus related to this paper: 9clot-r5t6k9

Title : Hydrolysis of synthetic polyesters by Clostridium botulinum esterases - Perz_2016_Biotechnol.Bioeng_113_1024
Author(s) : Perz V , Baumschlager A , Bleymaier K , Zitzenbacher S , Hromic A , Steinkellner G , Pairitsch A , Lyskowski A , Gruber K , Sinkel C , Kuper U , Ribitsch D , Guebitz GM
Ref : Biotechnol Bioeng , 113 :1024 , 2016
Abstract : Two novel esterases from the anaerobe Clostridium botulinum ATCC 3502 (Cbotu_EstA and Cbotu_EstB) were expressed in Escherichia coli BL21-Gold(DE3) and were found to hydrolyze the polyester poly(butylene adipate-co-butylene terephthalate) (PBAT). The active site residues (triad Ser, Asp, His) are present in both enzymes at the same location only with some amino acid variations near the active site at the surrounding of aspartate. Yet, Cbotu_EstA showed higher kcat values on para-nitrophenyl butyrate and para-nitrophenyl acetate and was considerably more active (sixfold) on PBAT. The entrance to the active site of the modeled Cbotu_EstB appears more narrowed compared to the crystal structure of Cbotu_EstA and the N-terminus is shorter which could explain its lower activity on PBAT. The Cbotu_EstA crystal structure consists of two regions that may act as movable cap domains and a zinc metal binding site. Biotechnol. Bioeng. 2016;113: 1024-1034. (c) 2015 Wiley Periodicals, Inc.
ESTHER : Perz_2016_Biotechnol.Bioeng_113_1024
PubMedSearch : Perz_2016_Biotechnol.Bioeng_113_1024
PubMedID: 26524601
Gene_locus related to this paper: clobh-A5I3I2 , clobh-A51055

Title : Crystal structure of the Saccharomyces cerevisiae monoglyceride lipase Yju3p - Aschauer_2016_Biochim.Biophys.Acta_1861_462
Author(s) : Aschauer P , Rengachari S , Lichtenegger J , Schittmayer M , Das KM , Mayer N , Breinbauer R , Birner-Gruenberger R , Gruber CC , Zimmermann R , Gruber K , Oberer M
Ref : Biochimica & Biophysica Acta , 1861 :462 , 2016
Abstract : Monoglyceride lipases (MGLs) are a group of alpha/beta-hydrolases that catalyze the hydrolysis of monoglycerides (MGs) into free fatty acids and glycerol. This reaction serves different physiological functions, namely in the last step of phospholipid and triglyceride degradation, in mammalian endocannabinoid and arachidonic acid metabolism, and in detoxification processes in microbes. Previous crystal structures of MGLs from humans and bacteria revealed conformational plasticity in the cap region of this protein and gave insight into substrate binding. In this study, we present the structure of a MGL from Saccharomyces cerevisiae called Yju3p in its free form and in complex with a covalently bound substrate analog mimicking the tetrahedral intermediate of MG hydrolysis. These structures reveal a high conservation of the overall shape of the MGL cap region and also provide evidence for conformational changes in the cap of Yju3p. The complex structure reveals that, despite the high structural similarity, Yju3p seems to have an additional opening to the substrate binding pocket at a different position compared to human and bacterial MGL. Substrate specificities towards MGs with saturated and unsaturated alkyl chains of different lengths were tested and revealed highest activity towards MG containing a C18:1 fatty acid.
ESTHER : Aschauer_2016_Biochim.Biophys.Acta_1861_462
PubMedSearch : Aschauer_2016_Biochim.Biophys.Acta_1861_462
PubMedID: 26869448
Gene_locus related to this paper: yeast-mgll

Title : Structure of human dipeptidyl peptidase 10 (DPPY): a modulator of neuronal Kv4 channels - Bezerra_2015_Sci.Rep_5_8769
Author(s) : Bezerra GA , Dobrovetsky E , Seitova A , Fedosyuk S , Dhe-Paganon S , Gruber K
Ref : Sci Rep , 5 :8769 , 2015
Abstract : The voltage-gated potassium channel family (Kv) constitutes the most diverse class of ion channels in the nervous system. Dipeptidyl peptidase 10 (DPP10) is an inactive peptidase that modulates the electrophysiological properties, cell-surface expression and subcellular localization of voltage-gated potassium channels. As a consequence, DPP10 malfunctioning is associated with neurodegenerative conditions like Alzheimer and fronto-temporal dementia, making this protein an attractive drug target. In this work, we report the crystal structure of DPP10 and compare it to that of DPP6 and DPP4. DPP10 belongs to the S9B serine protease subfamily and contains two domains with two distinct folds: a beta-propeller and a classical alpha/beta-hydrolase fold. The catalytic serine, however, is replaced by a glycine, rendering the protein enzymatically inactive. Difference in the entrance channels to the active sites between DPP10 and DPP4 provide an additional rationale for the lack of activity. We also characterize the DPP10 dimer interface focusing on the alternative approach for designing drugs able to target protein-protein interactions.
ESTHER : Bezerra_2015_Sci.Rep_5_8769
PubMedSearch : Bezerra_2015_Sci.Rep_5_8769
PubMedID: 25740212
Gene_locus related to this paper: human-DPP10

Title : Surface engineering of a cutinase from Thermobifida cellulosilytica for improved polyester hydrolysis - Herrero_2013_Biotechnol.Bioeng_110_2581
Author(s) : Herrero Acero E , Ribitsch D , Dellacher A , Zitzenbacher S , Marold A , Steinkellner G , Gruber K , Schwab H , Guebitz GM
Ref : Biotechnol Bioeng , 110 :2581 , 2013
Abstract : Modeling and comparison of the structures of the two closely related cutinases Thc_Cut1 and Thc_Cut2 from Thermobifida cellulosilytica DSM44535 revealed that dissimilarities in their electrostatic and hydrophobic surface properties in the vicinity to the active site could be responsible for pronounced differences in hydrolysis efficiencies of polyester (i.e., PET, polyethyleneterephthalate). To investigate this hypothesis in more detail, selected amino acids of surface regions outside the active site of Thc_Cut2, which hydrolyzes PET much less efficiently than Thc_Cut1 were exchanged by site-directed mutagenesis. The mutants were expressed in E. coli BL21-Gold(DE3), purified and characterized regarding their specific activities and kinetic parameters on soluble substrates and their ability to hydrolyze PET and the PET model substrate bis(benzoyloxyethyl) terephthalate (3PET). Compared to Thc_Cut2, mutants carrying Arg29Asn and/or Ala30Val exchanges showed considerable higher specific activity and higher kcat /KM values on soluble substrates. Exchange of the positively charged arginine (Arg19 and Arg29) located on the enzyme surface to the non-charged amino acids serine and asparagine strongly increased the hydrolysis activity for 3PET and PET. In contrast, exchange of the uncharged glutamine (Glu65) by the negatively charged glutamic acid lead to a complete loss of hydrolysis activity on PET films. These findings clearly demonstrate that surface properties (i.e., amino acids located outside the active site on the protein surface) play an important role in PET hydrolysis. Biotechnol. Bioeng. 2013;110: 2581-2590. (c) 2013 Wiley Periodicals, Inc.
ESTHER : Herrero_2013_Biotechnol.Bioeng_110_2581
PubMedSearch : Herrero_2013_Biotechnol.Bioeng_110_2581
PubMedID: 23592055

