scholarly journals Considerations on activity determinants of fungal polyphenol oxidases based on mutational and structural studies

2021 ◽  
Author(s):  
Efstratios Nikolaivits ◽  
Alexandros Valmas ◽  
Grigorios Dedes ◽  
Evangelos Topakas ◽  
Maria Dimarogona
Keyword(s):  
Crystal Structure ◽  
Fruits And Vegetables ◽  
Structural Data ◽  
Structural Similarity ◽  
Site Directed Mutagenesis ◽  
Special Focus ◽  
Structural Determinants ◽  
Polyphenol Oxidases ◽  
High Structural Similarity ◽  
Tree Branch

ABSTRACTPolyphenol oxidases (PPOs) are an industrially relevant family of enzymes, being involved in the post-harvest browning of fruits and vegetables, as well as in human melanogenesis. Their involvement lies in their ability to oxidize phenolic or polyphenolic compounds, that subsequently form pigments. PPO family includes tyrosinases and catechol oxidases, which in spite of their high structural similarity, exhibit different catalytic activities. Long-standing research efforts have not yet managed to decipher the structural determinants responsible for this differentiation, as every new theory is disproved by a more recent study. In the present work, we combined biochemical along with structural data, in order to rationalize the function of a previously characterized PPO from Thermothelomyces thermophila (TtPPO). The crystal structure of a TtPPO variant, determined at 1.55 Å resolution, represents the second known structure of an ascomycete PPO. Kinetic data of structure-guided mutants prove the implication of “gate” residue L306, residue HB1+1 (G292) and HB2+1 (Y296) in TtPPO function against various substrates. Our findings demonstrate the role of L306 in the accommodation of bulky substrates and that residue HB1+1 is unlikely to determine monophenolase activity as suggested from previous studies.IMPORTANCEPPOs are enzymes of biotechnological interest. They have been extensively studied both biochemically and structurally, with a special focus on the plant-derived counterparts. Even so, explicit description of the molecular determinants of their substrate specificity is still pending. Especially for ascomycete PPOs, only one crystal structure has been determined so far, thus limiting our knowledge on this tree branch of the family. In the present study, we report the second crystal structure of an ascomycete PPO. Combined with site-directed mutagenesis and biochemical studies, we depict the amino acids in the vicinity of the active site that affect enzyme activity, and perform a detailed analysis on a variety of substrates. Our findings improve current understanding of structure-function relations of microbial PPOs, which is a prerequisite for the engineering of biocatalysts of desired properties.

scholarly journals Considerations on activity determinants of fungal polyphenol oxidases based on mutational and structural studies

2021 ◽  
Author(s):  
Efstratios Nikolaivits ◽  
Alexandros Valmas ◽  
Grigorios Dedes ◽  
Evangelos Topakas ◽  
Maria Dimarogona
Keyword(s):  
Crystal Structure ◽  
Fruits And Vegetables ◽  
Structural Data ◽  
Structural Similarity ◽  
Site Directed Mutagenesis ◽  
Special Focus ◽  
Structural Determinants ◽  
Polyphenol Oxidases ◽  
High Structural Similarity ◽  
Tree Branch

Polyphenol oxidases (PPOs) are an industrially relevant family of enzymes, being involved in the post-harvest browning of fruits and vegetables, as well as in human melanogenesis. Their involvement lies in their ability to oxidize phenolic or polyphenolic compounds, that subsequently form pigments. PPO family includes tyrosinases and catechol oxidases, which in spite of their high structural similarity, exhibit different catalytic activities. Long-standing research efforts have not yet managed to decipher the structural determinants responsible for this differentiation, as every new theory is disproved by a more recent study. In the present work, we combined biochemical along with structural data, in order to rationalize the function of a previously characterized PPO from Thermothelomyces thermophila (TtPPO). The crystal structure of a TtPPO variant, determined at 1.55 Å resolution, represents the second known structure of an ascomycete PPO. Kinetic data of structure-guided mutants prove the implication of “gate” residue L306, residue HB1+1 (G292) and HB2+1 (Y296) in TtPPO function against various substrates. Our findings demonstrate the role of L306 in the accommodation of bulky substrates and that residue HB1+1 is unlikely to determine monophenolase activity as suggested from previous studies. IMPORTANCE PPOs are enzymes of biotechnological interest. They have been extensively studied both biochemically and structurally, with a special focus on the plant-derived counterparts. Even so, explicit description of the molecular determinants of their substrate specificity is still pending. Especially for ascomycete PPOs, only one crystal structure has been determined so far, thus limiting our knowledge on this tree branch of the family. In the present study, we report the second crystal structure of an ascomycete PPO. Combined with site-directed mutagenesis and biochemical studies, we depict the amino acids in the vicinity of the active site that affect enzyme activity, and perform a detailed analysis on a variety of substrates. Our findings improve current understanding of structure-function relations of microbial PPOs, which is a prerequisite for the engineering of biocatalysts of desired properties.

