Structural Elements Involved in ATP Hydrolysis Inhibition and ATP Synthesis of Tuberculosis and Nontuberculous Mycobacterial F-ATP Synthase Decipher New Targets for Inhibitors.

The F1FO-ATP synthase is required for the viability of tuberculosis (TB) and nontuberculous mycobacteria (NTM) and has been validated as a drug target. Here, we present the cryo-EM structures of the Mycobacterium smegmatis F1-ATPase and the F1FO-ATP synthase with different nucleotide occupation within the catalytic sites and visualize critical elements for latent ATP hydrolysis and efficient ATP synthesis. Mutational studies reveal that the extended C-terminal domain (αCTD) of subunit α is the main element for the self-inhibition mechanism of ATP hydrolysis for TB and NTM bacteria. Rotational studies indicate that the transition between the inhibition state by the αCTD and the active state is a rapid process. We demonstrate that the unique mycobacterial γ-loop and subunit δ are critical elements required for ATP formation. The data underline that these mycobacterium-specific elements of α, γ, and δ are attractive targets, providing a platform for the discovery of species-specific inhibitors.

Digital Cascade Assays for ADP- or ATP-Producing Enzymes Using a Femtoliter Reactor Array Device.

Digital enzyme assays are emerging biosensing methods for highly sensitive quantitative analysis of biomolecules with single-molecule detection sensitivity. However, current digital enzyme assays require a fluorogenic substrate for detection, which limits the applicability of this method to certain enzymes. ATPases and kinases are representative enzymes for which fluorogenic substrates are not available; however, these enzymes form large domains and play a central role in biology. In this study, we implemented a fluorogenic cascade reaction in a femtoliter reactor array device to develop a digital bioassay platform for ATPases and kinases. The digital cascade assay enabled quantitative measurement of the single-molecule activity of F1-ATPase, the catalytic portion of ATP synthase. We also demonstrated a digital assay for human choline kinase α. Furthermore, we developed a digital cascade assay for ATP-synthesizing enzymes and demonstrated a digital assay for pyruvate kinase. These results show the high versatility of this assay platform. Thus, the digital cascade assay has great potential for the highly sensitive detection and accurate characterization of various ADP- and ATP-producing enzymes, such as kinases, which may serve as disease biomarkers.

Amplification of over 100 kbp DNA from Single Template Molecules in Femtoliter Droplets.

Reconstitution of the DNA amplification system in microcompartments is the primary step toward artificial cell construction through a bottom-up approach. However, amplification of >100 kbp DNA in micrometer-sized reactors has not yet been achieved. Here, implementing a fully reconstituted replisome of Escherichia coli in micrometer-sized water-in-oil droplets, we developed the in-droplet replication cycle reaction (RCR) system. For a 16 kbp template DNA, the in-droplet RCR system yielded positive RCR signals with a high success rate (82%) for the amplification from single molecule template DNA. The success rate for a 208 kbp template DNA was evidently lower (23%). This study establishes a platform for genome-sized DNA amplification from a single copy of template DNA with the potential to build more complex artificial cell systems comprising a large number of genes.