TY - JOUR
T1 - Conformational dynamics of 1-deoxy-D-xylulose 5-phosphate synthase on ligand binding revealed by H/D exchange MS
AU - Zhou, Jieyu
AU - Yang, Luying
AU - DeColli, Alicia
AU - Freel Meyers, Caren
AU - Nemeria, Natalia S.
AU - Jordan, Frank
N1 - Funding Information:
ACKNOWLEDGMENTS. This work was supported, in whole or in part, by NIH Grant R01 GM084998 (to A.D., C.F.M., N.S.N., and F.J.).
Publisher Copyright:
© 2017, National Academy of Sciences. All rights reserved.
PY - 2017/8/29
Y1 - 2017/8/29
N2 - The enzyme 1-deoxy-D-xylulose 5-phosphate synthase (DXPS) is a key enzyme in the methylerythritol 4-phosphate pathway and is a target for the development of antibiotics, herbicides, and antimalarial drugs. DXPS catalyzes the formation of 1-deoxy-D-xylulose 5-phosphate (DXP), a branch point metabolite in isoprenoid biosynthesis, and is also used in the biosynthesis of thiamin (vitamin B1) and pyridoxal (vitamin B6). Previously, we found that DXPS is unique among the superfamily of thiamin diphosphate (ThDP)-dependent enzymes in stabilizing the predecarboxylation intermediate, C2-alpha-lactyl-thiamin diphosphate (LThDP), which has subsequent decarboxylation that is triggered by D-glyceraldehyde 3-phosphate (GAP). Herein, we applied hydrogen–deuterium (H/D) exchange MS (HDX-MS) of full-length Escherichia coli DXPS to provide a snapshot of the conformational dynamics of this enzyme, leading to the following conclusions. (i) The high sequence coverage of DXPS allowed us to monitor structural changes throughout the entire enzyme, including two segments (spanning residues 183–238 and 292–317) not observed by X-ray crystallography. (ii) Three regions of DXPS (spanning residues 42–58, 183–199, and 278–298) near the active center displayed both EX1 (monomolecular) and EX2 (bimolecuar) H/D exchange (HDX) kinetic behavior in both ligand-free and ligand-bound states. All other peptides behaved according to the common EX2 kinetic mechanism. (iii) The observation of conformational changes on DXPS provides support for the role of conformational dynamics in the DXPS mechanism: The closed conformation of DXPS is critical for stabilization of LThDP, whereas addition of GAP converts DXPS to the open conformation that coincides with decarboxylation of LThDP and DXP release.
AB - The enzyme 1-deoxy-D-xylulose 5-phosphate synthase (DXPS) is a key enzyme in the methylerythritol 4-phosphate pathway and is a target for the development of antibiotics, herbicides, and antimalarial drugs. DXPS catalyzes the formation of 1-deoxy-D-xylulose 5-phosphate (DXP), a branch point metabolite in isoprenoid biosynthesis, and is also used in the biosynthesis of thiamin (vitamin B1) and pyridoxal (vitamin B6). Previously, we found that DXPS is unique among the superfamily of thiamin diphosphate (ThDP)-dependent enzymes in stabilizing the predecarboxylation intermediate, C2-alpha-lactyl-thiamin diphosphate (LThDP), which has subsequent decarboxylation that is triggered by D-glyceraldehyde 3-phosphate (GAP). Herein, we applied hydrogen–deuterium (H/D) exchange MS (HDX-MS) of full-length Escherichia coli DXPS to provide a snapshot of the conformational dynamics of this enzyme, leading to the following conclusions. (i) The high sequence coverage of DXPS allowed us to monitor structural changes throughout the entire enzyme, including two segments (spanning residues 183–238 and 292–317) not observed by X-ray crystallography. (ii) Three regions of DXPS (spanning residues 42–58, 183–199, and 278–298) near the active center displayed both EX1 (monomolecular) and EX2 (bimolecuar) H/D exchange (HDX) kinetic behavior in both ligand-free and ligand-bound states. All other peptides behaved according to the common EX2 kinetic mechanism. (iii) The observation of conformational changes on DXPS provides support for the role of conformational dynamics in the DXPS mechanism: The closed conformation of DXPS is critical for stabilization of LThDP, whereas addition of GAP converts DXPS to the open conformation that coincides with decarboxylation of LThDP and DXP release.
KW - Conformational dynamics
KW - DXP synthase
KW - EX1 kinetics
KW - H/D exchange MS
KW - ThDP-dependent enzymes
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U2 - 10.1073/pnas.1619981114
DO - 10.1073/pnas.1619981114
M3 - Article
C2 - 28808005
AN - SCOPUS:85028538248
SN - 0027-8424
VL - 114
SP - 9355
EP - 9360
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 35
ER -