Title : Fusion of binding domains to Thermobifida cellulosilytica cutinase to tune sorption characteristics and enhancing PET hydrolysis - Ribitsch_2013_Biomacromolecules_14_1769
Author(s) : Ribitsch D , Yebra AO , Zitzenbacher S , Wu J , Nowitsch S , Steinkellner G , Greimel K , Doliska A , Oberdorfer G , Gruber CC , Gruber K , Schwab H , Stana-Kleinschek K , Herrero Acero E , Guebitz GM
Ref : Biomacromolecules , 14 :1769 , 2013
Abstract : A cutinase from Thermomyces cellullosylitica (Thc_Cut1), hydrolyzing the synthetic polymer polyethylene terephthalate (PET), was fused with two different binding modules to improve sorption and thereby hydrolysis. The binding modules were from cellobiohydrolase I from Hypocrea jecorina (CBM) and from a polyhydroxyalkanoate depolymerase from Alcaligenes faecalis (PBM). Although both binding modules have a hydrophobic nature, it was possible to express the proteins in E. coli . Both fusion enzymes and the native one had comparable kcat values in the range of 311 to 342 s(-1) on pNP-butyrate, while the catalytic efficiencies kcat/Km decreased from 0.41 s(-1)/ muM (native enzyme) to 0.21 and 0.33 s(-1)/muM for Thc_Cut1+PBM and Thc_Cut1+CBM, respectively. The fusion enzymes were active both on the insoluble PET model substrate bis(benzoyloxyethyl) terephthalate (3PET) and on PET although the hydrolysis pattern was differed when compared to Thc_Cut1. Enhanced adsorption of the fusion enzymes was visible by chemiluminescence after incubation with a 6xHisTag specific horseradish peroxidase (HRP) labeled probe. Increased adsorption to PET by the fusion enzymes was confirmed with Quarz Crystal Microbalance (QCM-D) analysis and indeed resulted in enhanced hydrolysis activity (3.8x for Thc_Cut1+CBM) on PET, as quantified, based on released mono/oligomers.
ESTHER : Ribitsch_2013_Biomacromolecules_14_1769
PubMedSearch : Ribitsch_2013_Biomacromolecules_14_1769
PubMedID: 23718548
Gene_locus related to this paper: thefu-q6a0i4

Title : Conformational plasticity and ligand binding of bacterial monoacylglycerol lipase - Rengachari_2013_J.Biol.Chem_288_31093
Author(s) : Rengachari S , Aschauer P , Schittmayer M , Mayer N , Gruber K , Breinbauer R , Birner-Gruenberger R , Dreveny I , Oberer M
Ref : Journal of Biological Chemistry , 288 :31093 , 2013
Abstract : Monoacylglycerol lipases (MGLs) play an important role in lipid catabolism across all kingdoms of life by catalyzing the release of free fatty acids from monoacylglycerols. The three-dimensional structures of human and a bacterial MGL were determined only recently as the first members of this lipase family. In addition to the alpha/beta-hydrolase core, they showed unexpected structural similarities even in the cap region. Nevertheless, the structural basis for substrate binding and conformational changes of MGLs is poorly understood. Here, we present a comprehensive study of five crystal structures of MGL from Bacillus sp. H257 in its free form and in complex with different substrate analogs and the natural substrate 1-lauroylglycerol. The occurrence of different conformations reveals a high degree of conformational plasticity of the cap region. We identify a specific residue, Ile-145, that might act as a gatekeeper restricting access to the binding site. Site-directed mutagenesis of Ile-145 leads to significantly reduced hydrolase activity. Bacterial MGLs in complex with 1-lauroylglycerol, myristoyl, palmitoyl, and stearoyl substrate analogs enable identification of the binding sites for the alkyl chain and the glycerol moiety of the natural ligand. They also provide snapshots of the hydrolytic reaction of a bacterial MGL at different stages. The alkyl chains are buried in a hydrophobic tunnel in an extended conformation. Binding of the glycerol moiety is mediated via Glu-156 and water molecules. Analysis of the structural features responsible for cap plasticity and the binding modes of the ligands suggests conservation of these features also in human MGL.
ESTHER : Rengachari_2013_J.Biol.Chem_288_31093
PubMedSearch : Rengachari_2013_J.Biol.Chem_288_31093
PubMedID: 24014019
Gene_locus related to this paper: bac25-mglp

Title : Hydroxynitrile lyases with alpha\/beta-hydrolase fold: two enzymes with almost identical 3D structures but opposite enantioselectivities and different reaction mechanisms - Andexer_2012_Chembiochem_13_1932
Author(s) : Andexer JN , Staunig N , Eggert T , Kratky C , Pohl M , Gruber K
Ref : Chembiochem , 13 :1932 , 2012
Abstract : Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrins to yield hydrocyanic acid (HCN) and the respective carbonyl compound and are key enzymes in the process of cyanogenesis in plants. In organic syntheses, HNLs are used as biocatalysts for the formation of enantiopure cyanohydrins. We determined the structure of the recently identified, R-selective HNL from Arabidopsis thaliana (AtHNL) at a crystallographic resolution of 2.5 A. The structure exhibits an alpha/beta-hydrolase fold, very similar to the homologous, but S-selective, HNL from Hevea brasiliensis (HbHNL). The similarities also extend to the active sites of these enzymes, with a Ser-His-Asp catalytic triad present in all three cases. In order to elucidate the mode of substrate binding and to understand the unexpected opposite enantioselectivity of AtHNL, complexes of the enzyme with both (R)- and (S)-mandelonitrile were modeled using molecular docking simulations. Compared to the complex of HbHNL with (S)-mandelonitrile, the calculations produced an approximate mirror image binding mode of the substrate with the phenyl rings located at very similar positions, but with the cyano groups pointing in opposite directions. A catalytic mechanism for AtHNL is proposed, in which His236 from the catalytic triad acts as a general base and the emerging negative charge on the cyano group is stabilized by main-chain amide groups and an alpha-helix dipole very similar to alpha/beta-hydrolases. This mechanistic proposal is additionally supported by mutagenesis studies.
ESTHER : Andexer_2012_Chembiochem_13_1932
PubMedSearch : Andexer_2012_Chembiochem_13_1932
PubMedID: 22851196
Gene_locus related to this paper: arath-HNL

