scholarly journals Cell-Free Biosynthesis to Evaluate Lasso Peptide Formation and Enzyme–Substrate Tolerance

2021 ◽  
Author(s):  
Yuanyuan Si ◽  
Ashley M. Kretsch ◽  
Laura M. Daigh ◽  
Mark J. Burk ◽  
Douglas A. Mitchell
Keyword(s):  
Lasso Peptide ◽  
Enzyme Substrate ◽  
Peptide Formation ◽  
Substrate Tolerance

scholarly journals Cell-Free Biosynthesis to Evaluate Lasso Peptide Formation and Enzyme-Substrate Tolerance

2020 ◽  
Author(s):  
Yuanyuan Si ◽  
Ashley M. Kretsch ◽  
Laura M. Daigh ◽  
Mark J. Burk ◽  
Douglas A. Mitchell
Keyword(s):  
Genome Mining ◽  
Efficient Production ◽  
Surface Receptors ◽  
Lasso Peptide ◽  
Anticancer Properties ◽  
Lasso Peptides ◽  
Isolation And Characterization ◽  
Precursor Peptide ◽  
A Cell ◽  
Substrate Tolerance

AbstractLasso peptides are ribosomally synthesized and post-translationally modified peptide (RiPP) natural products that display a unique lariat-like structure. Owing to a rigid topology, lasso peptides are unusually stable towards heat and proteolytic degradation. Some lasso peptides have been shown to bind human cell-surface receptors and exhibit anticancer properties, while others display antibacterial or antiviral activities. Known lasso peptides are produced by bacteria and genome-mining studies indicate that lasso peptides are a relatively prevalent RiPP class; however, the discovery, isolation, and characterization of lasso peptides are constrained by the lack of an efficient production system. In this study, we employ a cell-free biosynthesis (CFB) strategy to address the longstanding challenges associated with lasso peptide production. We report the successful formation of a diverse array of lasso peptides that include known examples as well as a new predicted lasso peptide from Thermobifida halotolerans. We further demonstrate the utility of CFB to rapidly generate and characterize multisite precursor peptide variants in order to evaluate the substrate tolerance of the biosynthetic pathway. We show that the lasso-forming cyclase from the fusilassin pathway can produce millions of sequence-diverse lasso peptides via CFB with an extraordinary level of sequence permissiveness within the ring region of the lasso peptide. These data lay a firm foundation for the creation of large lasso peptide libraries using CFB to identify new variants with unique properties.

Cell-Specific Chemical Delivery Using a Selective Nitroreductase–Nitroaryl Pair

2018 ◽  
Author(s):  
Todd D. Gruber ◽  
Chithra Krishnamurthy ◽  
Jonathan B. Grimm ◽  
Michael R. Tadross ◽  
Laura M. Wysocki ◽  
...  
Keyword(s):  
Small Molecules ◽  
Mammalian Cells ◽  
Target Cells ◽  
Specific Chemical ◽  
E Coli ◽  
Enzyme Substrate ◽  
Cell Specificity ◽  
General Chemical ◽  
Increased Activity ◽  
Enzyme Variant

<p>The utility of<b> </b>small molecules to probe or perturb biological systems is limited by the lack of cell-specificity. ‘Masking’ the activity of small molecules using a general chemical modification and ‘unmasking’ it only within target cells could overcome this limitation. To this end, we have developed a selective enzyme–substrate pair consisting of engineered variants of <i>E. coli</i> nitroreductase (NTR) and a 2‑nitro-<i>N</i>-methylimidazolyl (NM) masking group. To discover and optimize this NTR–NM system, we synthesized a series of fluorogenic substrates containing different nitroaromatic masking groups, confirmed their stability in cells, and identified the best substrate for NTR. We then engineered the enzyme for improved activity in mammalian cells, ultimately yielding an enzyme variant (enhanced NTR, or eNTR) that possesses up to 100-fold increased activity over wild-type NTR. These improved NTR enzymes combined with the optimal NM masking group enable rapid, selective unmasking of dyes, indicators, and drugs to genetically defined populations of cells.</p>

The role of secondary alcoholic groups of D-galacturonan in its degradation with exo-D-galacturonanase

