scholarly journals Semirational design and engineering of grapevine glucosyltransferases for enhanced activity and modified product selectivity

Glycobiology ◽  
2019 ◽  
Vol 29 (11) ◽  
pp. 765-775 ◽  
Author(s):  
Rakesh Joshi ◽  
Johanna Trinkl ◽  
Annika Haugeneder ◽  
Katja Härtl ◽  
Katrin Franz-Oberdorf ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Structural Changes ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Uridine Diphosphate ◽  
Amino Acid Residues ◽  
Product Selectivity ◽  
Active Site Residues ◽  
Product Specificity ◽  
Increased Activity

Abstract Uridine diphosphate-dependent glycosyltransferases (UGTs) catalyze the transfer of a diversity of sugars to several acceptor molecules and often exhibit distinct substrate specificity. Modulation of glycosyltransferases for increased catalytic activity and altered substrate or product specificity are the key manipulations for the biotechnological use of glycosyltransferases in various biosynthetic processes. Here, we have engineered the binding pocket of three previously characterized Vitis vinifera glycosyltransferases, UGT88F12, UGT72B27 and UGT92G6, by structure-guided in silico mutagenesis to facilitate the interactions of active site residues with flavonol glucosides and thus modify substrate specificity and activity. Site-directed mutagenesis at selected sites, followed with liquid chromatography–mass spectrometry based activity assays, exhibited that mutant UGTs were altered in product selectivity and activity as compared to the wild-type enzymes. Mutant UGTs produced larger amounts of flavonol di-monosaccharide glucosides, which imply that the mutations led to structural changes that increased the volume of the binding pocket to accommodate a larger substrate and to release larger products at ease. Mutants showed increased activity and modified product specificity. Thus, structure-based systematic mutations of the amino acid residues in the binding pocket can be explored for the generation of engineered UGTs for diverse biotechnological applications.

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 Identification of Key Amino Acid Residues Determining Product Specificity of 2,3-Oxidosqualene Cyclase in Siraitia grosvenorii

Catalysts ◽  
2018 ◽  
Vol 8 (12) ◽  
pp. 577 ◽  
Author(s):  
Jing Qiao ◽  
Jiushi Liu ◽  
Jingjing Liao ◽  
Zuliang Luo ◽  
Xiaojun Ma ◽  
...  
Keyword(s):  
Amino Acid ◽  
Site Directed Mutagenesis ◽  
Bioactive Molecules ◽  
Amino Acid Residues ◽  
Product Specificity ◽  
Oxidosqualene Cyclase ◽  
Siraitia Grosvenorii ◽  
Initial Substrate ◽  
New Strategy ◽  
Key Residues

Sterols and triterpenes are structurally diverse bioactive molecules generated through cyclization of linear 2,3-oxidosqualene. Based on carbocationic intermediates generated during the initial substrate preorganization step, oxidosqualene cyclases (OSCs) are roughly segregated into a dammarenyl cation group that predominantly catalyzes triterpenoid precursor products and a protosteryl cation group which mostly generates sterol precursor products. The mechanism of conversion between two scaffolds is not well understood. Previously, we have characterized a promiscuous OSC from Siraitia grosvenorii (SgCS) that synthesizes a novel cucurbitane-type triterpene cucurbitadienol as its main product. By integration of homology modeling, molecular docking and site-directed mutagenesis, we discover that five key amino acid residues (Asp486, Cys487, Cys565, Tyr535, and His260) may be responsible for interconversions between chair–boat–chair and chair–chair–chair conformations. The discovery of euphol, dihydrolanosterol, dihydroxyeuphol and tirucallenol unlocks a new path to triterpene diversity in nature. Our findings also reveal mechanistic insights into the cyclization of oxidosqualene into cucurbitane-type and lanostane-type skeletons, and provide a new strategy to identify key residues determining OSC specificity.

scholarly journals Main Structural Targets for Engineering Lipase Substrate Specificity

Catalysts ◽  
2020 ◽  
Vol 10 (7) ◽  
pp. 747
Author(s):  
Samah Hashim Albayati ◽  
Malihe Masomian ◽  
Siti Nor Hasmah Ishak ◽  
Mohd Shukuri bin Mohamad Ali ◽  
Adam Leow Thean ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Rational Design ◽  
Molecular Mechanisms ◽  
Experimental Studies ◽  
Random Mutagenesis ◽  
Binding Pocket ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Saturation Mutagenesis ◽  
Process Conditions