Title : Tailoring a stabilized variant of hydroxynitrile lyase from Arabidopsis thaliana - Okrob_2012_Chembiochem_13_797
Author(s) : Okrob D , Metzner J , Wiechert W , Gruber K , Pohl M
Ref : Chembiochem , 13 :797 , 2012
Abstract : The R-selective hydroxynitrile lyase from Arabidopsis thaliana (AtHNL) cannot be applied for stereoselective cyanohydrin syntheses in aqueous media because of its limited stability at pH<5, which is required in order to suppress the uncatalyzed racemic cyanohydrin formation. To stabilize AtHNL we designed a surface-modified variant incorporating 11 changes in the amino acids on the protein surface. Comparative characterization of the variant and the wild-type enzyme showed a broadened pH optimum towards the acidic range, along with enhancement of activity by up to twofold and significantly increased pH- and thermostabilities. The effect can most probably be explained by a shift of the isoelectic point from pH 5.1 to 4.8. Application of the variant for the synthesis of (R)-cyanohydrins in an aqueous/organic two-phase system at pH 4.5 demonstrated the high stereoselectivity and robustness of the variant relative to the wild-type enzyme, which is immediately inactivated under these conditions.
ESTHER : Okrob_2012_Chembiochem_13_797
PubMedSearch : Okrob_2012_Chembiochem_13_797
PubMedID: 22378361
Gene_locus related to this paper: arath-HNL

Title : The structure of monoacylglycerol lipase from Bacillus sp. H257 reveals unexpected conservation of the cap architecture between bacterial and human enzymes - Rengachari_2012_Biochim.Biophys.Acta_1821_1012
Author(s) : Rengachari S , Bezerra GA , Riegler-Berket L , Gruber CC , Sturm C , Taschler U , Boeszoermenyi A , Dreveny I , Zimmermann R , Gruber K , Oberer M
Ref : Biochimica & Biophysica Acta , 1821 :1012 , 2012
Abstract : Monoacylglycerol lipases (MGLs) catalyse the hydrolysis of monoacylglycerol into free fatty acid and glycerol. MGLs have been identified throughout all genera of life and have adopted different substrate specificities depending on their physiological role. In humans, MGL plays an integral part in lipid metabolism affecting energy homeostasis, signalling processes and cancer cell progression. In bacteria, MGLs degrade short-chain monoacylglycerols which are otherwise toxic to the organism. We report the crystal structures of MGL from the bacterium Bacillus sp. H257 (bMGL) in its free form at 1.2 and in complex with phenylmethylsulfonyl fluoride at 1.8 resolution. In both structures, bMGL adopts an alpha/beta hydrolase fold with a cap in an open conformation. Access to the active site residues, which were unambiguously identified from the protein structure, is facilitated by two different channels. The larger channel constitutes the highly hydrophobic substrate binding pocket with enough room to accommodate monoacylglycerol. The other channel is rather small and resembles the proposed glycerol exit hole in human MGL. Molecular dynamics simulation of bMGL yielded open and closed states of the entrance channel and the glycerol exit hole. Despite differences in the number of residues, secondary structure elements, and low sequence identity in the cap region, this first structure of a bacterial MGL reveals striking structural conservation of the overall cap architecture in comparison with human MGL. Thus it provides insight into the structural conservation of the cap amongst MGLs throughout evolution and provides a framework for rationalising substrate specificities in each organism.
ESTHER : Rengachari_2012_Biochim.Biophys.Acta_1821_1012
PubMedSearch : Rengachari_2012_Biochim.Biophys.Acta_1821_1012
PubMedID: 22561231
Gene_locus related to this paper: bac25-mglp

Title : Extracellular serine proteases from Stenotrophomonas maltophilia: Screening, isolation and heterologous expression in E. coli - Ribitsch_2012_J.Biotechnol_157_140
Author(s) : Ribitsch D , Heumann S , Karl W , Gerlach J , Leber R , Birner-Gruenberger R , Gruber K , Eiteljoerg I , Remler P , Siegert P , Lange J , Maurer KH , Berg G , Guebitz GM , Schwab H
Ref : J Biotechnol , 157 :140 , 2012
Abstract : A large strain collection comprising antagonistic bacteria was screened for novel detergent proteases. Several strains displayed protease activity on agar plates containing skim milk but were inactive in liquid media. Encapsulation of cells in alginate beads induced protease production. Stenotrophomonas maltophilia emerged as best performer under washing conditions. For identification of wash-active proteases, four extracellular serine proteases called StmPr1, StmPr2, StmPr3 and StmPr4 were cloned. StmPr2 and StmPr4 were sufficiently overexpressed in E. coli. Expression of StmPr1 and StmPr3 resulted in unprocessed, insoluble protein. Truncation of most of the C-terminal domain which has been identified by enzyme modeling succeeded in expression of soluble, active StmPr1 but failed in case of StmPr3. From laundry application tests StmPr2 turned out to be a highly wash-active protease at 45 degrees C. Specific activity of StmPr2 determined with suc-L-Ala-L-Ala-L-Pro-l-Phe-p-nitroanilide as the substrate was 17+/-2U/mg. In addition we determined the kinetic parameters and cleavage preferences of protease StmPr2.
ESTHER : Ribitsch_2012_J.Biotechnol_157_140
PubMedSearch : Ribitsch_2012_J.Biotechnol_157_140
PubMedID: 21983234

Title : Crystallization and preliminary X-ray diffraction analysis of human dipeptidyl peptidase 10 (DPPY), a component of voltage-gated potassium channels - Bezerra_2012_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_68_214
Author(s) : Bezerra GA , Dobrovetsky E , Seitova A , Dhe-Paganon S , Gruber K
Ref : Acta Crystallographica Sect F Struct Biol Cryst Commun , 68 :214 , 2012
Abstract : Dipeptidyl peptidase 10 (DPP10, DPPY) is an inactive peptidase associated with voltage-gated potassium channels, acting as a modulator of their electrophysiological properties, cell-surface expression and subcellular localization. Because potassium channels are important disease targets, biochemical and structural characterization of their interaction partners was sought. DPP10 was cloned and expressed using an insect-cell system and the protein was purified via His-tag affinity and size-exclusion chromatography. Crystals obtained by the sitting-drop method were orthorhombic, belonging to space group P2(1)2(1)2(1) with unit-cell parameters a = 80.91, b = 143.73, c = 176.25 A. A single solution with two molecules in the asymmetric unit was found using the structure of DPP6 (also called DPPX; PDB entry 1xfd) as the search model in a molecular replacement protocol.
ESTHER : Bezerra_2012_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_68_214
PubMedSearch : Bezerra_2012_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_68_214
PubMedID: 22298003
Gene_locus related to this paper: human-DPP10