1980 ◽  
Vol 45 (2) ◽  
pp. 427-434 ◽  
Author(s):  
Kveta Heinrichová ◽  
Rudolf Kohn
Keyword(s):  
Acetyl Derivative ◽  
Competitive Inhibitor ◽  
Hydroxyl Groups ◽  
Pectic Acid ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
The Individual ◽  
Degradation Degree ◽  
Derivatives Of

The effect of exo-D-galacturonanase from carrot on O-acetyl derivatives of pectic acid of variousacetylation degree was studied. Substitution of hydroxyl groups at C(2) and C(3) of D-galactopyranuronic acid units influences the initial rate of degradation, degree of degradation and its maximum rate, the differences being found also in the time of limit degradations of the individual O-acetyl derivatives. Value of the apparent Michaelis constant increases with increase of substitution and value of Vmax changes. O-Acetyl derivatives act as a competitive inhibitor of degradation of D-galacturonan. The extent of the inhibition effect depends on the degree of substitution. The only product of enzymic reaction is D-galactopyranuronic acid, what indicates that no degradation of the terminal substituted unit of O-acetyl derivative of pectic acid takes place. Substitution of hydroxyl groups influences the affinity of the enzyme towards the modified substrate. The results let us presume that hydroxyl groups at C(2) and C(3) of galacturonic unit of pectic acid are essential for formation of the enzyme-substrate complex.

Mass transfer in heterogeneous system enzyme-substrate; Approximate analytical model for the case of liquid substrate-immobilized enzyme

1982 ◽  
Vol 47 (11) ◽  
pp. 3013-3018
Author(s):  
František Kaštánek ◽  
Jindřich Zahradník ◽  
Germanico Ocampo
Keyword(s):  
Mass Transfer ◽  
Heterogeneous System ◽  
Immobilized Enzyme ◽  
Enzymatic Reaction ◽  
Analytical Form ◽  
Approximate Model ◽  
Basic Type ◽  
Reaction Interface ◽  
Enzyme Substrate ◽  
Flow Intensity

Calculation procedure is suggested for flow intensity of substrate toward reaction interface of immobilized enzyme at simultaneous effect of enzymatic reaction and internal diffusion. The approximate model is presented in an analytical form for the basic type of Michaelis-Menten kinetics and for the case of inhibition in excess of substrate.

A new method for the in situ assay of α-glucosidase activity and inhibitor screening through an enzyme substrate-mediated DNA hybridization chain reaction

2019 ◽  
Vol 43 (24) ◽  
pp. 9458-9465
Author(s):  
Xiquan Yue ◽  
Lihong Su ◽  
Xu Chen ◽  
Junfeng Liu ◽  
Longpo Zheng ◽  
...  
Keyword(s):  
Small Molecule ◽  
Dna Hybridization ◽  
New Method ◽  
Hybridization Chain Reaction ◽  
Chain Reaction ◽  
Inhibitor Screening ◽  
Glucosidase Activity ◽  
Enzyme Substrate ◽  
In Situ Assay

The strategy is based on small molecule-mediated hybridization chain reaction.

scholarly journals Spontaneous Cleavages of a Heterologous Protein, the CenA Endoglucanase of Cellulomonas fimi, in Escherichia coli

2021 ◽  
Vol 14 ◽  
pp. 117863612110246
Author(s):  
Cheuk Yin Lai ◽  
Ka Lun Ng ◽  
Hao Wang ◽  
Chui Chi Lam ◽  
Wan Keung Raymond Wong
Keyword(s):  
Escherichia Coli ◽  
Cellulolytic Bacterium ◽  
Systematic Screening ◽  
Cellulomonas Fimi ◽  
E Coli ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Glycosylation Sites ◽  
Endogenous Proteins ◽  
Cellulose Binding

