scholarly journals Engineering of Saccharomyces pastorianus Old Yellow Enzyme 1 for the Synthesis of Pharmacologically Active (S)-Profen Derivatives

2020 ◽  
Author(s):  
Guigao Liu ◽  
Shang Li ◽  
Qinghua Shi ◽  
Hengyu Li ◽  
Jiyang Guo ◽  
...  
Keyword(s):  
Asymmetric Reduction ◽  
Methyl Esters ◽  
Pichia Stipitis ◽  
Site Directed Mutagenesis ◽  
Old Yellow Enzyme ◽  
Saccharomyces Pastorianus ◽  
Mutation Strategy ◽  
Arylpropionic Acid ◽  
Pharmacologically Active ◽  
Key Residues

<a>2-Arylpropionic acid </a><a>derivatives</a>, such as ibuprofen, constitute an important group of non-steroidal anti-inflammatory drugs (NSAIDs). Biocatalytic asymmetric reduction of<a> 2-arylacrylic acid</a> derivatives by ene reductases (EREDs) is a valuable approach for synthesis of these derivatives. However, previous bioreduction of <a>2-arylacrylic acid derivatives</a> by either ERED wild-types or variants resulted solely in nonpharmacological (<i>R</i>)-enantiomers as the products. <a></a><a>Here, </a>we present the engineering of <i>Saccharomyces pastorianus</i> old yellow enzyme 1 (OYE1) into (<i>S</i>)-stereoselective enzymes, which afford pharmacologically active (<i>S</i>)-profen derivatives. By structural comparison of substrate recognition in related EREDs and analysis of non-covalent contacts in the pro-<i>S</i> model of OYE1, the key residues of OYE1 that switch its stereoselectivity to an (<i>S</i>)-stereopreference were identified. Systematic site-directed mutagenesis screening at these positions successfully provided the (<i>S</i>)-stereoselective OYE1 variants, which catalyzed stereoselective bioreduction of various profen precursors to afford pharmacologically active (<i>S</i>)-derivatives including (<i>S</i>)-ibuprofen and (<i>S</i>)-naproxen methyl esters with up to >99% <i>ee</i> values. <a>Moreover, the key residues and mutation strategy obtained from OYE1 </a>could be further transferred to OYE 2.6 (from <i>Pichia stipitis</i>) and KnOYE1 (from <i>Kazachstania naganishii</i>) to create the (<i>S</i>)-stereoselective EREDs. Our results may provide a generalizable strategy for stereocontrol of OYEs and set the basis for biocatalytic production of (<i>S</i>)-profens.

scholarly journals Engineering of Saccharomyces pastorianus Old Yellow Enzyme 1 for the Synthesis of Pharmacologically Active (S)-Profen Derivatives

2020 ◽  
Author(s):  
Guigao Liu ◽  
Shang Li ◽  
Qinghua Shi ◽  
Hengyu Li ◽  
Jiyang Guo ◽  
...  
Keyword(s):  
Asymmetric Reduction ◽  
Methyl Esters ◽  
Pichia Stipitis ◽  
Site Directed Mutagenesis ◽  
Old Yellow Enzyme ◽  
Saccharomyces Pastorianus ◽  
Mutation Strategy ◽  
Arylpropionic Acid ◽  
Pharmacologically Active ◽  
Key Residues

<a>2-Arylpropionic acid </a><a>derivatives</a>, such as ibuprofen, constitute an important group of non-steroidal anti-inflammatory drugs (NSAIDs). Biocatalytic asymmetric reduction of<a> 2-arylacrylic acid</a> derivatives by ene reductases (EREDs) is a valuable approach for synthesis of these derivatives. However, previous bioreduction of <a>2-arylacrylic acid derivatives</a> by either ERED wild-types or variants resulted solely in nonpharmacological (<i>R</i>)-enantiomers as the products. <a></a><a>Here, </a>we present the engineering of <i>Saccharomyces pastorianus</i> old yellow enzyme 1 (OYE1) into (<i>S</i>)-stereoselective enzymes, which afford pharmacologically active (<i>S</i>)-profen derivatives. By structural comparison of substrate recognition in related EREDs and analysis of non-covalent contacts in the pro-<i>S</i> model of OYE1, the key residues of OYE1 that switch its stereoselectivity to an (<i>S</i>)-stereopreference were identified. Systematic site-directed mutagenesis screening at these positions successfully provided the (<i>S</i>)-stereoselective OYE1 variants, which catalyzed stereoselective bioreduction of various profen precursors to afford pharmacologically active (<i>S</i>)-derivatives including (<i>S</i>)-ibuprofen and (<i>S</i>)-naproxen methyl esters with up to >99% <i>ee</i> values. <a>Moreover, the key residues and mutation strategy obtained from OYE1 </a>could be further transferred to OYE 2.6 (from <i>Pichia stipitis</i>) and KnOYE1 (from <i>Kazachstania naganishii</i>) to create the (<i>S</i>)-stereoselective EREDs. Our results may provide a generalizable strategy for stereocontrol of OYEs and set the basis for biocatalytic production of (<i>S</i>)-profens.