scholarly journals Versatile Fungal Polyphenol Oxidase with Chlorophenol Bioremediation Potential: Characterization and Protein Engineering

2018 ◽  
Vol 84 (23) ◽  
Author(s):  
Efstratios Nikolaivits ◽  
Maria Dimarogona ◽  
Ioanna Karagiannaki ◽  
Angelina Chalima ◽  
Ayelet Fishman ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Fruits And Vegetables ◽  
Homology Model ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Gene Encoding ◽  
Content Type ◽  
Polyphenol Oxidases ◽  
Increased Activity ◽  
Work Knowledge

ABSTRACTPolyphenol oxidases (PPOs) have been mostly associated with the undesirable postharvest browning in fruits and vegetables and have implications in human melanogenesis. Nonetheless, they are considered useful biocatalysts in the food, pharmaceutical, and cosmetic industries. The aim of the present work was to characterize a novel PPO and explore its potential as a bioremediation agent. A gene encoding an extracellular tyrosinase-like enzyme was amplified from the genome ofThermothelomyces thermophilaand expressed inPichia pastoris. The recombinant enzyme (TtPPO) was purified and biochemically characterized. Its production reached 40 mg/liter, and it appeared to be a glycosylated and N-terminally processed protein.TtPPO showed broad substrate specificity, as it could oxidize 28/30 compounds tested, including polyphenols, substituted phenols, catechols, and methoxyphenols. Its optimum temperature was 65°C, with a half-life of 18.3 h at 50°C, while its optimum pH was 7.5. The homology model ofTtPPO was constructed, and site-directed mutagenesis was performed in order to increase its activity on mono- and dichlorophenols (di-CPs). The G292N/Y296V variant ofTtPPO 5.3-fold increased activity on 3,5-dichlorophenol (3,5-diCP) compared to the wild type.IMPORTANCEA novel fungal PPO was heterologously expressed and biochemically characterized. Construction of single and double mutants led to the generation of variants with altered specificity against CPs. Through this work, knowledge is gained regarding the effect of mutations on the substrate specificity of PPOs. This work also demonstrates that more potent biocatalysts for the bioremediation of harmful CPs can be developed by applying site-directed mutagenesis.

scholarly journals Crystal structure of gluconate 5-dehydrogenase from Lentibacter algarum

2020 ◽  
Vol 76 (5) ◽  
pp. 228-234
Author(s):  
Dengke Tian ◽  
Xueqi Fu ◽  
Wenqiang Cao ◽  
Hong Yuan
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Structural Similarity ◽  
Unit Cell ◽  
Unit Cell Parameters ◽  
Monoclinic System ◽  
Space Group ◽  
Cell Parameters ◽  
High Structural Similarity

Gluconate 5-dehydrogenase (Ga5DH; EC 1.1.1.69) from Lentibacter algarum (LaGa5DH) was recombinantly expressed in Escherichia coli and purified to homogeneity. The protein was crystallized and the crystal structure was solved at 2.1 Å resolution. The crystal belonged to the monoclinic system, with space group P1 and unit-cell parameters a = 55.42, b = 55.48, c = 79.16 Å, α = 100.51, β = 105.66, γ = 97.99°. The structure revealed LaGaDH to be a tetramer, with each subunit consisting of six α-helices and three antiparallel β-hairpins. LaGa5DH has high structural similarity to other Ga5DH proteins, demonstrating that this enzyme is highly conserved.

scholarly journals The crystal structure of mycobacterial epoxide hydrolase A

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Eike C. Schulz ◽  
Sara R. Henderson ◽  
Boris Illarionov ◽  
Thomas Crosskey ◽  
Stacey M. Southall ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Substrate Specificity ◽  
Drug Targets ◽  
Epoxide Hydrolase ◽  
Sequence Similarity ◽  
Structural Similarity ◽  
High Sequence Similarity ◽  
High Structural Similarity ◽  
Potential Drug Targets ◽  
Insight Into

