Abstract
Pyridoxal 5′-phosphate (PLP), the biologically active form of vitamin B6, is an essential cofactor in many biosynthetic pathways. The emergence of PLP-dependent enzymes as drug targets and biocatalysts, such as tryptophan synthase (TS), has underlined the demand to understand PLP-dependent catalysis and reaction specificity. The ability of neutron diffraction to resolve the positions of hydrogen atoms makes it an ideal technique to understand how the electrostatic environment and selective protonation of PLP regulates PLP-dependent activities. Facilitated by microgravity crystallization of TS with the Toledo Crystallization Box, we report the 2.1 Å joint X-ray/neutron (XN) structure of TS with PLP in the internal aldimine form. Positions of hydrogens were directly determined in both the α- and β-active sites, including PLP cofactor. The joint XN structure thus provides insight into the selective protonation of the internal aldimine and the electrostatic environment of TS necessary to understand the overall catalytic mechanism.
| Original language | English |
|---|---|
| Article number | 101827 |
| Journal | Cell Reports Physical Science |
| Volume | 5 |
| Issue number | 2 |
| DOIs | |
| State | Published - Feb 21 2024 |
Funding
We are especially grateful for the assistance of April Spinale, Ray Polniak, and Marc Giulianotti, International Space Station (ISS) National Laboratory, for flight preparation. We thank SpaceX for access to CRS-15 and CRS-18 and ESA Astronaut Alexander Gerst and NASA astronauts Christina Koch and Nicklaus Hague for sample handling at the International Space Station. For beamline access, we thank the Life Sciences Collaborative Team (LS-CAT) for access to beamline 21-ID-F at APS. We also thank Lisa Keefe and Kevin Battaile for access and assistance at APS IMCA-CAT 17-ID-B used in early stages of this study. Additionally, thank you to Rob Phillips, Len Mueller, and Mike Toney for helpful discussion and suggestions. V.T.F. acknowledges the UK Engineering and Physical Sciences Research Council for grants EP/C015452/1 and GR/R99393/01 under which the Deuteration Laboratory was created within ILL’s Life Sciences Group. Funding was provided by the Center for the Advancement of Science in Space (contract GA2017–251, T.C.M.) and the National Institutes of Health ( 1R01GM137008-01A1 , T.C.M. and A.K.). The research at ORNL’s High Flux Isotope Reactor (IMAGINE beamline) was sponsored by the Scientific User Facilities Division , Office of Basic Energy Sciences , US Department of Energy . The authors thank the Institut Laue–Langevin for provision of neutron beam time on the LADI-III beamline. We are especially grateful for the assistance of April Spinale, Ray Polniak, and Marc Giulianotti, International Space Station (ISS) National Laboratory, for flight preparation. We thank SpaceX for access to CRS-15 and CRS-18 and ESA Astronaut Alexander Gerst and NASA astronauts Christina Koch and Nicklaus Hague for sample handling at the International Space Station. For beamline access, we thank the Life Sciences Collaborative Team (LS-CAT) for access to beamline 21-ID-F at APS. We also thank Lisa Keefe and Kevin Battaile for access and assistance at APS IMCA-CAT 17-ID-B used in early stages of this study. Additionally, thank you to Rob Phillips, Len Mueller, and Mike Toney for helpful discussion and suggestions. V.T.F. acknowledges the UK Engineering and Physical Sciences Research Council for grants EP/C015452/1 and GR/R99393/01 under which the Deuteration Laboratory was created within ILL's Life Sciences Group. Funding was provided by the Center for the Advancement of Science in Space (contract GA2017–251, T.C.M.) and the National Institutes of Health (1R01GM137008-01A1, T.C.M. and A.K.). The research at ORNL's High Flux Isotope Reactor (IMAGINE beamline) was sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, US Department of Energy. The authors thank the Institut Laue–Langevin for provision of neutron beam time on the LADI-III beamline. V.N.D. T.C.M. and A.K. wrote the paper. V.N.D. and T.C.M. conducted X-ray diffraction data collection at APS. V.N.D. and A.K. collected room temperature X-ray diffraction data at ORNL. A.K. conducted preliminary TS neutron diffraction data collection at ORNL. V.N.D. and A.K. conducted joint X-ray/neutron structure refinement, and V.N.D. performed X-ray structure refinements. M.P.B. conducted the neutron diffraction data collection at ILL. J.M.D. and V.N.D. contributed to perdeuterated protein preparation, and J.M.D. provided all the crystal handling at ILL. V.N.D. and J.M.P. completed the computational analysis. The manuscript was written using data contributions of all authors. The authors declare no competing interests.
| Funders | Funder number |
|---|---|
| ESA Astronaut Alexander Gerst | |
| Institut Laue | |
| Scientific User Facilities Division | |
| National Institutes of Health | 1R01GM137008-01A1 |
| U.S. Department of Energy | |
| National Aeronautics and Space Administration | |
| Basic Energy Sciences | |
| Engineering and Physical Sciences Research Council | EP/C015452/1, GR/R99393/01, GA2017–251 |
Keywords
- enzyme mechanism
- fold-type II
- macromolecular crystallography
- microgravity crystallization
- neutron crystallography
- neutron diffraction
- pyridoxal 5’-phosphate