scholarly journals Engineering of Saccharomyces pastorianus old yellow enzyme 1 for the synthesis of pharmacologically active (S)-profen derivatives

2021 ◽  
Vol 507 ◽  
pp. 111568
Author(s):  
Guigao Liu ◽  
Shang Li ◽  
Qinghua Shi ◽  
Hengyu Li ◽  
Jiyang Guo ◽  
...  
Keyword(s):  
Old Yellow Enzyme ◽  
Saccharomyces Pastorianus ◽  
Pharmacologically Active

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.

Cel48A fromThermobifida fusca: Structure and site directed mutagenesis of key residues

2013 ◽  
Vol 111 (4) ◽  
pp. 664-673 ◽  
Author(s):  
Maxim Kostylev ◽  
Markus Alahuhta ◽  
Mo Chen ◽  
Roman Brunecky ◽  
Michael E. Himmel ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Key Residues

Structure analysis and site‐directed mutagenesis of defined key residues and motives for pilus‐related sortase C1 in group B Streptococcus

2011 ◽  
Vol 25 (6) ◽  
pp. 1874-1886 ◽  
Author(s):  
Roberta Cozzi ◽  
Enrico Malito ◽  
Annalisa Nuccitelli ◽  
Mariapina D'Onofrio ◽  
Manuele Martinelli ◽  
...  
Keyword(s):  
Structure Analysis ◽  
Group B Streptococcus ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Group B ◽  
Key Residues

scholarly journals Location of the Permeation Pathway in the Recombinant Type 1 Inositol 1,4,5-Trisphosphate Receptor

1999 ◽  
Vol 114 (2) ◽  
pp. 243-250 ◽  
Author(s):  
Josefina Ramos-Franco ◽  
Daniel Galvan ◽  
Gregory A. Mignery ◽  
Michael Fill
Keyword(s):  
Lipid Bilayers ◽  
Mammalian Cells ◽  
Single Channel ◽  
Site Directed Mutagenesis ◽  
Recombinant Type ◽  
Mutation Strategy ◽  
Inositol 1,4,5 Trisphosphate ◽  
Trisphosphate Receptor ◽  
Permeation Properties

The inositol 1,4,5-trisphosphate receptor (InsP3R) forms ligand-regulated intracellular Ca2+ release channels in the endoplasmic reticulum of all mammalian cells. The InsP3R has been suggested to have six transmembrane regions (TMRs) near its carboxyl terminus. A TMR-deletion mutation strategy was applied to define the location of the InsP3R pore. Mutant InsP3Rs were expressed in COS-1 cells and single channel function was defined in planar lipid bilayers. Mutants having the fifth and sixth TMR (and the interceding lumenal loop), but missing all other TMRs, formed channels with permeation properties similar to wild-type channels (gCs = 284; gCa = 60 pS; PCa/PCs = 6.3). These mutant channels bound InsP3, but ligand occupancy did not regulate the constitutively open pore (Po &gt; 0.80). We propose that a region of 191 amino acids (including the fifth and sixth TMR, residues 2398–2589) near the COOH terminus of the protein forms the InsP3R pore. Further, we have produced a constitutively open InsP3R pore mutant that is ideal for future site-directed mutagenesis studies of the structure–function relationships that define Ca2+ permeation through the InsP3R channel.

scholarly journals Protein engineering of the 2-haloacid halidohydrolase IVa from Pseudomonas cepacia MBA4

1993 ◽  
Vol 292 (1) ◽  
pp. 69-74 ◽  
Author(s):  
W Asmara ◽  
U Murdiyatmo ◽  
A J Baines ◽  
A T Bull ◽  
D J Hardman
Keyword(s):  
Amino Acid ◽  
Rational Design ◽  
Catalytic Mechanism ◽  
Protonated Form ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Pseudomonas Cepacia ◽  
Amino Acid Residues ◽  
Substrate Complex ◽  
Key Residues