Abstract The human pathogen Mycobacterium tuberculosis is the causative agent of tuberculosis resulting in over 1 million fatalities every year, despite decades of research into the development of new anti-TB compounds. Unlike most other organisms M. tuberculosis has six putative genes for epoxide hydrolases (EH) of the α/β-hydrolase family with little known about their individual substrates, suggesting functional significance for these genes to the organism. Due to their role in detoxification, M. tuberculosis EH’s have been identified as potential drug targets. Here, we demonstrate epoxide hydrolase activity of M. thermoresistibile epoxide hydrolase A (Mth-EphA) and report its crystal structure in complex with the inhibitor 1,3-diphenylurea at 2.0 Å resolution. Mth-EphA displays high sequence similarity to its orthologue from M. tuberculosis and generally high structural similarity to α/β-hydrolase EHs. The structure of the inhibitor bound complex reveals the geometry of the catalytic residues and the conformation of the inhibitor. Comparison to other EHs from mycobacteria allows insight into the active site plasticity with respect to substrate specificity. We speculate that mycobacterial EHs may have a narrow substrate specificity providing a potential explanation for the genetic repertoire of epoxide hydrolase genes in M. tuberculosis.

Crystal structure and functional characterization of a glucosamine-6-phosphate N-acetyltransferase from Arabidopsis thaliana

2012 ◽  
Vol 443 (2) ◽  
pp. 427-437 ◽  
Author(s):  
Heike Riegler ◽  
Thomas Herter ◽  
Irina Grishkovskaya ◽  
Anja Lude ◽  
Malgorzata Ryngajllo ◽  
...  
Keyword(s):  
Crystal Structure ◽  
De Novo ◽  
Homo Sapiens ◽  
Functional Characterization ◽  
Catalytic Efficiency ◽  
Structural Similarity ◽  
Highly Active ◽  
Glycan Chain ◽  
High Structural Similarity ◽  
Protein Sequence Identity

GlcNAc (N-acetylglucosamine) is an essential part of the glycan chain in N-linked glycoproteins. It is a building block for polysaccharides such as chitin, and several glucosaminoglycans and proteins can be O-GlcNAcylated. The deacetylated form, glucosamine, is an integral part of GPI (glycosylphosphatidylinositol) anchors. Both are incorporated into polymers by glycosyltransferases that utilize UDP-GlcNAc. This UDP-sugar is synthesized in a short pathway comprising four steps starting from fructose 6-phosphate. GNA (glucosamine-6-phosphate N-acetyltransferase) catalyses the second of these four reactions in the de novo synthesis in eukaryotes. A phylogenetic analysis revealed that only one GNA isoform can be found in most of the species investigated and that the most likely Arabidopsis candidate is encoded by the gene At5g15770 (AtGNA). qPCR (quantitative PCR) revealed the ubiquitous expression of AtGNA in all organs of Arabidopsis plants. Heterologous expression of AtGNA showed that it is highly active between pH 7 and 8 and at temperatures of 30–40°C. It showed Km values of 231 μM for glucosamine 6-phosphate and 33 μM for acetyl-CoA respectively and a catalytic efficiency comparable with that of other GNAs characterized. The solved crystal structure of AtGNA at a resolution of 1.5 Å (1 Å=0.1 nm) revealed a very high structural similarity to crystallized GNA proteins from Homo sapiens and Saccharomyces cerevisiae despite less well conserved protein sequence identity.

scholarly journals Differential Contribution of Transmembrane Domains IV, V, VI, and VII to Human Angiotensin II Type 1 Receptor Homomer Formation

2017 ◽  
Vol 292 (8) ◽  
pp. 3341-3350 ◽  
Author(s):  
Brent M. Young ◽  
Elaine Nguyen ◽  
Matthew A. J. Chedrawe ◽  
Jan K. Rainey ◽  
Denis J. Dupré
Keyword(s):  
Crystal Structure ◽  
Angiotensin Ii ◽  
Drug Targets ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Structural Data ◽  
Transmembrane Domains ◽  
Site Directed Mutagenesis ◽  
Hydrophobic Residues