CenA is an endoglucanase secreted by the Gram-positive cellulolytic bacterium, Cellulomonas fimi, to the environment as a glycosylated protein. The role of glycosylation in CenA is unclear. However, it seems not crucial for functional activity and secretion since the unglycosylated counterpart, recombinant CenA (rCenA), is both bioactive and secretable in Escherichia coli. Using a systematic screening approach, we have demonstrated that rCenA is subjected to spontaneous cleavages (SC) in both the cytoplasm and culture medium of E. coli, under the influence of different environmental factors. The cleavages were found to occur in both the cellulose-binding (CellBD) and catalytic domains, with a notably higher occurring rate detected in the former than the latter. In CellBD, the cleavages were shown to occur close to potential N-linked glycosylation sites, suggesting that these sites might serve as ‘attributive tags’ for differentiating rCenA from endogenous proteins and the points of initiation of SC. It is hypothesized that glycosylation plays a crucial role in protecting CenA from SC when interacting with cellulose in the environment. Subsequent to hydrolysis, SC would ensure the dissociation of CenA from the enzyme-substrate complex. Thus, our findings may help elucidate the mechanisms of protein turnover and enzymatic cellulolysis.

scholarly journals Tuning a bi-enzymatic cascade reaction in Escherichia coli to facilitate NADPH regeneration for ε-caprolactone production

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Jinghui Xiong ◽  
Hefeng Chen ◽  
Ran Liu ◽  
Hao Yu ◽  
Min Zhuo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Regeneration System ◽  
Nadph Regeneration ◽  
Whole Cell ◽  
Cyclohexanone Monooxygenase ◽  
Ribosome Binding ◽  
Ribosome Binding Sites ◽  
Whole Cell Biocatalyst ◽  
Substrate Tolerance

Abstractε-Caprolactone is a monomer of poly(ε-caprolactone) which has been widely used in tissue engineering due to its biodegradability and biocompatibility. To meet the massive demand for this monomer, an efficient whole-cell biocatalytic approach was constructed to boost the ε-caprolactone production using cyclohexanol as substrate. Combining an alcohol dehydrogenase (ADH) with a cyclohexanone monooxygenase (CHMO) in Escherichia coli, a self-sufficient NADPH-cofactor regeneration system was obtained. Furthermore, some improved variants with the better substrate tolerance and higher catalytic ability to ε-caprolactone production were designed by regulating the ribosome binding sites. The best mutant strain exhibited an ε-caprolactone yield of 0.80 mol/mol using 60 mM cyclohexanol as substrate, while the starting strain only got a conversion of 0.38 mol/mol when 20 mM cyclohexanol was supplemented. The engineered whole-cell biocatalyst was used in four sequential batches to achieve a production of 126 mM ε-caprolactone with a high molar yield of 0.78 mol/mol.

scholarly journals Targeting of GLUT5 for Transporter-Mediated Drug-Delivery Is Contingent upon Substrate Hydrophilicity

2021 ◽  
Vol 22 (10) ◽  
pp. 5073
Author(s):  
Nazanin Nahrjou ◽  
Avik Ghosh ◽  
Marina Tanasova
Keyword(s):  
Cancer Cells ◽  
Normal Breast ◽  
Proof Of Concept ◽  
Drug Conjugates ◽  
High Fructose ◽  
Specific Cytotoxicity ◽  
Breast Cells ◽  
Substrate Tolerance ◽  
Bioactive Agents ◽  
Positive Breast Cancer

Specific link between high fructose uptake and cancer development and progression highlighted fructose transporters as potential means to achieve GLUT-mediated discrimination between normal and cancer cells. The gained expression of fructose-specific transporter GLUT5 in various cancers offers a possibility for developing cancer-specific imaging and bioactive agents. Herein, we explore the feasibility of delivering a bioactive agent through cancer-relevant fructose-specific transporter GLUT5. We employed specific targeting of GLUT5 by 2,5-anhydro-D-mannitol and investigated several drug conjugates for their ability to induce cancer-specific cytotoxicity. The proof-of-concept analysis was carried out for conjugates of chlorambucil (CLB) in GLUT5-positive breast cancer cells and normal breast cells. The cytotoxicity of conjugates was assessed over 24 h and 48 h, and significant dependence between cancer-selectivity and conjugate size was observed. The differences were found to relate to the loss of GLUT5-mediated uptake upon increased conjugate size and hydrophobicity. The findings provide information on the substrate tolerance of GLUT5 and highlight the importance of maintaining appropriate hydrophilicity for GLUT-mediated delivery.

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