The chemical modification of L-2-haloacid halidohydrolase IVa (Hdl IVa), originally identified in Pseudomonas cepacia MBA4, produced as a recombinant protein in Escherichia coli DH5 alpha, led to the identification of histidine and arginine as amino acid residues likely to play a part in the catalytic mechanism of the enzyme. These results, together with DNA sequence and analyses [Murdiyatmo, Asmara, Baines, Bull and Hardman (1992) Biochem. J. 284, 87-93] provided the basis for the rational design of a series of random- and site-directed-mutagenesis experiments of the Hdl IVa structural gene (hdl IVa). Subsequent apparent kinetic analyses of purified mutant enzymes identified His-20 and Arg-42 as the key residues in the activity of this halidohydrolase. It is also proposed that Asp-18 is implicated in the functioning of the enzyme, possibly by positioning the correct tautomer of His-20 for catalysis in the enzyme-substrate complex and stabilizing the protonated form of His-20 in the transition-state complex. Comparison of conserved amino acid sequences between the Hdl IVa and other halidohydrolases suggests that L-2-haloacid halidohydrolases contain conserved amino acid sequences that are not found in halidohydrolases active towards both D- and L-2-monochloropropionate.

scholarly journals Mycobacterial OtsA structures unveil substrate preference mechanism and allosteric regulation by 2-oxoglutarate and 2-phosphoglycerate

2019 ◽  
Author(s):  
Vítor Mendes ◽  
Marta Acebrón-García-de-Eulate ◽  
Nupur Verma ◽  
Michal Blaszczyk ◽  
Márcio V. B. Dias ◽  
...  
Keyword(s):  
Cell Wall ◽  
Feedback Inhibition ◽  
Structural Information ◽  
Site Directed Mutagenesis ◽  
Substrate Preference ◽  
Directed Mutagenesis ◽  
Allosteric Site ◽  
Trehalose Biosynthesis ◽  
Key Residues ◽  
Allosteric Regulators

AbstractTrehalose is an essential disaccharide for mycobacteria and a key constituent of several cell wall glycolipids with fundamental roles in pathogenesis. Mycobacteria possess two pathways for trehalose biosynthesis. However, only the OtsAB pathway was found to be essential inM. tuberculosis, with marked growth and virulence defects of OtsA mutants and strict essentiality of OtsB2. Herein, we report the first mycobacterial OtsA structures fromM. thermoresistibilein both apo and ligand-bound forms. Structural information reveals three key residues in the mechanism of substrate preference that were further confirmed by site-directed mutagenesis. Additionally, we identify 2-oxoglutarate and 2-phosphoglycerate as allosteric regulators of OtsA. The structural analysis in this work strongly contributed to define the mechanisms for feedback inhibition, show different conformational states of the enzyme and map a new allosteric site.

scholarly journals Structure–function analysis of the nsp14 N7–guanine methyltransferase reveals an essential role in Betacoronavirus replication

2021 ◽  
Vol 118 (49) ◽  
pp. e2108709118
Author(s):  
Natacha S. Ogando ◽  
Priscila El Kazzi ◽  
Jessika C. Zevenhoven-Dobbe ◽  
Brenda W. Bontes ◽  
Alice Decombe ◽  
...  
Keyword(s):  
Function Analysis ◽  
Nonstructural Protein ◽  
Site Directed Mutagenesis ◽  
Messenger Rnas ◽  
Mtase Activity ◽  
Mrna Capping ◽  
Activity Spectrum ◽  
Immune Sensing ◽  
Key Residues

As coronaviruses (CoVs) replicate in the host cell cytoplasm, they rely on their own capping machinery to ensure the efficient translation of their messenger RNAs (mRNAs), protect them from degradation by cellular 5′ exoribonucleases (ExoNs), and escape innate immune sensing. The CoV nonstructural protein 14 (nsp14) is a bifunctional replicase subunit harboring an N-terminal 3′-to-5′ ExoN domain and a C-terminal (N7-guanine)–methyltransferase (N7-MTase) domain that is presumably involved in viral mRNA capping. Here, we aimed to integrate structural, biochemical, and virological data to assess the importance of conserved N7-MTase residues for nsp14’s enzymatic activities and virus viability. We revisited the crystal structure of severe acute respiratory syndrome (SARS)–CoV nsp14 to perform an in silico comparative analysis between betacoronaviruses. We identified several residues likely involved in the formation of the N7-MTase catalytic pocket, which presents a fold distinct from the Rossmann fold observed in most known MTases. Next, for SARS-CoV and Middle East respiratory syndrome CoV, site-directed mutagenesis of selected residues was used to assess their importance for in vitro enzymatic activity. Most of the engineered mutations abolished N7-MTase activity, while not affecting nsp14-ExoN activity. Upon reverse engineering of these mutations into different betacoronavirus genomes, we identified two substitutions (R310A and F426A in SARS-CoV nsp14) abrogating virus viability and one mutation (H424A) yielding a crippled phenotype across all viruses tested. Our results identify the N7-MTase as a critical enzyme for betacoronavirus replication and define key residues of its catalytic pocket that can be targeted to design inhibitors with a potential pan-coronaviral activity spectrum.

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