Biochemical and Mutational Analysis of Radical S-Adenosyl-L-Methionine Adenosylhopane Synthase HpnH from Zymomonas mobilis Reveals that the Conserved Residue Cysteine-106 Reduces a Radical Intermediate and Determines the Stereochemistry

Biochemistry ◽

10.1021/acs.biochem.1c00536 ◽

2021 ◽

Author(s):

Shusuke Sato ◽

Fumitaka Kudo ◽

Michel Rohmer ◽

Tadashi Eguchi

Keyword(s):

Zymomonas Mobilis ◽

Mutational Analysis ◽

Radical Intermediate ◽

Conserved Residue

Download Full-text

Elements in nucleotide sensing and hydrolysis of the AAA+ disaggregation machine ClpB: a structure-based mechanistic dissection of a molecular motor

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004713030629 ◽

2014 ◽

Vol 70 (2) ◽

pp. 582-595 ◽

Cited By ~ 16

Author(s):

Cathleen Zeymer ◽

Thomas R. M. Barends ◽

Nicolas D. Werbeck ◽

Ilme Schlichting ◽

Jochen Reinstein

Keyword(s):

Atp Hydrolysis ◽

Mutational Analysis ◽

Molecular Motor ◽

Nucleotide Binding ◽

Side Chain ◽

Side Chain Conformation ◽

Proposed Model ◽

Hydrolysis Of ◽

Conserved Residue

ATPases of the AAA+ superfamily are large oligomeric molecular machines that remodel their substrates by converting the energy from ATP hydrolysis into mechanical force. This study focuses on the molecular chaperone ClpB, the bacterial homologue of Hsp104, which reactivates aggregated proteins under cellular stress conditions. Based on high-resolution crystal structures in different nucleotide states, mutational analysis and nucleotide-binding kinetics experiments, the ATPase cycle of the C-terminal nucleotide-binding domain (NBD2), one of the motor subunits of this AAA+ disaggregation machine, is dissected mechanistically. The results provide insights into nucleotide sensing, explaining how the conserved sensor 2 motif contributes to the discrimination between ADP and ATP binding. Furthermore, the role of a conserved active-site arginine (Arg621), which controls binding of the essential Mg2+ion, is described. Finally, a hypothesis is presented as to how the ATPase activity is regulated by a conformational switch that involves the essential Walker A lysine. In the proposed model, an unusual side-chain conformation of this highly conserved residue stabilizes a catalytically inactive state, thereby avoiding unnecessary ATP hydrolysis.

Download Full-text