Microbial lipases represent one of the most important groups of biotechnological biocatalysts. However, the high-level production of lipases requires an understanding of the molecular mechanisms of gene expression, folding, and secretion processes. Stable, selective, and productive lipase is essential for modern chemical industries, as most lipases cannot work in different process conditions. However, the screening and isolation of a new lipase with desired and specific properties would be time consuming, and costly, so researchers typically modify an available lipase with a certain potential for minimizing cost. Improving enzyme properties is associated with altering the enzymatic structure by changing one or several amino acids in the protein sequence. This review detailed the main sources, classification, structural properties, and mutagenic approaches, such as rational design (site direct mutagenesis, iterative saturation mutagenesis) and direct evolution (error prone PCR, DNA shuffling), for achieving modification goals. Here, both techniques were reviewed, with different results for lipase engineering, with a particular focus on improving or changing lipase specificity. Changing the amino acid sequences of the binding pocket or lid region of the lipase led to remarkable enzyme substrate specificity and enantioselectivity improvement. Site-directed mutagenesis is one of the appropriate methods to alter the enzyme sequence, as compared to random mutagenesis, such as error-prone PCR. This contribution has summarized and evaluated several experimental studies on modifying the substrate specificity of lipases.

scholarly journals Amino Acid Residues That Contribute to Substrate Specificity of Class A β-Lactamase SME-1

2005 ◽  
Vol 49 (8) ◽  
pp. 3421-3427 ◽  
Author(s):  
Fahd K. Majiduddin ◽  
Timothy Palzkill
Keyword(s):  
Substrate Specificity ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Class A ◽  
Study Support ◽  
Functional Mutants ◽  
Hydrolysis Of ◽  
Traditional Class

ABSTRACT Carbapenem antibiotics are used as antibiotics of last resort because they possess a broad spectrum of antimicrobial activity and are not easily hydrolyzed by β-lactamases. Recently, class A enzymes, such as the SME-1, NMC-A, and IMI-1 β-lactamases, have been identified with the capacity to hydrolyze carbapenem antibiotics. Traditional class A β-lactamases, such as TEM-1 and SHV-1, are unable to hydrolyze carbapenem antibiotics and exhibit some differences in sequence from those that are able to hydrolyze carbapenem antibiotics. The positions that differ may contribute to the unique substrate specificity of the class A carbapenemase SME-1. Codons in the SME-1 gene representing residues 104, 105, 132, 167, 237, and 241 were randomized by site-directed mutagenesis, and functional mutants were selected for the ability to hydrolyze imipenem, ampicillin, or cefotaxime. Although several positions are important for hydrolysis of β-lactam antibiotics, no single position was found to uniquely contribute to carbapenem hydrolysis. The results of this study support a model whereby the carbapenemase activity of SME-1 is due to a highly distributed set of interactions that subtly alter the structure of the active-site pocket.

scholarly journals Evidence that Asn542 of neprilysin (EC 3.4.24.11) is involved in binding of the P2′ residue of substrates and inhibitors

1995 ◽  
Vol 311 (2) ◽  
pp. 623-627 ◽  
Author(s):  
N Dion ◽  
H Le Moual ◽  
M C Fournié-Zaluski ◽  
B P Roques ◽  
P Crine ◽  
...  
Keyword(s):  
Active Site ◽  
Substrate Binding ◽  
Biologically Active ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Active Site Residues ◽  
Hydrophobic Cluster Analysis ◽  
Active Peptides ◽  
Ic50 Values

Neprilysin (EC 3.4.24.11) is a Zn2+ metallopeptidase involved in the degradation of biologically active peptides, e.g. enkephalins and atrial natriuretic peptide. The substrate specificity and catalytic activity of neprilysin resemble those of thermolysin, a crystallized bacterial Zn2+ metalloprotease. Despite little overall homology between the primary structures of thermolysin and neprilysin, many of the amino acid residues involved in catalysis, as well as Zn2+ and substrate binding, are highly conserved. Most of the active-site residues of neprilysin have their homologues in thermolysin and have been characterized by site-directed mutagenesis. Furthermore, hydrophobic cluster analysis has revealed some other analogies between the neprilysin and thermolysin sequences [Benchetrit, Bissery, Mornon, Devault, Crine and Roques (1988) Biochemistry 27, 592-596]. According to this analysis the role of Asn542 in the neprilysin active site is analogous to that of Asn112 of thermolysin, which is to bind the substrate. Site-directed mutagenesis was used to change Asn542 to Gly or Gln residues. The effect of these mutations on substrate catalysis and inhibitor binding was examined with a series of thiorphan-like compounds containing various degrees of methylation at the P2′ residue. For both mutated enzymes, determination of kinetic parameters with [D-Ala2,Leu5]enkephalin as substrate showed that the large decrease in activity was attributable to an increase in Km (14-16-fold) whereas kcat values were only slightly affected (2-3-fold decrease). This is in agreement with Asn542 being involved in substrate binding rather than directly in catalysis. Finally, the IC50 values for thiorphan and substituted thiorphans strongly suggest that Asn542 of neprilysin binds the substrate on the amino side of the P2′ residue by formation of a unique hydrogen bond.

