Production and Characterization of Bifunctional Enzymes. Domain Swapping To Produce New Bifunctional Enzymes in the Aspartate Pathway†

Biochemistry ◽  
2002 ◽  
Vol 41 (11) ◽  
pp. 3720-3725 ◽  
Author(s):  
Cindy L. James ◽  
Ronald E. Viola
Keyword(s):  
Domain Swapping ◽  
Bifunctional Enzymes ◽  
Aspartate Pathway

scholarly journals Biochemical Characterization of a Bifunctional Enzyme Constructed by the Fusion of a Glucuronan Lyase and a Chitinase from Trichoderma sp.

Life ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 234
Author(s):  
Zeineb Baklouti ◽  
Cédric Delattre ◽  
Guillaume Pierre ◽  
Christine Gardarin ◽  
Slim Abdelkafi ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Biochemical Properties ◽  
Cell Wall Degrading Enzymes ◽  
Promising Candidate ◽  
Structural Modifications ◽  
Chimeric Enzymes ◽  
Bifunctional Enzymes ◽  
Trichoderma Sp ◽  
Ph Variations

Bifunctional enzymes created by the fusion of a glucuronan lyase (TrGL) and a chitinase (ThCHIT42) from Trichoderma sp. have been constructed with the aim to validate a proof of concept regarding the potential of the chimera lyase/hydrolase by analyzing the functionality and the efficiency of the chimeric constructions compared to parental enzymes. All the chimeric enzymes, including or nor linker (GGGGS), were shown functional with activities equivalent or higher to native enzymes. The velocity of glucuronan lyase was considerably increased for chimeras, and may involved structural modifications at the active site. The fusion has induced a slightly decrease of the thermostability of glucuronan lyase, without modifying its catalytic activity regarding pH variations ranging from 5 to 8. The biochemical properties of chitinase seemed to be more disparate between the different fusion constructions suggesting an impact of the linkers or structural interactions with the linked glucuronan lyase. The chimeric enzymes displayed a decreased stability to temperature and pH variations, compared to parental one. Overall, TrGL-ThCHIT42 offered the better compromise in terms of biochemical stability and enhanced activity, and could be a promising candidate for further experiments in the field of fungi Cell Wall-Degrading Enzymes (CWDEs).

A cautionary tale of structure-guided inhibitor development against an essential enzyme in the aspartate-biosynthetic pathway

2014 ◽  
Vol 70 (12) ◽  
pp. 3244-3252 ◽  
Author(s):  
Alexander G. Pavlovsky ◽  
Bharani Thangavelu ◽  
Pravin Bhansali ◽  
Ronald E. Viola
Keyword(s):  
Active Site ◽  
Enzyme Inhibitor ◽  
Bacterial Survival ◽  
Cautionary Tale ◽  
Lead Compounds ◽  
Semialdehyde Dehydrogenase ◽  
Fragment Library ◽  
Aspartate Pathway ◽  
Essential Enzyme

The aspartate pathway is essential for the production of the amino acids required for protein synthesis and of the metabolites needed in bacterial development. This pathway also leads to the production of several classes of quorum-sensing molecules that can trigger virulence in certain microorganisms. The second enzyme in this pathway, aspartate β-semialdehyde dehydrogenase (ASADH), is absolutely required for bacterial survival and has been targeted for the design of selective inhibitors. Fragment-library screening has identified a new set of inhibitors that, while they do not resemble the substrates for this reaction, have been shown to bind at the active site of ASADH. Structure-guided development of these lead compounds has produced moderate inhibitors of the target enzyme, with some selectivity observed between the Gram-negative and Gram-positive orthologs of ASADH. However, many of these inhibitor analogs and derivatives have not yet achieved the expected enhanced affinity. Structural characterization of these enzyme–inhibitor complexes has provided detailed explanations for the barriers that interfere with optimal binding. Despite binding in the same active-site region, significant changes are observed in the orientation of these bound inhibitors that are caused by relatively modest structural alterations. Taken together, these studies present a cautionary tale for issues that can arise in the systematic approach to the modification of lead compounds that are being used to develop potent inhibitors.