G protein-coupled receptors (GPCRs) play an important role in drug therapy and represent one of the largest families of drug targets. The angiotensin II type 1 receptor (AT1R) is notable as it has a central role in the treatment of cardiovascular disease. Blockade of AT1R signaling has been shown to alleviate hypertension and improve outcomes in patients with heart failure. Despite this, it has become apparent that our initial understanding of AT1R signaling is oversimplified. There is considerable evidence to suggest that AT1R signaling is highly modified in the presence of receptor-receptor interactions, but there is very little structural data available to explain this phenomenon even with the recent elucidation of the AT1R crystal structure. The current study investigates the involvement of transmembrane domains in AT1R homomer assembly with the goal of identifying hydrophobic interfaces that contribute to receptor-receptor affinity. A recently published crystal structure of the AT1R was used to guide site-directed mutagenesis of outward-facing hydrophobic residues within the transmembrane region of the AT1R. Bioluminescence resonance energy transfer was employed to analyze how receptor mutation affects the assembly of AT1R homomers with a specific focus on hydrophobic residues. Mutations within transmembrane domains IV, V, VI, and VII had no effect on angiotensin-mediated β-arrestin1 recruitment; however, they exhibited differential effects on the assembly of AT1R into oligomeric complexes. Our results demonstrate the importance of hydrophobic amino acids at the AT1R transmembrane interface and provide the first glimpse of the requirements for AT1R complex assembly.

scholarly journals Crystal structure of the N-terminal anticodon-binding domain of the nondiscriminating aspartyl-tRNA synthetase fromHelicobacter pylori

2017 ◽  
Vol 73 (2) ◽  
pp. 62-69 ◽  
Author(s):  
Chomphunuch Songsiriritthigul ◽  
Suwimon Suebka ◽  
Chun-Jung Chen ◽  
Pitchayada Fuengfuloy ◽  
Pitak Chuawong
Keyword(s):  
Crystal Structure ◽  
Crucial Role ◽  
Structural Similarity ◽  
Trna Synthetase ◽  
Binding Domain ◽  
Trna Recognition ◽  
Binding Domains ◽  
Human Pathogenic Bacterium ◽  
High Structural Similarity ◽  
H Pylori

The N-terminal anticodon-binding domain of the nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) plays a crucial role in the recognition of both tRNAAspand tRNAAsn. Here, the first X-ray crystal structure of the N-terminal domain of this enzyme (ND-AspRS1–104) from the human-pathogenic bacteriumHelicobacter pyloriis reported at 2.0 Å resolution. The apo form ofH. pyloriND-AspRS1–104shares high structural similarity with the N-terminal anticodon-binding domains of the discriminating aspartyl-tRNA synthetase (D-AspRS) fromEscherichia coliand ND-AspRS fromPseudomonas aeruginosa, allowing recognition elements to be proposed for tRNAAspand tRNAAsn. It is proposed that a long loop (Arg77–Lys90) in thisH. pyloridomain influences its relaxed tRNA specificity, such that it is classified as nondiscriminating. A structural comparison between D-AspRS fromE. coliand ND-AspRS fromP. aeruginosasuggests that turns E and F (78GAGL81and83NPKL86) inH. pyloriND-AspRS play a crucial role in anticodon recognition. Accordingly, the conserved Pro84 in turn F facilitates the recognition of the anticodons of tRNAAsp(34GUC36) and tRNAAsn(34GUU36). The absence of the amide H atom allows both C and U bases to be accommodated in the tRNA-recognition site.

scholarly journals Complexed Crystal Structure of Saccharomyces cerevisiae Dihydroorotase with Inhibitor 5-Fluoroorotate Reveals a New Binding Mode

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Hong-Hsiang Guan ◽  
Yen-Hua Huang ◽  
En-Shyh Lin ◽  
Chun-Jung Chen ◽  
Cheng-Yang Huang
Keyword(s):  
Crystal Structure ◽  
Saccharomyces Cerevisiae ◽  
Active Site ◽  
Structural Similarity ◽  
Binding Mode ◽  
Site Directed Mutagenesis ◽  
Molecular Evidence ◽  
Binding Modes ◽  
Pyrimidine Nucleotides ◽  
Quenching Method

