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Construing recombinant ZFP160 from Aspergillus terreus as pterin deaminase enzyme
Sajitha Bijukumar1, Thandeeswaran Murugesan2, Anand Raj Dhanapal3
1Cancer Therapeutics Laboratory, Department of Microbial Biotechnology, Bharathiar University, Coimbatore, Tamil Nadu, India.
Abstract:
Pterin deaminase stands as a metalloenzyme and exhibits both antitumor and anticancer activities. Therefore, this study aimed to explore the molecular function of zinc finger protein-160 (zfp160) from Aspergillus terreus with its enzyme mechanism in detail. Subsequently, preliminary molecular docking studies on zfp160 from A. terreus were done. Next, the cloning and expression of zfp160 protein were carried out. Following, protein expression was induced and purified through nickel NTA column with imidazole gradient elution. Through the Mascot search engine tool, the expressed protein of MALDI-TOF was confirmed by 32 kDa bands of SDS-PAGE. Furthermore, its enzymatic characterization and biochemical categorization were also explored. The optimum conditions for enzyme were determined to be pH 8, temperature 35°C, km 50 μm with folic acid as substrate, and Vmax of 24.16 (IU/mL). Further, in silico analysis tried to explore the interactions and binding affinity of various substrates to the modeled pterin deaminase from A. terreus. Our results revealed the binding mode of different substrate molecules with pterin deaminase using the approximate scoring functions that possibly correlate with actual experimental binding affinities. Following this, molecular dynamic simulations provided the in-depth knowledge on deciphering functional mechanisms of pterin deaminase over other drugs.
Insights
This study details the molecular function and enzyme mechanism of pterin deaminase from Aspergillus terreus, revealing its potential anticancer properties. Research explored protein expression, purification, and substrate interactions for therapeutic applications.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Pterin deaminase is a metalloenzyme with known antitumor and anticancer activities.
- Understanding its molecular function is crucial for developing novel therapeutic agents.
Purpose of the Study:
- To investigate the molecular function and enzyme mechanism of zinc finger protein-160 (zfp160) from Aspergillus terreus.
- To explore the interactions and binding affinities of substrates with pterin deaminase.
- To provide insights into the functional mechanisms of pterin deaminase for potential drug development.
Main Methods:
- Cloning, expression, and purification of zfp160 protein.
- Enzymatic characterization including determination of optimal pH, temperature, Km, and Vmax.
- In silico analysis, including molecular docking and molecular dynamic simulations.
Main Results:
- Successfully cloned, expressed, and purified zfp160 protein (32 kDa).
- Determined optimal enzymatic conditions: pH 8, 35°C, Km 50 μm (folic acid substrate), Vmax 24.16 (IU/mL).
- In silico studies revealed substrate binding modes and affinities, correlating with experimental data.
Conclusions:
- The study elucidates the detailed molecular function and biochemical properties of Aspergillus terreus pterin deaminase.
- Findings provide a foundation for understanding its anticancer potential and developing it as a therapeutic target.
- In silico analyses offer valuable insights into enzyme-substrate interactions and mechanism of action.