scholarly journals Structural and functional characterization of engineered bifunctional fusion proteins of CD39 and CD73 ectonucleotidases

2021 ◽  
Vol 320 (1) ◽  
pp. C15-C29 ◽  
Author(s):  
Elizabeth H. Zhong ◽  
Carola Ledderose ◽  
Paola De Andrade Mello ◽  
Keiichi Enjyoji ◽  
Justin Mark Lunderberg ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
High Performance ◽  
Inflammatory Diseases ◽  
Functional Characterization ◽  
Bifunctional Enzymes ◽  
Human Acid ◽  
Inflammatory Processes ◽  
Single Polypeptide

Extracellular diphosphate and triphosphate nucleotides are released from activated or injured cells to trigger vascular and immune P2 purinergic receptors, provoking inflammation and vascular thrombosis. These metabokines are scavenged by ectonucleoside triphosphate diphosphohydrolase-1 (E-NTPDase1 or CD39). Further degradation of the monophosphate nucleoside end products occurs by surface ecto-5′-nucleotidase (NMPase) or CD73. These ectoenzymatic processes work in tandem to promote adenosinergic responses, which are immunosuppressive and antithrombotic. These homeostatic ectoenzymatic mechanisms are lost in the setting of oxidative stress, which exacerbates inflammatory processes. We have engineered bifunctional enzymes made up from ectodomains (ECDs) of CD39 and CD73 within a single polypeptide. Human alkaline phosphatase-ectodomain (ALP-ECD) and human acid phosphatase-ectodomain (HAP-ECD) fusion proteins were also generated, characterized, and compared with these CD39-ECD, CD73-ECD, and bifunctional fusion proteins. Through the application of colorimetrical functional assays and high-performance liquid chromatography kinetic assays, we demonstrate that the bifunctional ectoenzymes express high levels of CD39-like NTPDase activity and CD73-like NMPase activity. Chimeric CD39-CD73-ECD proteins were superior in converting triphosphate and diphosphate nucleotides into nucleosides when compared with ALP-ECD and HAP-ECD. We also note a pH sensitivity difference between the bifunctional fusion proteins and parental fusions, as well as ectoenzymatic property distinctions. Intriguingly, these innovative reagents decreased platelet activation to exogenous agonists in vitro. We propose that these chimeric fusion proteins could serve as therapeutic agents in inflammatory diseases, acting to scavenge proinflammatory ATP and also generate anti-inflammatory adenosine.

Morphological Characterization of Mycobacteriophage R1

1974 ◽  
Vol 32 ◽  
pp. 254-255
Author(s):  
B. L. Soloff ◽  
T. A. Rado
Keyword(s):  
Carbon Source ◽  
Stationary Phase ◽  
Growth Phase ◽  
Minimal Medium ◽  
Morphological Characterization ◽  
Logarithmic Growth Phase ◽  
Complete Lysis ◽  
Lysogenic Culture ◽  
Logarithmic Growth

Mycobacteriophage R1 was originally isolated from a lysogenic culture of M. butyricum. The virus was propagated on a leucine-requiring derivative of M. smegmatis, 607 leu−, isolated by nitrosoguanidine mutagenesis of typestrain ATCC 607. Growth was accomplished in a minimal medium containing glycerol and glucose as carbon source and enriched by the addition of 80 μg/ ml L-leucine. Bacteria in early logarithmic growth phase were infected with virus at a multiplicity of 5, and incubated with aeration for 8 hours. The partially lysed suspension was diluted 1:10 in growth medium and incubated for a further 8 hours. This permitted stationary phase cells to re-enter logarithmic growth and resulted in complete lysis of the culture.

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