Title : A New Esterase from Thermobifida halotolerans Hydrolyses Polyethylene Terephthalate (PET) and Polylactic Acid (PLA) - Ribitsch_2012_Polymers_4_617
Author(s) : Ribitsch D , Herrero Acero E , Greimel K , Dellacher A , Zitzenbacher S , Marold A , Rodriguez RD , Steinkellner G , Gruber K , Schwab H , Guebitz GM
Ref : Polymers , 4 :617 , 2012
Abstract : A new esterase from Thermobifida halotolerans (Thh_Est) was cloned and expressed in E. coli and investigated for surface hydrolysis of polylactic acid (PLA) and polyethylene terephthalate (PET). Thh_Est is a member of the serine hydrolases superfamily containing the -GxSxG- motif with 8587% homology to an esterase from T. alba, to an acetylxylan esterase from T. fusca and to various Thermobifida cutinases. Thh_Est hydrolyzed the PET model substrate bis(benzoyloxyethyl)terephthalate and PET releasing terephthalic acid and mono-(2-hydroxyethyl) terephthalate in comparable amounts (19.8 and 21.5 mmol/mol of enzyme) while no higher oligomers like bis-(2-hydroxyethyl) terephthalate were detected. Similarly, PLA was hydrolyzed as indicated by the release of lactic acid. Enzymatic surface hydrolysis of PET and PLA led to a strong hydrophilicity increase, as quantified with a WCA decrease from 90.8 and 75.5 to 50.4 and to a complete spread of the water drop on the surface, respectively
ESTHER : Ribitsch_2012_Polymers_4_617
PubMedSearch : Ribitsch_2012_Polymers_4_617
PubMedID:
Gene_locus related to this paper: 9actn-h6wx58

Title : Structures of human DPP7 reveal the molecular basis of specific inhibition and the architectural diversity of proline-specific peptidases - Bezerra_2012_PLoS.One_7_e43019
Author(s) : Bezerra GA , Dobrovetsky E , Dong A , Seitova A , Crombett L , Shewchuk LM , Hassell AM , Sweitzer SM , Sweitzer TD , McDevitt PJ , Johanson KO , Kennedy-Wilson KM , Cossar D , Bochkarev A , Gruber K , Dhe-Paganon S
Ref : PLoS ONE , 7 :e43019 , 2012
Abstract : Proline-specific dipeptidyl peptidases (DPPs) are emerging targets for drug development. DPP4 inhibitors are approved in many countries, and other dipeptidyl peptidases are often referred to as DPP4 activity- and/or structure-homologues (DASH). Members of the DASH family have overlapping substrate specificities, and, even though they share low sequence identity, therapeutic or clinical cross-reactivity is a concern. Here, we report the structure of human DPP7 and its complex with a selective inhibitor Dab-Pip (L-2,4-diaminobutyryl-piperidinamide) and compare it with that of DPP4. Both enzymes share a common catalytic domain (alpha/beta-hydrolase). The catalytic pocket is located in the interior of DPP7, deep inside the cleft between the two domains. Substrates might access the active site via a narrow tunnel. The DPP7 catalytic triad is completely conserved and comprises Ser162, Asp418 and His443 (corresponding to Ser630, Asp708 and His740 in DPP4), while other residues lining the catalytic pockets differ considerably. The "specificity domains" are structurally also completely different exhibiting a beta-propeller fold in DPP4 compared to a rare, completely helical fold in DPP7. Comparing the structures of DPP7 and DPP4 allows the design of specific inhibitors and thus the development of less cross-reactive drugs. Furthermore, the reported DPP7 structures shed some light onto the evolutionary relationship of prolyl-specific peptidases through the analysis of the architectural organization of their domains.
ESTHER : Bezerra_2012_PLoS.One_7_e43019
PubMedSearch : Bezerra_2012_PLoS.One_7_e43019
PubMedID: 22952628
Gene_locus related to this paper: human-DPP7

Title : Enzymatic Surface Hydrolysis of PET: Effect of Structural Diversity on Kinetic Properties of Cutinases from Thermobifida. - Herrero-Acero_2011_Macromolecules_44_4632
Author(s) : Herrero Acero E , Ribitsch D , Steinkellner G , Gruber K , Greimel K , Eiteljoerg I , Trotscha E , Wei R , Zimmermann W , Zinn M , Cavaco-Paulo A , Freddi G , Schwab H , Guebitz G
Ref : Macromolecules , 44 :4632 , 2011
Abstract : In this study cutinases from Thermobifida cellulosilytica DSM44535 (Thc_Cut1 and Thc_Cut2) and Thermobifida fusca DSM44342 (Thf42_Cut1) hydrolyzing poly(ethylene terephthalate) (PET) were successfully cloned and expressed in E.coli BL21-Gold(DE3). Their ability to hydrolyze PET was compared with other enzymes hydrolyzing natural polyesters, including the PHA depolymerase (ePhaZmcl) from Pseudomonas fluorescens and two cutinases from T. fusca KW3. The three isolated Thermobifida cutinases are very similar (only a maximum of 18 amino acid differences) but yet had different kinetic parameters on soluble substrates. Their kcat and Km values on pNP-acetate were in the ranges 2.4-211.9 s-1 and 127-200 micoM while on pNP-butyrate they showed kcat and Km values between 5.3 and 195.1 s-1 and between 1483 and 2133 microM. Thc_Cut1 released highest amounts of MHET and terephthalic acid from PET and bis(benzoyloxyethyl) terephthalate (3PET) with the highest concomitant increase in PET hydrophilicity as indicated by water contact angle (WCA) decreases. FTIR-ATR analysis revealed an increase in the crystallinity index A1340/A1410 upon enzyme treatment and an increase of the amount of carboxylic and hydroxylic was measured using derivatization with 2-(bromomethyl)naphthalene. Modeling the covalently bound tetrahedral intermediate consisting of cutinase and 3PET indicated that the active site His-209 is in the proximity of the O of the substrate thus allowing hydrolysis. On the other hand, the models indicated that regions of Thc_Cut1 and Thc_Cut2 which differed in electrostatic and in hydrophobic surface properties were able to reach/interact with PET which may explain their different hydrolysis efficiencies.
ESTHER : Herrero-Acero_2011_Macromolecules_44_4632
PubMedSearch : Herrero-Acero_2011_Macromolecules_44_4632
PubMedID:
Gene_locus related to this paper: bacsu-pnbae , thefu-q6a0i4 , thefu-q6a0i3

