Acta Scientific Medical Sciences (ISSN: 2582-0931)

Research Article Volume 4 Issue 2

A Novel Mechanism for the Target-Based Inhibition of Cdc14 Phosphatase Activity in F. Oxysporum Through Analysis of Catalytic Specificity and Enzyme Kinetics

Sia Cho*, Darrell Fan* and Harry G. Heiberger*

SSP in Biochemistry, Department of Natural Sciences, University of California San Diego, La Jolla, USA

*Corresponding Author: Sia Cho, Darrell Fan and Harry G. Heiberger, SSP in Biochemistry, Department of Natural Sciences, University of California San Diego, La Jolla, USA.

Received: December 31, 2019; Published: January 22, 2020

×

Abstract

  The prevalence of fungal pathogenicity in the agricultural industry poses enormous hazards toward the world population. Preventative measures have thus far been unsuccessful, underscoring the need for new fungicide research. Cdc14 is a protein tyrosine phosphatase (PTP) that is critical for cell cycle regulation and virulence in a variety of fungal species. In this study, we investigate the enzymatic properties of Cdc14 in Fusarium oxysporum, a soilborne plant pathogen that causes a pernicious vascular wilt disease called Fusarium Wilt. By analyzing the catalytic mechanisms of FoCdc14, we identified optimal characteristics for targeted inhibitor development. Our enzyme underwent multiple stages of biochemical and computational analyses to extract data on its specificity, including inhibitor screenings both In vitro and In silico. IC50 values and reversibility mechanisms were also tested using para-nitrophenyl phosphate (pNPP) to determine optimal binding properties for multiple peptide substrates. The integration of data from all tested parameters was then used to identify a baseline inhibitor for further optimization. By applying modifications, we were able to create an optimized inhibitor with a binding affinity increase of nearly 150%. These modifications included the addition of functional groups that maximized intermolecular interactions at the FoCdc14 binding site around residues Cys337 and Arg343. Throughout this process, we confirmed the conservation of FoCdc14’s enzyme kinetics and catalytic specificity to other Cdc14 orthologs. Because of such conservation, this novel inhibitor holds great potential to counteract virulence in F. oxysporum and other fungi, possibly paving the path for potent new fungicides. 

 

Keywords: F. Oxysporum; Cdc14; Inhibition Mechanism; Enzyme Kinetics; Molecular Docking; Substrate Specificity; Protein-Drug Interaction; Comparative Modeling; Ligand Optimization

×

References

  1. Almeida F., et al. “The Still Underestimated Problem of Fungal Diseases Worldwide”. Frontiers in Microbiology (2019): 10. 
  2. World Food Programme. Hunger Statistics (2018). 
  3. Hahn M. “The rising threat of fungicide resistance in plant pathogenic fungi: Botrytis as a case study”. Journal of Chemical Biology 7.4 (2014): 133-141. 
  4. Michielse CB and Rep M. “Pathogen profile update: Fusarium oxysporum”. Molecular Plant Pathology 10 (2009): 311-324. 
  5. Husaini AM., et al. “Host–Pathogen Interaction in Fusarium oxysporum Infections: Where Do We Stand?” Molecular Plant-Microbe Interactions 31.9 (2018): 889-898. 
  6. Gordon TR. “Fusarium oxysporum and the Fusarium Wilt Syndrome”. Annual Review of Phytopathology 55 (2017): 23-39. 
  7. Pérez-Vicente., et al. “Technical Manual Prevention and diagnostic of Fusarium Wilt (Panama disease) of banana caused by Fusarium oxysporum f. sp”. cubense Tropical Race 4 (TR4) (2014). 
  8. Nucci M and Anaissie E. “Fusarium infections in immunocompromised patients”. Clinical Microbiology Reviews 20.4 (2007): 695-704. 
  9. Jain P., et al. “Current Status of Fusarium Infection in Human and Animal”. Asian Journal of Animal and Veterinary Advances 6.3 (2011): 201-227. 
  10. Gray CH., et al. “The structure of the cell cycle protein Cdc14 reveals a proline-directed protein phosphatase”. The EMBO Journal 22.14 (2003): 3524-3535. 
  11. Kerk D., et al. “Evolutionary Radiation Pattern of Novel Protein Phosphatases Revealed by Analysis of Protein Data from the Completely Sequenced Genomes of Humans, Green Algae, and Higher Plants”. Plant Physiology 146.2 (2007): 351-367. 
  12. Bremmer SC., et al. “Cdc14 phosphatases preferentially dephosphorylate a subset of cyclin-dependent kinase (Cdk) sites containing phosphoserine”. The Journal of Biological Chemistry 287.3 (2012): 1662–1669. 
  13. Wang W., et al. “Kinetic and Mechanistic Studies of a Cell Cycle Protein Phosphatase Cdc14”. Journal of Biological Chemistry 279.29 (2004): 30459-30468. 
  14. Powers BL., et al. “A Substrate Trapping Method for Identification of Direct Cdc14 Phosphatase Targets”. Methods in Molecular Biology the Mitotic Exit Network (2016): 119-132. 
  15. Gray CH and Barford D. “Getting in the Ring: Proline-Directed Substrate Specificity in the Cell Cycle Proteins Cdc14 and CDK2-CyclinA3”. Cell Cycle 2.6 (2003): 500-502. 
×

Citation

Citation: Sia Cho, Darrell Fan and Harry G. Heiberger. “A Novel Mechanism for the Target-Based Inhibition of Cdc14 Phosphatase Activity in F. Oxysporum Through Analysis of Catalytic Specificity and Enzyme Kinetics". Acta Scientific Medical Sciences 4.2 (2020): 01-16.



Member In




News and Events


  • Certification for Review
    Acta Scientific certifies the Editors/reviewers for their review done towards the assigned articles of the respective journals.
  • Submission Timeline for March Issue
    The last date for submission of articles for regular Issues is February 29, 2020.
  • Publication Certificate
    Authors will be issued a "Publication Certificate" as a mark of appreciation for publishing their work.
  • Best Article of the Issue
    The Editors will elect one Best Article after each issue release. The authors of this article will be provided with a certificate of “Best Article of the Issue”.
  • Welcoming Article Submission
    Acta Scientific delightfully welcomes active researchers for submission of articles towards the upcoming issue of respective journals.
  • Contact US