Dihydroorotase (DHOase) possesses a binuclear metal center in which two Zn ions are bridged by a posttranslationally carbamylated lysine. DHOase catalyzes the reversible cyclization of N-carbamoyl aspartate (CA-asp) to dihydroorotate (DHO) in the third step of the pathway for the biosynthesis of pyrimidine nucleotides and is an attractive target for potential anticancer and antimalarial chemotherapy. Crystal structures of ligand-bound DHOase show that the flexible loop extends toward the active site when CA-asp is bound (loop-in mode) or moves away from the active site, facilitating the product DHO release (loop-out mode). DHOase binds the product-like inhibitor 5-fluoroorotate (5-FOA) in a similar mode to DHO. In the present study, we report the crystal structure of DHOase from Saccharomyces cerevisiae (ScDHOase) complexed with 5-FOA at 2.5 Å resolution (PDB entry 7CA0). ScDHOase shares structural similarity with Escherichia coli DHOase (EcDHOase). However, our complexed structure revealed that ScDHOase bound 5-FOA differently from EcDHOase. 5-FOA ligated the Zn atoms in the active site of ScDHOase. In addition, 5-FOA bound to ScDHOase through the loop-in mode. We also characterized the binding of 5-FOA to ScDHOase by using the site-directed mutagenesis and fluorescence quenching method. Based on these lines of molecular evidence, we discussed whether these different binding modes are species- or crystallography-dependent.

scholarly journals Identification of V6.51L as a selectivity hotspot in stereoselective A2B adenosine receptor antagonist recognition

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Xuesong Wang ◽  
Willem Jespers ◽  
Rubén Prieto-Díaz ◽  
Maria Majellaro ◽  
Adriaan P. IJzerman ◽  
...  
Keyword(s):  
Radioligand Binding ◽  
Structural Data ◽  
Binding Mode ◽  
Selective Recognition ◽  
Chiral Hplc ◽  
Binding Assays ◽  
Structural Determinants ◽  
X Ray Crystallography ◽  
Adenosine Receptor Antagonist ◽  
Energy Perturbation

AbstractThe four adenosine receptors (ARs) A1AR, A2AAR, A2BAR, and A3AR are G protein-coupled receptors (GPCRs) for which an exceptional amount of experimental and structural data is available. Still, limited success has been achieved in getting new chemical modulators on the market. As such, there is a clear interest in the design of novel selective chemical entities for this family of receptors. In this work, we investigate the selective recognition of ISAM-140, a recently reported A2BAR reference antagonist. A combination of semipreparative chiral HPLC, circular dichroism and X-ray crystallography was used to separate and unequivocally assign the configuration of each enantiomer. Subsequently affinity evaluation for both A2A and A2B receptors demonstrate the stereospecific and selective recognition of (S)-ISAM140 to the A2BAR. The molecular modeling suggested that the structural determinants of this selectivity profile would be residue V2506.51 in A2BAR, which is a leucine in all other ARs including the closely related A2AAR. This was herein confirmed by radioligand binding assays and rigorous free energy perturbation (FEP) calculations performed on the L249V6.51 mutant A2AAR receptor. Taken together, this study provides further insights in the binding mode of these A2BAR antagonists, paving the way for future ligand optimization.

scholarly journals Deep learning for visualization and novelty detection in large X-ray diffraction datasets

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Lars Banko ◽  
Phillip M. Maffettone ◽  
Dennis Naujoks ◽  
Daniel Olds ◽  
Alfred Ludwig
Keyword(s):  
Thin Film ◽  
Crystal Structure ◽  
Artificial Intelligence ◽  
Deep Learning ◽  
Novelty Detection ◽  
Structural Similarity ◽  
X Ray Diffraction ◽  
X Ray ◽  
Diffraction Patterns ◽  
Post Hoc

AbstractWe apply variational autoencoders (VAE) to X-ray diffraction (XRD) data analysis on both simulated and experimental thin-film data. We show that crystal structure representations learned by a VAE reveal latent information, such as the structural similarity of textured diffraction patterns. While other artificial intelligence (AI) agents are effective at classifying XRD data into known phases, a similarly conditioned VAE is uniquely effective at knowing what it doesn’t know: it can rapidly identify data outside the distribution it was trained on, such as novel phases and mixtures. These capabilities demonstrate that a VAE is a valuable AI agent for aiding materials discovery and understanding XRD measurements both ‘on-the-fly’ and during post hoc analysis.

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