Title : Uneven twins: comparison of two enantiocomplementary hydroxynitrile lyases with alpha\/beta-hydrolase fold - Guterl_2009_J.Biotechnol_141_166
Author(s) : Guterl JK , Andexer JN , Sehl T , von Langermann J , Frindi-Wosch I , Rosenkranz T , Fitter J , Gruber K , Kragl U , Eggert T , Pohl M
Ref : J Biotechnol , 141 :166 , 2009
Abstract : Hydroxynitrile lyases (HNLs) are applied in technical processes for the synthesis of chiral cyanohydrins. Here we describe the thorough characterization of the recently discovered R-hydroxynitrile lyase from Arabidopsis thaliana and its S-selective counterpart from Manihot esculenta (MeHNL) concerning their properties relevant for technical applications. The results are compared to available data of the structurally related S-HNL from Hevea brasiliensis (HbHNL), which is frequently applied in technical processes. Whereas substrate ranges are highly similar for all three enzymes, the stability of MeHNL with respect to higher temperature and low pH-values is superior to the other HNLs with alpha/beta-hydrolase fold. This enhanced stability is supposed to be due to the ability of MeHNL to form tetramers in solution, while HbHNL and AtHNL are dimers. The different inactivation pathways, deduced by means of circular dichroism, tryptophan fluorescence and static light scattering further support these results. Our data suggest different possibilities to stabilize MeHNL and AtHNL for technical applications: whereas the application of crude cell extracts is appropriate for MeHNL, AtHNL is stabilized by addition of polyols. In addition, the molecular reason for the inhibition of MeHNL and HbHNL by acetate could be elucidated, whereas no such inhibition was observed with AtHNL.
ESTHER : Guterl_2009_J.Biotechnol_141_166
PubMedSearch : Guterl_2009_J.Biotechnol_141_166
PubMedID: 19433222
Gene_locus related to this paper: arath-HNL , hevbr-hnl , manes-hnl

Title : Epoxide-hydrolase-initiated hydrolysis\/rearrangement cascade of a methylene-interrupted bis-epoxide yields chiral THF moieties without involvement of a cyclase - Ueberbacher_2009_Chembiochem_10_1697
Author(s) : Ueberbacher BT , Oberdorfer G , Gruber K , Faber K
Ref : Chembiochem , 10 :1697 , 2009
Abstract : In contrast with electrophilic enzyme-catalysed cyclisations in terpenoid biosynthesis, cyclisations of tetrahydrofuran moieties found in several groups of natural products, such as annonaceous acetogenins, neurofurans and phytooxylipins, appear to proceed through a nucleophilic cascade mechanism starting from bis-epoxy fatty acid precursors. This hypothesis was verified by epoxide-hydrolase-catalysed hydrolytic ring-opening/cyclisation cascades starting from a methylene-interrupted meso-bis-epoxide model substrate, which furnished the corresponding THF products with excellent de and ee values. Molecular modelling showed that the points of enzyme attack were consistent with the stereospecificities of the enzymes, whereas the stereochemical courses of the cyclisation were solely governed by Baldwin's rules and did not invoke the involvements of a "cyclase".
ESTHER : Ueberbacher_2009_Chembiochem_10_1697
PubMedSearch : Ueberbacher_2009_Chembiochem_10_1697
PubMedID: 19496106

Title : Substrate binding in the FAD-dependent hydroxynitrile lyase from almond provides insight into the mechanism of cyanohydrin formation and explains the absence of dehydrogenation activity - Dreveny_2009_Biochemistry_48_3370
Author(s) : Dreveny I , Andryushkova AS , Glieder A , Gruber K , Kratky C
Ref : Biochemistry , 48 :3370 , 2009
Abstract : In a large number of plant species hydroxynitrile lyases catalyze the decomposition of cyanohydrins in order to generate hydrogen cyanide upon tissue damage. Hydrogen cyanide serves as a deterrent against herbivores and fungi. In vitro hydroxynitrile lyases are proficient biocatalysts for the stereospecific synthesis of cyanohydrins. Curiously, hydroxynitrile lyases from different species are completely unrelated in structure and substrate specificity despite catalyzing the same reaction. The hydroxynitrile lyase from almond shows close resemblance to flavoproteins of the glucose-methanol-choline oxidoreductase family. We report here 3D structural data of this lyase with the reaction product benzaldehyde bound within the active site, which allow unambiguous assignment of the location of substrate binding. Based on the binding geometry, a reaction mechanism is proposed that involves one of the two conserved active site histidine residues acting as a general base abstracting the proton from the cyanohydrin hydroxyl group. Site-directed mutagenesis shows that both active site histidines are required for the reaction to occur. There is no evidence that the flavin cofactor directly participates in the reaction. Comparison with other hydroxynitrile lyases reveals a large diversity of active site architectures, which, however, share the common features of a general active site base and a nearby patch with positive electrostatic potential. On the basis of the difference in substrate binding geometry between the FAD-dependent HNL from almond and the related oxidases, we can rationalize why the HNL does not act as an oxidase.
ESTHER : Dreveny_2009_Biochemistry_48_3370
PubMedSearch : Dreveny_2009_Biochemistry_48_3370
PubMedID: 19256550

Title : Atomic resolution crystal structures and quantum chemistry meet to reveal subtleties of hydroxynitrile lyase catalysis - Schmidt_2008_J.Biol.Chem_283_21827
Author(s) : Schmidt A , Gruber K , Kratky C , Lamzin VS
Ref : Journal of Biological Chemistry , 283 :21827 , 2008
Abstract : Hydroxynitrile lyases are versatile enzymes that enantiospecifically cope with cyanohydrins, important intermediates in the production of various agrochemicals or pharmaceuticals. We determined four atomic resolution crystal structures of hydroxynitrile lyase from Hevea brasiliensis: one native and three complexes with acetone, isopropyl alcohol, and thiocyanate. We observed distinct distance changes among the active site residues related to proton shifts upon substrate binding. The combined use of crystallography and ab initio quantum chemical calculations allowed the determination of the protonation states in the enzyme active site. We show that His(235) of the catalytic triad must be protonated in order for catalysis to proceed, and we could reproduce the cyanohydrin synthesis in ab initio calculations. We also found evidence for the considerable pK(a) shifts that had been hypothesized earlier. We envision that this knowledge can be used to enhance the catalytic properties and the stability of the enzyme for industrial production of enantiomerically pure cyanohydrins.
ESTHER : Schmidt_2008_J.Biol.Chem_283_21827
PubMedSearch : Schmidt_2008_J.Biol.Chem_283_21827
PubMedID: 18524775
Gene_locus related to this paper: hevbr-hnl

