Overexpression, purification, enzymatic and microscopic characterization of recombinant mycobacterial F-ATP synthase

• Main Authors: Saw, Wuan-Geok, Wong, Chui-Fann, Dick, Thomas, Grüber, Gerhard
• Other Authors: School of Biological Sciences
• Format: Article
• Language: English
• Published: 2020
• Subjects: Science::Biological sciences::Biochemistry; Science::Biological sciences::Molecular biology; F-ATP Synthase; Bioenergetics
• Online Access: https://hdl.handle.net/10356/139317
• Institution: Nanyang Technological University

The F-ATP synthase is an essential enzyme in mycobacteria, including the pathogenic Mycobacterium tuberculosis. Several new compounds in the TB-drug pipeline target the F-ATP synthase. In light of the importance and pharmacological attractiveness of this novel antibiotic target, tools have to be developed to generate a recombinant mycobacterial F1FO ATP synthase to achieve atomic insight and mutants for mechanistic and regulatory understanding as well as structure-based drug design. Here, we report the first genetically engineered, purified and enzymatically active recombinant M. smegmatis F1FO ATP synthase. The projected 2D- and 3D structures of the recombinant enzyme derived from negatively stained electron micrographs are presented. Furthermore, the first 2D projections from cryo-electron images are revealed, paving the way for an atomic resolution structure determination.