Structural and functional characterization of mycobacterial 2,4-dienoyl-CoA reductase

Abstract

Tuberculosis (TB) remains one of the leading causes of death worldwide as an infectious disease. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a very significant pathogen within the genus Mycobacterium. The most concerning issue is that Mtb is becoming increasingly drug resistant, making the development of new drugs an urgent necessity. Lipids play a very crucial role in the pathogenesis and survival of Mtb. The enzyme 2,4-dienoyl-CoA reductase (DCR) is a very important enzyme for bacterial lipid metabolism, helping to catabolize unsaturated fatty acids via β-oxidation cycle. Therefore, DCR might be a potential drug target of tuberculosis. To develop a new drug, structural and functional characterization of the drug target is a prerequisite and important step. The structure and exact function of mycobacterial DCR is still unrevealed. In this study, structural and functional characterization of Mycobacterium smegmatis DCR was carried out as M. smegmatis is the model organism of mycobacterial research and is easy to handle in laboratory experiments.

For protein expression, both heterologous expression and expression in native host (M. smegmatis) was tried. After a few optimization steps, optimal expression was achieved through heterologous expression in E. coli BL21(DE3) Star pLysS using codon optimized M. smegmatis fadH gene that encodes DCR. After purification, the full length DCR was confirmed by MALDI-TOF mass spectrometry. Size exclusion chromatography-Multi angle laser light scattering studies suggested that M. smegmatis DCR is predominantly dimeric. Circular dichroism spectrum of M. smegmatis DCR suggested that it is well folded. By NanoDSF analysis, the melting temperature (Tm) of the protein was estimated to be 50 ºC. The presence of iron-sulfur (Fe-S) cluster in the M. smegmatis DCR was confirmed by UV-Vis spectrometry. The activity of purified DCR was examined using the substrate 2E,4E-decadienoyl-CoA and specific activity was calculated to be 0.12 µmole/mgmin. To reveal the structure of target protein, crystallization trials were performed. It showed one drop with small crystal and several drops with phase separation of the protein using the SaltRx screen. In addition, fadH knockout strain of M. smegmatis were created to explore the role of fadH in their life cycle. Further study will enhance our understanding of its significance. In conclusion, M. smegmatis DCR was successfully purified and characterized in this study. Hopefully this experiment might be useful in further study to understand the potentiality of mycobacterial DCR as a drug target.