Title : Inverting enantioselectivity of Burkholderia gladioli esterase EstB by directed and designed evolution - Ivancic_2007_J.Biotechnol_129_109
Author(s) : Ivancic M , Valinger G , Gruber K , Schwab H
Ref : J Biotechnol , 129 :109 , 2007
Abstract : Esterase EstB from Burkholderia gladioli, showing moderate S-enantioselectivity (E(S)=6.1) in the hydrolytic kinetic resolution of methyl-beta-hydroxyisobutyrate, was subjected to directed evolution in order to reverse its enantioselectivity. After one round of ep-PCR, saturation mutagenesis and high-throughput screening, it was found that different mutations at position 152 (in the vicinity of the active site) increase, decrease and even reverse the natural enantioselectivity of this enzyme. The newly created R-enantioselectivity of the esterase mutein (E(Rapp)=1.5) has been further enhanced by a designed evolution strategy involving random mutations close to the active site. Based on the three-dimensional structure nineteen amino acid residues have been selected as mutation sites for saturation mutagenesis. Mutations at three sites (135, 253 and 351) were found to increase R-enantioselectivity. Successive rounds of saturation mutagenesis at these "hot spots" resulted in an increase in R-enantioselectivity from E(Rapp)=1.5 for the parent mutant to E(Rapp)=28.9 for the best variant which carried four amino acid substitutions. Our results prove designed evolution followed by high-throughput screening to be an efficient strategy for engineering enzyme enantioselectivity.
ESTHER : Ivancic_2007_J.Biotechnol_129_109
PubMedSearch : Ivancic_2007_J.Biotechnol_129_109
PubMedID: 17147964

Title : Structural determinants of the enantioselectivity of the hydroxynitrile lyase from Hevea brasiliensis - Gartler_2007_J.Biotechnol_129_87
Author(s) : Gartler G , Kratky C , Gruber K
Ref : J Biotechnol , 129 :87 , 2007
Abstract : The hydroxynitrile lyase from the tropical rubber tree Hevea brasiliensis (HbHNL) is utilized as a biocatalyst in stereospecific syntheses of alpha-hydroxynitriles from aldehydes and methyl-ketones. The catalyzed reaction represents one of the few industrially relevant examples of enzyme mediated C-C coupling reactions. In this work, we determined the X-ray crystal structures (at 1.54 and 1.76 Angstroms resolution) of HbHNL complexes with two chiral substrates -- mandelonitrile and 2,3-dimethyl-2-hydroxy-butyronitrile -- by soaking and rapid freeze quenching techniques. This is the first structural observation of the complex between a HNL and chiral substrates. Consistent with the known selectivity of the enzyme, only the S-enantiomers of the two substrates were observed in the active site. The binding modes of the chiral substrates were identical to that observed for the biological substrate acetone cyanohydrin. This indicates that the transformation of these non-natural substrates follows the same mechanism. A large hydrophobic pocket was identified in the active site of HbHNL which accommodates the more voluminous substituents of the two substrates. A three-point binding mode of the substrates -- hydrophobic pocket, hydrogen bonds between the hydroxyl group and Ser80 and Thr11, electrostatic interaction of the cyano group with Lys236 -- offers a likely structural explanation for the enantioselectivity of the enzyme. The structural data rationalize the observed (S)-enantioselectivity and form the basis for modifying the stereospecificity through rational design. The structures also revealed the necessity of considerable flexibility of the sidechain of Trp128 in order to bind and transform larger substrates.
ESTHER : Gartler_2007_J.Biotechnol_129_87
PubMedSearch : Gartler_2007_J.Biotechnol_129_87
PubMedID: 17250917
Gene_locus related to this paper: hevbr-hnl

Title : Serine scanning: a tool to prove the consequences of N-glycosylation of proteins - Weis_2007_J.Biotechnol_129_50
Author(s) : Weis R , Gaisberger R , Gruber K , Glieder A
Ref : J Biotechnol , 129 :50 , 2007
Abstract : N-Glycosylation of proteins is a common posttranslational modification in eukaryotes. Often this results in enhanced protein stability through protection by the attached sugar moieties. Due to its 13 potential N-glycosylation motifs (N-X-T/S), recombinant hydroxynitrile lyase isoenzyme 5 from almonds (PaHNL5) is secreted by the heterologous host Pichia pastoris in a massively glycosylated form, and it shows extraordinary stability at low pH. The importance of N-glycosylation in general, and individual glycosylation sites in particular for stability at low pH were investigated. To identify especially important glycosylation sites asparagine from all N-X-S/T-motifs was replaced by serine. Thus, critical sites, which contributed to overall enzyme activity and/or stability, were identified individually. One glycosylation site revealed to be essential for stability at low pH. After enzymatic deglycosylation, leaving only one acetylglucosamine attached to asparagines, PaHNL5 retained most of its stability at low pH. Protonation effects in the active site as well as higher-order aggregational events upon incubation in low pH were excluded. This study provides evidence for the interconnection of N-glycosylation and stability at low pH for PaHNL5. Moreover, serine scanning was proven to be applicable for quick identification of critical glycosylation sites.
ESTHER : Weis_2007_J.Biotechnol_129_50
PubMedSearch : Weis_2007_J.Biotechnol_129_50
PubMedID: 17224199

Title : Stereoselective biocatalytic synthesis of (S)-2-hydroxy-2-methylbutyric acid via substrate engineering by using thio-disguised precursors and oxynitrilase catalysis - Fechter_2007_Chemistry_13_3369
Author(s) : Fechter MH , Gruber K , Avi M , Skranc W , Schuster C , Pochlauer P , Klepp KO , Griengl H
Ref : Chemistry , 13 :3369 , 2007
Abstract : 3-Tetrahydrothiophenone (4) and 4-phenylthiobutan-2-one (7) were used as masked 2-butanone equivalents to give the corresponding cyanohydrins 5 (79 % yield, 91 % ee) and 8 (95 % yield, 96 % ee) in an enzymatic cyanohydrin reaction applying the hydroxynitrile lyase (HNL) from Hevea brasiliensis. After hydrolysis and desulphurisation the desired intermediate (S)-2-hydroxy-2-methylbutyric acid (10) was obtained with 99 % ee. Interestingly, when applying (R)-selective HNL from Prunus amygdalus again the (S)-cyanohydrin 5 was formed (62 % ee). The absolute configuration of 5 was verified by crystal structure determination of the corresponding hydrolysis derived carboxylate. The fact that both enzymes yield the same enantiomer was analysed and interpreted by molecular modelling calculations.
ESTHER : Fechter_2007_Chemistry_13_3369
PubMedSearch : Fechter_2007_Chemistry_13_3369
PubMedID: 17226866

Title : A biocatalytic Henry reaction--the hydroxynitrile lyase from Hevea brasiliensis also catalyzes nitroaldol reactions -
Author(s) : Purkarthofer T , Gruber K , Gruber-Khadjawi M , Waich K , Skranc W , Mink D , Griengl H
Ref : Angew Chem Int Ed Engl , 45 :3454 , 2006
PubMedID: 16634109

