skip to main content

DOE PAGESDOE PAGES

Title: Enzyme intermediates captured “on the fly” by mix-and-inject serial crystallography

Background: 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. Results: Here, we demonstrate a general method for capturing enzyme catalysis “in action” by mix-and-inject serial crystallography (MISC). Specifically, we follow the catalytic reaction of the Mycobacterium tuberculosis β-lactamase with the third-generation antibiotic ceftriaxone by time-resolved serial femtosecond crystallography. The results reveal, in near atomic detail, antibiotic cleavage and inactivation from 30 ms to 2 s. Conclusions: MISC is a versatile and generally applicable method to investigate reactions of biological macromolecules, some of which are of immense biological significance and might be, in addition, important targets for structure-based drug design. With megahertz X-ray pulse rates expected at the Linac Coherent Light Source II and the European X-ray free-electron laser, multiple, finely spaced time delays can be collected rapidly, allowing a comprehensive description of biomolecular reactions in terms of structure and kinetics frommore » the same set of X-ray data.« less
Authors:
 [1] ;  [2] ;  [3] ;  [4] ;  [4] ;  [5] ;  [2] ;  [6] ;  [6] ;  [7] ;  [7] ;  [5] ;  [8] ;  [3] ;  [9] ;  [7] ;  [1] ;  [7] ;  [3] ;  [3] more »;  [3] ;  [3] ;  [10] ;  [2] ;  [2] ;  [1] ;  [6] ;  [6] ;  [3] ;  [11] ;  [12] ;  [7] ;  [13] ;  [13] ;  [14] ;  [3] ;  [2] ;  [2] ;  [15] ;  [7] ;  [13] ;  [13] ;  [13] ;  [3] ;  [16] ;  [4] ;  [17] ;  [2] ;  [1] ; ORCiD logo [2] « less
  1. Rice Univ., Houston, TX (United States). Dept. of BioSciences
  2. Univ. of Wisconsin, Milwaukee, WI (United States). Dept. of Physics
  3. Arizona State Univ., Tempe, AZ (United States). School of Molecular Sciences and Biodesign Center for Applied Structural Discovery
  4. Cornell Univ., Ithaca, NY (United States). School of Applied and Engineering Physics
  5. Deutsches Elektronen-Synchrotron (DESY), Hamburg (Germany). Center for Free-Electron Laser Science; Univ. of Hamburg (Germany)
  6. SLAC National Accelerator Lab., Menlo Park, CA (United States). Linac Coherent Light Source (LCLS)
  7. Deutsches Elektronen-Synchrotron (DESY), Hamburg (Germany). Center for Free-Electron Laser Science
  8. Max Planck Inst. fuer Biochemie, Planegg (Germany)
  9. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  10. Univ. of Wisconsin, Milwaukee, WI (United States). Dept. of Physics; Milwaukee School of Engineering, Milwaukee, WI (United States)
  11. Deutsches Elektronen-Synchrotron (DESY), Hamburg (Germany). Photon Science
  12. Univ. of New York, Buffalo, NY (United States). Hauptman-Woodward Inst.
  13. Arizona State Univ., Tempe, AZ (United States). Dept. of Physics
  14. Seoul National Univ. (Korea, Republic of). Dept. of Agricultural Biotechnology
  15. Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
  16. Deutsches Elektronen-Synchrotron (DESY), Hamburg (Germany). Center for Free-Electron Laser Science; Univ. of Hamburg (Germany); Centre for Ultrafast Imaging, Hamburg, (Germany)
  17. 4Marbles Inc., Westfield, IN (United States); GlaxoSmithKline, Gunnels Wood Road (United Kingdom)
Publication Date:
Grant/Contract Number:
AC02-05CH11231; SC0002164; AC02-76SF00515; AC52-07NA27344
Type:
Accepted Manuscript
Journal Name:
BMC Biology
Additional Journal Information:
Journal Volume: 16; Journal Issue: 1; Journal ID: ISSN 1741-7007
Publisher:
BioMed Central
Research Org:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22); National Science Foundation (NSF)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES
OSTI Identifier:
1460337