scholarly journals Functional Diversity of Four Glycoside Hydrolase Family 3 Enzymes from the Rumen Bacterium Prevotella bryantii B14

2010 ◽  
Vol 192 (9) ◽  
pp. 2335-2345 ◽  
Author(s):  
Dylan Dodd ◽  
Shinichi Kiyonari ◽  
Roderick I. Mackie ◽  
Isaac K. O. Cann
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Amino Acid Residue ◽  
Glycoside Hydrolase ◽  
Site Directed Mutagenesis ◽  
Rumen Bacterium ◽  
Glycoside Hydrolase Family ◽  
Active Site Residues ◽  
Sequence Alignments ◽  
Prevotella Bryantii

ABSTRACT Prevotella bryantii B14 is a member of the phylum Bacteroidetes and contributes to the degradation of hemicellulose in the rumen. The genome of P. bryantii harbors four genes predicted to encode glycoside hydrolase (GH) family 3 (GH3) enzymes. To evaluate whether these genes encode enzymes with redundant biological functions, each gene was cloned and expressed in Escherichia coli. Biochemical analysis of the recombinant proteins revealed that the enzymes exhibit different substrate specificities. One gene encoded a cellodextrinase (CdxA), and three genes encoded β-xylosidase enzymes (Xyl3A, Xyl3B, and Xyl3C) with different specificities for either para-nitrophenyl (pNP)-linked substrates or substituted xylooligosaccharides. To identify the amino acid residues that contribute to catalysis and substrate specificity within this family of enzymes, the roles of conserved residues (R177, K214, H215, M251, and D286) in Xyl3B were probed by site-directed mutagenesis. Each mutation led to a severely decreased catalytic efficiency without a change in the overall structure of the mutant enzymes. Through amino acid sequence alignments, an amino acid residue (E115) that, when mutated to aspartic acid, resulted in a 14-fold decrease in the k cat/Km for pNP-β-d-xylopyranoside (pNPX) with a concurrent 1.1-fold increase in the k cat/Km for pNP-β-d-glucopyranoside (pNPG) was identified. Amino acid residue E115 may therefore contribute to the discrimination between β-xylosides and β-glucosides. Our results demonstrate that each of the four GH3 enzymes has evolved to perform a specific role in lignopolysaccharide hydrolysis and provide insight into the role of active-site residues in catalysis and substrate specificity for GH3 enzymes.

scholarly journals Genome Mining, Phylogenetic and Structural Analysis of Bacterial Nitrilases for the Biodegradation of Nitrile Compounds

2021 ◽  
Author(s):  
Richa Salwan ◽  
Vivek Sharma ◽  
Surajit Das
Keyword(s):  
Structural Analysis ◽  
Substrate Specificity ◽  
Active Site ◽  
Structural Information ◽  
Genome Mining ◽  
Binding Pocket ◽  
Catalytic Triad ◽  
Vital Role ◽  
Active Site Residues ◽  
Motif Analysis

Abstract Microbial nitrilases play vital role in biodegradation of nitrile-containing contaminants in pollutant and effluents treatments in chemical and textile industries as well as the biosynthesis of IAA from tryptophan in plants. However, the lack of structural information hinders the correlation of its activity and substrate specificity. Here, we have identified bacterial genomes for nitrilases bearing unassigned functions including hypothetical, uncharacterized, or putative role. The genomic annotations revealed four predicted nitrilases encoding genes as uncharacterized subgroup of the nitrilase superfamily. Further, the annotation of these nitrilases revealed relatedness with nitrilase hydratases and cyanoalanine hydratases. The characterization of motif analysis of these protein sequences, predicted a single motif of 20-28 aa, and glutamate (E), lysine (K) and cysteine (C) residues as a part of catalytic triad along with several active site residues. The structural analysis of the modeled nitrilases revealed geometrical and close conformation of α-helices and β-sheets arranged in a sandwich structure. The catalytic residues constituted the substrate binding pocket and exhibited the wide nitrile substrate spectra for both aromatic and aliphatic nitriles containing compounds. The aromatic amino acid residues Y159 in active site were predicted to show importance for substrate specificity. The substitution of non-aromatic alanine residue in place of Y159 completely disrupted the catalytic activity for indole-3-acetonitrile. The present study reports several uncharacterized nitrilases which have not been reported so far for their role in the biodegradation of pollutants, xenobiotics which could find applications in industries.

scholarly journals Identification of Active Site Residues of the Siderophore Synthesis Enzyme PvdF and Evidence for Interaction of PvdF with a Substrate-Providing Enzyme

2021 ◽  
Vol 22 (4) ◽  
pp. 2211
Author(s):  
Priya Philem ◽  
Torsten Kleffmann ◽  
Sinan Gai ◽  
Bill C. Hawkins ◽  
Sigurd M. Wilbanks ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Active Site ◽  
Opportunistic Pathogen ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Active Site Residues ◽  
Infection Models ◽  
Key Factor