Title : Observation of a short, strong hydrogen bond in the active site of hydroxynitrile lyase from Hevea brasiliensis explains a large pKa shift of the catalytic base induced by the reaction intermediate - Stranzl_2004_J.Biol.Chem_279_3699
Author(s) : Stranzl GR , Gruber K , Steinkellner G , Zangger K , Schwab H , Kratky C
Ref : Journal of Biological Chemistry , 279 :3699 , 2004
Abstract : The hydroxynitrile lyase from Hevea brasiliensis (HbHNL) uses a catalytic triad consisting of Ser(80)-His(235)-Asp(207) to enhance the basicity of Ser(80)-O gamma for abstracting a proton from the OH group of the substrate cyanohydrin. Following the observation of a relatively short distance between a carboxyl oxygen of Asp(207) and the N delta(1)(His(235)) in a 1.1 A crystal structure of HbHNL, we here show by (1)H and (15)N-NMR spectroscopy that a short, strong hydrogen bond (SSHB) is formed between the two residues upon binding of the competitive inhibitor thiocyanate to HbHNL: the proton resonance of H-N delta 1(His(235)) moves from 15.41 ppm in the free enzyme to 19.35 ppm in the complex, the largest downfield shift observed so far upon inhibitor binding. Simultaneously, the D/H fractionation factor decreases from 0.98 to 0.35. In the observable pH range, i.e. between pH 4 and 10, no significant changes in chemical shifts (and therefore hydrogen bond strength) were observed for free HbHNL. For the complex with thiocyanate, the 19.35 ppm signal returned to 15.41 ppm at approximately pH 8, which indicates a pK(a) near this value for the H-N epsilon(2)(His(235)). These NMR results were analyzed on the basis of finite difference Poisson-Boltzmann calculations, which yielded the relative free energies of four protonation states of the His(235)-Asp(207) pair in solution as well as in the protein environment with and without bound inhibitor. The calculations explain all the NMR features, i.e. they suggest why a short, strong hydrogen bond is formed upon inhibitor binding and why this short, strong hydrogen bond reverts back to a normal one at approximately pH 8. Importantly, the computations also yield a shift of the free energy of the anionic state relative to the zwitterionic reference state by about 10.6 kcal/mol, equivalent to a shift in the apparent pK(a) of His(235) from 2.5 to 10. This huge inhibitor-induced increase in basicity is a prerequisite for His(235) to act as general base in the HbHNL-catalyzed cyanohydrin reaction.
ESTHER : Stranzl_2004_J.Biol.Chem_279_3699
PubMedSearch : Stranzl_2004_J.Biol.Chem_279_3699
PubMedID: 14597632
Gene_locus related to this paper: manes-hnl

Title : Reaction mechanism of hydroxynitrile lyases of the alpha\/beta-hydrolase superfamily: the three-dimensional structure of the transient enzyme-substrate complex certifies the crucial role of LYS236 - Gruber_2004_J.Biol.Chem_279_20501
Author(s) : Gruber K , Gartler G , Krammer B , Schwab H , Kratky C
Ref : Journal of Biological Chemistry , 279 :20501 , 2004
Abstract : The hydroxynitrile lyases (HNLs) from Hevea brasiliensis (HbHNL) and from Manihot esculenta (MeHNL) are both members of the alpha/beta-hydrolase superfamily. Mechanistic proposals have been put forward in the past for both enzymes; they differed with respect to the role of the active-site lysine residue for which a catalytic function was claimed for the Hevea enzyme but denied for the Manihot enzyme. We applied a freeze-quench method to prepare crystals of the complex of HbHNL with the biological substrate acetone cyanohydrin and determined its three-dimensional structure. Site-directed mutagenesis was used to prepare the mutant K236L, which is inactive although its three-dimensional structure is similar to the wild-type enzyme. However, the structure of the K236L-acetone cyanohydrin complex shows the substrate in a different orientation from the wild-type complex. Finite difference Poisson-Boltzmann calculations show that in the absence of Lys(236) the catalytic base His(235) would be protonated at neutral pH. All of this suggests that Lys(236) is instrumental for catalysis in several ways, i.e. by correctly positioning the substrate, by stabilizing the negatively charged reaction product CN(-), and by modulating the basicity of the catalytic base. These data complete the elucidation of the reaction mechanism of alpha/beta-hydrolase HNLs, in which the catalytic triad acts as a general base rather than as a nucleophile; proton abstraction from the substrate is performed by the serine, and reprotonation of the product cyanide is performed by the histidine residues. Together with a threonine side chain, the active-site serine and lysine are also involved in substrate binding.
ESTHER : Gruber_2004_J.Biol.Chem_279_20501
PubMedSearch : Gruber_2004_J.Biol.Chem_279_20501
PubMedID: 14998991
Gene_locus related to this paper: hevbr-hnl

Title : Comprehensive step-by-step engineering of an (R)-hydroxynitrile lyase for large-scale asymmetric synthesis -
Author(s) : Glieder A , Weis R , Skranc W , Poechlauer P , Dreveny I , Majer S , Wubbolts M , Schwab H , Gruber K
Ref : Angew Chem Int Ed Engl , 42 :4815 , 2003
PubMedID: 14562357

Title : The active site of hydroxynitrile lyase from Prunus amygdalus: modeling studies provide new insights into the mechanism of cyanogenesis - Dreveny_2002_Protein.Sci_11_292
Author(s) : Dreveny I , Kratky C , Gruber K
Ref : Protein Science , 11 :292 , 2002
Abstract : The FAD-dependent hydroxynitrile lyase from almond (Prunus amygdalus, PaHNL) catalyzes the cleavage of R-mandelonitrile into benzaldehyde and hydrocyanic acid. Catalysis of the reverse reaction-the enantiospecific formation of alpha-hydroxynitriles--is now widely utilized in organic syntheses as one of the few industrially relevant examples of enzyme-mediated C-C bond formation. Starting from the recently determined X-ray crystal structure, systematic docking calculations with the natural substrate were used to locate the active site of the enzyme and to identify amino acid residues involved in substrate binding and catalysis. Analysis of the modeled substrate complexes supports an enzymatic mechanism that includes the flavin cofactor as a mere "spectator" of the reaction and relies on general acid/base catalysis by the conserved His-497. Stabilization of the negative charge of the cyanide ion is accomplished by a pronounced positive electrostatic potential at the binding site. PaHNL activity requires the FAD cofactor to be bound in its oxidized form, and calculations of the pKa of enzyme-bound HCN showed that the observed inactivation upon cofactor reduction is largely caused by the reversal of the electrostatic potential within the active site. The suggested mechanism closely resembles the one proposed for the FAD-independent, and structurally unrelated HNL from Hevea brasiliensis. Although the actual amino acid residues involved in the catalytic cycle are completely different in the two enzymes, a common motif for the mechanism of cyanogenesis (general acid/base catalysis plus electrostatic stabilization of the cyanide ion) becomes evident.
ESTHER : Dreveny_2002_Protein.Sci_11_292
PubMedSearch : Dreveny_2002_Protein.Sci_11_292
PubMedID: 11790839

