Biochemical and biophysical characterization of biosynthetic arginine decarboxylase from Thermus thermophilus

Abstract

The biosynthetic arginine decarboxylase in Thermus thermophilus is responsible for producing spermidine, a polyamine with numerous biological applications in humans. The arginine decarboxylase has significant applications in biotechnology industries, suggesting the need to evaluate its biochemical and biophysical characteristics at the molecular level. In this study, both in vitro and in silico methods were employed to investigate the structural and functional behavior of the arginine decarboxylase protein. In in vitro, MALDI-TOF, size exclusion, and assay studies were performed to examine the nature and activity of the protein. The MALDI-TOF analysis confirmed the purified protein as biosynthetic arginine decarboxylase. The assay results revealed that the Pyridoxal 5’-Phosphate (PLP) cofactor plays a crucial role in enhancing enzyme activity by producing agmatine (a by-product of spermidine). Further, optimum enzyme activity was observed at 50 °C, suggesting the extremophilic nature of the enzyme. Unlike other proteins, this enzyme displayed optimal activity at both acidic and basic pH, demonstrating its sensitivity to pH changes. Furthermore, the addition of divalent ions like Mg ²⁺ increased the rate of reaction. In in silico, structure modeling, and comparative molecular dynamics simulation studies were used to assess the protein stability and behavior at different pH and temperature conditions. The findings of this study could be applied to improve enzyme production in the industry.

Communicated by Ramaswamy H. Sarma

Keywords:

• Thermus thermophilus
• biosynthetic arginine decarboxylase
• MALDI-TOF
• Activity assay
• molecular dynamics simulation

Authors contribution

VM, KA, HN have designed the research. VM and KA conducted the in vitro experiments and VM, HN and KA have done in silico analysis. KA and MJ have wrote the paper. All the authors have read and approved the manuscript.

Data availability statement

Despite the reason of sensitivity, the data supporting this study are not openly, but are available upon reasonable request to the corresponding author.

Acknowledgment

JJ acknowledges DST INDO-TAIWAN (GITA/DST/TWN/P-86/2019), Department of Biotechnology- Bioinformatics Centre (BIC)-No.BT/PR40154/BTIS/137/34/2021, DBT-NNP N0.BT/PR40156/BTIS/137/54/2023, TANSCHE (RGP/2019-20/ALU/HECP-0049dated:27/04/2021), DST- Fund for Improvement of S&T Infrastructure (FIST) (SR/FST/LSI-667/2016) (C), and DST-Promotion of University Research and Scientific Excellence (PURSE phase II) (No. SR/PURSE Phase 2/38(G), 2017), MHRD-RUSA 2.0, New Delhi (F.24e51/2014-U, Policy (TNMulti-Gen). VM thank ICMR for providing financial assistance through ICMR/SRF Fellowship (BMI/11(103)/2022).

Disclosure statement

The authors declare no conflict of interest.

Additional information

Funding

JJ acknowledges DST INDO-TAIWAN (GITA/DST/TWN/P-86/2019), Department of Biotechnology- Bioinformatics Centre (BIC)-No.BT/PR40154/BTIS/137/34/2021, DBT-NNP N0.BT/PR40156/BTIS/137/54/2023, TANSCHE (RGP/2019-20/ALU/HECP-0049 dated:27/04/2021), DST- Fund for Improvement of S&T Infrastructure (FIST) (SR/FST/LSI-667/2016) (C), and DST-Promotion of University Research and Scientific Excellence (PURSE phase II) (No. SR/PURSE Phase 2/38(G), 2017), MHRD-RUSA 2.0, New Delhi (F.24e51/2014-U, Policy (TNMulti-Gen). VM thank ICMR for providing financial assistance through ICMR/SRF Fellowship (BMI/11(103)/2022).