The problematic opportunistic pathogen Pseudomonas aeruginosa secretes a siderophore, pyoverdine. Pyoverdine scavenges iron needed by the bacteria for growth and for pathogenicity in a range of different infection models. PvdF, a hydroxyornithine transformylase enzyme, is essential for pyoverdine synthesis, catalysing synthesis of formylhydroxyornithine (fOHOrn) that forms part of the pyoverdine molecule and provides iron-chelating hydroxamate ligands. Using a mass spectrometry assay, we confirm that purified PvdF catalyses synthesis of fOHOrn from hydroxyornithine and formyltetrahydrofolate substrates. Site directed mutagenesis was carried out to investigate amino acid residues predicted to be required for enzymatic activity. Enzyme variants were assayed for activity in vitro and also in vivo, through measuring their ability to restore pyoverdine production to a pvdF mutant strain. Variants at two putative catalytic residues N168 and H170 greatly reduced enzymatic activity in vivo though did not abolish activity in vitro. Change of a third residue D229 abolished activity both in vivo and in vitro. A change predicted to block entry of N10-formyltetrahydrofolate (fTHF) to the active site also abolished activity both in vitro and in vivo. A co-purification assay showed that PvdF binds to an enzyme PvdA that catalyses synthesis of hydroxyornithine, with this interaction likely to increase the efficiency of fOHOrn synthesis. Our findings advance understanding of how P. aeruginosa synthesises pyoverdine, a key factor in host–pathogen interactions.

scholarly journals Structural insights on the effects of mutation of a charged binding pocket residue on phosphopeptide binding to 14-3-3 ζ Protein

2021 ◽  
Author(s):  
Sreevidya T S ◽  
Somavally Dalvi ◽  
Prasanna Venkatraman ◽  
Satyavani Vemparala
Keyword(s):  
Electrostatic Interactions ◽  
Structural Changes ◽  
Structural Integrity ◽  
Peptide Binding ◽  
Limited Proteolysis ◽  
Binding Pocket ◽  
Mutant Protein ◽  
Amino Acid Residues ◽  
Salt Bridges ◽  
Dynamics Simulations

Mutation of an invariant aspartate residue in the binding pocket of 14-3-3ζ isoform to alanine dramatically reduced phosphopeptide binding and induced opening of the binding pocket. Here we use extensive molecular dynamics simulations to understand the role of D124 residue in ligand binding. The simulations show that in the absence of phosphopeptide, the D124A mutation leads to binding pocket reorganization including widening up of the binding pocket at the major groove and repositioning of N173, a key residue that interacts with the main chain of phosphopeptide. These structural changes would interfere with the efficient binding of the peptide, corroborating the experimental observations. Both gain and loss of electrostatic interactions in the form of salt bridges strongly indicate a rearrangement of the network of interactions within the binding pocket. Limited proteolysis coupled mass spectrometry (lip-MS) of the apo and holo forms of WT and mutant protein shows a peptide binding helix otherwise buried in the WT protein was particularly accessible to trypsin in the apo form of the mutant protein and the region was mapped to 158-186 amino acid residues of 14-3-3ζ. These results further confirm the dynamic nature of D124A mutant. Unlike other basic residues, the invariant D124 facilitates peptide binding by maintaining the geometry of interacting residues and by enforcing the structural integrity of amphipathic pocket.

scholarly journals Support for a three-dimensional structure predicting a Cys-Glu-Lys catalytic triad for Pseudomonas aeruginosa amidase comes from site-directed mutagenesis and mutations altering substrate specificity

2002 ◽  
Vol 365 (3) ◽  
pp. 731-738 ◽  
Author(s):  
Carlos NOVO ◽  
Sebastien FARNAUD ◽  
Renée TATA ◽  
Alda CLEMENTE ◽  
Paul R. BROWN
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Substrate Specificity ◽  
Catalytic Mechanism ◽  
Three Dimensional ◽  
Data Bank ◽  
Catalytic Triad ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Three Dimensional Models

The aliphatic amidase from Pseudomonas aeruginosa belongs to the nitrilase superfamily, and Cys166 is the nucleophile of the catalytic mechanism. A model of amidase was built by comparative modelling using the crystal structure of the worm nitrilase—fragile histidine triad fusion protein (NitFhit; Protein Data Bank accession number 1EMS) as a template. The amidase model predicted a catalytic triad (Cys-Glu-Lys) situated at the bottom of a pocket and identical with the presumptive catalytic triad of NitFhit. Three-dimensional models for other amidases belonging to the nitrilase superfamily also predicted Cys-Glu-Lys catalytic triads. Support for the structure for the P. aeruginosa amidase came from site-direct mutagenesis and from the locations of amino acid residues that altered substrate specificity or binding when mutated.

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