Title : Elucidation of the mode of substrate binding to hydroxynitrile lyase from Hevea brasiliensis - Gruber_2001_Proteins_44_26
Author(s) : Gruber K
Ref : Proteins , 44 :26 , 2001
Abstract : The hydroxynitrile lyase from Hevea brasiliensis (Hb-HNL) is used as a catalyst in enantiospecific syntheses of alpha-hydroxynitriles from aldehydes and methyl-ketones. The catalyzed reaction represents one of the few industrially relevant examples of enzyme mediated C-C coupling reactions. In this work, we modeled Hb-HNL substrate complexes that have as yet proven inaccessible to experimental structure determination and were able to identify two binding modes for the natural substrate acetone cyanohydrin in docking simulations. Discrimination of the two alternatives was achieved by modeling complexes with two different chiral cyanohydrins followed by an analysis of the respective relative binding energies from molecular mechanics and thermodynamic integration. Only for one of the alternative binding modes the experimentally established S-selectivity of the enzyme was correctly predicted. Our results yielded further support for an enzymatic mechanism involving the catalytic triad Ser80, His235, and Asp207 as a general acid/base. A pivotal role was ascribed to Lys236, which seems to be crucial for enzymatic activity at low pH values. In addition, the modeling calculations provided possible explanations for the observed substrate and enantioselectivity of the enzyme that rationalize available mutational data and will be the basis for future protein engineering efforts.
ESTHER : Gruber_2001_Proteins_44_26
PubMedSearch : Gruber_2001_Proteins_44_26
PubMedID: 11354003

Title : The hydroxynitrile lyase from almond: a lyase that looks like an oxidoreductase - Dreveny_2001_Structure_9_803
Author(s) : Dreveny I , Gruber K , Glieder A , Thompson A , Kratky C
Ref : Structure , 9 :803 , 2001
Abstract : BACKGROUND: Cyanogenesis is a defense process of several thousand plant species. Hydroxynitrile lyase, a key enzyme of this process, cleaves a cyanohydrin into hydrocyanic acid and the corresponding aldehyde or ketone. The reverse reaction constitutes an important tool in biocatalysis. Different classes of hydroxynitrile lyases have convergently evolved from FAD-dependent oxidoreductases, alpha/beta hydrolases, and alcohol dehydrogenases. The FAD-dependent hydroxynitrile lyases (FAD-HNLs) carry a flavin cofactor whose redox properties appear to be unimportant for catalysis.
RESULTS: We have determined the crystal structure of a 61 kDa hydroxynitrile lyase isoenzyme from Prunus amygdalus (PaHNL1) to 1.5 A resolution. Clear electron density originating from four glycosylation sites could be observed. As concerns the overall protein fold including the FAD cofactor, PaHNL1 belongs to the family of GMC oxidoreductases. The active site for the HNL reaction is probably at a very similar position as the active sites in homologous oxidases.
CONCLUSIONS: There is strong evidence from the structure and the reaction product that FAD-dependent hydroxynitrile lyases have evolved from an aryl alcohol oxidizing precursor. Since key residues implicated in oxidoreductase activity are also present in PaHNL1, it is not obvious why this enzyme shows no oxidase activity. Similarly, features proposed to be relevant for hydroxy-nitrile lyase activity in other hydroxynitrile lyases, i.e., a general base and a positive charge to stabilize the cyanide, are not obviously present in the putative active site of PaHNL1. Therefore, the reason for its HNL activity is far from being well understood at this point.
ESTHER : Dreveny_2001_Structure_9_803
PubMedSearch : Dreveny_2001_Structure_9_803
PubMedID: 11566130

Title : Atomic resolution crystal structure of hydroxynitrile lyase from Hevea brasiliensis - Gruber_1999_Biol.Chem_380_993
Author(s) : Gruber K , Gugganig M , Wagner UG , Kratky C
Ref : Biol Chem , 380 :993 , 1999
Abstract : The X-ray crystal structure of native hydroxynitrile lyase from Hevea brasiliensis (Hb-HNL) has been determined at 1.1 A resolution. It refined to a final R of 11.5% for all data and an Rfree of 14.4%. The favorable data-to-parameter ratio at atomic resolution made the refinement of individual anisotropic displacement parameters possible. The data also allowed a clear distinction of the alternate orientations of all histidine and the majority of asparagine and glutamine side chains. A number of hydrogen atoms, including one on the imidazole of the mechanistically important His-235, became visible as peaks in a difference electron density map. The structure revealed a discretely disordered sidechain of Ser-80, which is part of the putative catalytic triad. Analysis of the anisotropy indicated an increased mobility of residues near the entrance to the active site and within the active site.
ESTHER : Gruber_1999_Biol.Chem_380_993
PubMedSearch : Gruber_1999_Biol.Chem_380_993
PubMedID: 10494852

Title : Three-dimensional structures of enzyme-substrate complexes of the hydroxynitrile lyase from Hevea brasiliensis - Zuegg_1999_Protein.Sci_8_1990
Author(s) : Zuegg J , Gruber K , Gugganig M , Wagner UG , Kratky C
Ref : Protein Science , 8 :1990 , 1999
Abstract : The 3D structures of complexes between the hydroxynitrile lyase from Hevea brasiliensis (Hb-HNL) and several substrate and/or inhibitor molecules, including trichloracetaldehyde, hexafluoracetone, acetone, and rhodanide, were determined by X-ray crystallography. The complex with trichloracetaldehyde showed a covalent linkage between the protein and the inhibitor, which had apparently resulted from nucleophilic attack of the catalytic Ser80-Ogamma. All other complexes showed the substrate or inhibitor molecule merely hydrogen bonded to the protein. In addition, the native crystal structure of Hb-HNL was redetermined at cryo-temperature and at room temperature, eliminating previous uncertainties concerning residual electron density within the active site, and leading to the observation of two conserved water molecules. One of them was found to be conserved in all complex structures and appears to have mainly structural significance. The other water molecule is conserved in all structures except for the complex with rhodanide; it is hydrogen bonded to the imidazole of the catalytic His235 and appears to affect the Hb-HNL catalyzed reaction. The observed 3D structural data suggest implications for the enzyme mechanism. It appears that the enzyme-catalyzed cyanohydrin formation is unlikely to proceed via a hemiacetal or hemiketal intermediate covalently attached to the enzyme, despite the observation of such an intermediate for the complex with trichloracetaldehyde. Instead, the data are consistent with a mechanism where the incoming substrate is activated by hydrogen bonding with its carbonyl oxygen to the Ser80 and Thr11 hydroxy groups. A hydrogen cyanide molecule subsequently replaces a water molecule and is deprotonated presumably by the His235 base. Deprotonation is facilitated by the proximity of the positive charge of the Lys236 side chain.
ESTHER : Zuegg_1999_Protein.Sci_8_1990
PubMedSearch : Zuegg_1999_Protein.Sci_8_1990
PubMedID: 10548044
Gene_locus related to this paper: hevbr-hnl