Enzyme Intermediates Captured "on-the-fly" by Mix-and-Inject Serial Crystallography

By Jose Olmos, Suraj Pandey1, Jose M. Martin-Garcia, George Douglas Calvey, Andrea Katz, Juray Knoska, Christopher Kupitz2, Mark S. Hunter2, Mengning Liang, Dominik Oberthuer, Oleksandr Yefanov, Max Oliver Wiedorn3, Michael Heymann, Mark Holl, Kanupriya Pande1, Anton Barty, Mitchell Miller4, Stephan Stern, Shatabdi Roy-Chowdhury5, Jesse David Coe5, Nirupa Nagaratnam5, James D Zook5, Jacob Christopher Verburgt6, Tyler Norwood, Ishwor Poudyal1, David Xu, Jason Koglin, Matt Seaberg, Yun Zhao5, Sasa Bajt, Thomas Grant, Valerio Mariani, Garrett Charles Nelson5, GANESH SUBRAMANIAN5, Euiyoung Bae, Raimund Fromme, Russel Fung, Peter Schwander7, Matthias Frank8, Thomas White, Uwe Weierstall5, Nadia Zatsepin5, John Spence5, Petra Fromme5, Henry Chapman3, Lois Pollack9, Lee Tremblay10, Abbas Ourmazd7, George Phillips4, Marius Schmidt1

1. University of Wisconsin - Milwaukee 2. SLAC National Accelerator Laboratory 3. Center for Free-Electron Laser Science 4. Rice University 5. Arizona State University 6. BioXFEL Undergraduate Summer Intern 7. University of Wisconsin-Milwaukee 8. Lawrence Livermore National Laboratory 9. Cornell University 10. 4Marbles Inc.

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Type

posted-content

Author

Jose Olmos and Suraj Pandey and Jose M. Martin-Garcia and George Calvey and Andrea Katz and Juray Knoska and Christopher Kupitz and Mark S. Hunter and Mengning Liang and Dominik Oberthuer and Oleksandr Yefanov and Max Wiedorn and Michael Heymann and Mark Holl and Kanupriya Pande and Anton Barty and Mitchell D. Miller and Stephan Stern and Shatabdi Roy-Chowdhury and Jesse D. Coe and Nirupa Nagaratnam and James Zook and Jacob Verburgt and Tyler Norwood and Ishwor Poudyal and David Xu and Jason Koglin and Matt Seaberg and Yun Zhao and Sasa Bajt and Thomas Grant and Valerio Mariani and Garrett Nelson and Ganesh Subramanian and Euiyoung Bae and Raimund Fromme and Russel Fung and Peter Schwander and Matthias Frank and Thomas White and Uwe Weierstall and Nadia Zatsepin and John Spence and Petra Fromme and Henry N. Chapman and Lois Pollack and Lee Tremblay and Abbas Ourmazd and George N. Phillips and Marius Schmidt

Citation

Olmos, J. et al., 2017. Enzyme Intermediates Captured “on-the-fly” by Mix-and-Inject Serial Crystallography. Available at: http://dx.doi.org/10.1101/202432.

Abstract

Ever since the first atomic structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction has been possible only in rare and exceptional cases. Here, we demonstrate a general method for capturing enzyme catalysis in-action by mix-and-inject serial crystallography. Specifically, we follow the catalytic reaction of the Mycobacterium tuberculosis beta-lactamase with the 3rd generation antibiotic ceftriaxone by time-resolved serial femtosecond crystallography. The results reveal, in near atomic detail, antibiotic cleavage and inactivation on the millisecond to second time scales including the crossover from transition state kinetics to steady-state kinetics.

DOI

Funding

NSF-STC Biology with X-ray Lasers (NSF-1231306)