TY - JOUR
T1 - X-ray crystallography–based structural elucidation of enzyme-bound intermediates along the 1-deoxy-D-xylulose 5-phosphate synthase reaction coordinate
AU - Chen, Percival Yang Ting
AU - DeColli, Alicia A.
AU - Freel Meyers, Caren L.
AU - Drennan, Catherine L.
N1 - Funding Information:
This work was supported by National Institutes of Health Grants R35 GM126982 (to C. L. D.) and R01 GM084998 (C. L. F. M.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Funding Information:
Acknowledgments—We thank Lauren Bambarger for assistance in synthesizing MAP and Sara Sanders for assistance in synthesizing BAP. We thank the Center for Molecular Biophysics at The Johns Hopkins University for access to the CD spectrometer. This work is based upon research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by NIGMS, National Institutes of Health (NIH) Grant P30 GM124165. The Pilatus 6M detector on 24-ID-C beam line is funded by NIH Office of Research Infrastructure High-End Instrumentation Grant S10 RR029205. This research used resources of the Advanced Photon Source, a United States Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract DE-AC02-06CH11357.
Publisher Copyright:
© 2019 Chen et al.
PY - 2019/8/16
Y1 - 2019/8/16
N2 - 1-Deoxy-D-xylulose 5-phosphate synthase (DXPS) uses thiamine diphosphate (ThDP) to convert pyruvate and D-glyceraldehyde 3-phosphate (D-GAP) into 1-deoxy-D-xylulose 5-phos-phate (DXP), an essential bacterial metabolite. DXP is not utilized by humans; hence, DXPS has been an attractive antibacterial target. Here, we investigate DXPS from Deinococcus radiodurans (DrDXPS), showing that it has similar kinetic parameters Km D-GAP AND Km pyruvate (54 3 and 11 1 M, respectively) and comparable catalytic activity (kcat 45 2 min1) with previously studied bacterial DXPS enzymes and employing it to obtain missing structural data on this enzyme family. In particular, we have determined crystallographic snapshots of DrDXPS in two states along the reaction coordinate: a structure of DrDXPS bound to C2-phosphonolactylThDP (PLThDP), mimicking the native pre-decarboxylation intermediate C2-lactylThDP (LThDP), and a native post-decarboxylation state with a bound enamine intermediate. The 1.94-Å-res-olution structure of PLThDP-bound DrDXPS delineates how two active-site histidine residues stabilize the LThDP intermediate. Meanwhile, the 2.40-Å-resolution structure of an enamine intermediate-bound DrDXPS reveals how a previously unknown 17-Å conformational change removes one of the two histidine residues from the active site, likely triggering LThDP decarboxylation to form the enamine intermediate. These results provide insight into how the bi-substrate enzyme DXPS limits side reactions by arresting the reaction on the less reactive LThDP intermediate when its cosubstrate is absent. They also offer a molecular basis for previous low-resolution experimental observations that correlate decarboxylation of LThDP with protein conformational changes.
AB - 1-Deoxy-D-xylulose 5-phosphate synthase (DXPS) uses thiamine diphosphate (ThDP) to convert pyruvate and D-glyceraldehyde 3-phosphate (D-GAP) into 1-deoxy-D-xylulose 5-phos-phate (DXP), an essential bacterial metabolite. DXP is not utilized by humans; hence, DXPS has been an attractive antibacterial target. Here, we investigate DXPS from Deinococcus radiodurans (DrDXPS), showing that it has similar kinetic parameters Km D-GAP AND Km pyruvate (54 3 and 11 1 M, respectively) and comparable catalytic activity (kcat 45 2 min1) with previously studied bacterial DXPS enzymes and employing it to obtain missing structural data on this enzyme family. In particular, we have determined crystallographic snapshots of DrDXPS in two states along the reaction coordinate: a structure of DrDXPS bound to C2-phosphonolactylThDP (PLThDP), mimicking the native pre-decarboxylation intermediate C2-lactylThDP (LThDP), and a native post-decarboxylation state with a bound enamine intermediate. The 1.94-Å-res-olution structure of PLThDP-bound DrDXPS delineates how two active-site histidine residues stabilize the LThDP intermediate. Meanwhile, the 2.40-Å-resolution structure of an enamine intermediate-bound DrDXPS reveals how a previously unknown 17-Å conformational change removes one of the two histidine residues from the active site, likely triggering LThDP decarboxylation to form the enamine intermediate. These results provide insight into how the bi-substrate enzyme DXPS limits side reactions by arresting the reaction on the less reactive LThDP intermediate when its cosubstrate is absent. They also offer a molecular basis for previous low-resolution experimental observations that correlate decarboxylation of LThDP with protein conformational changes.
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U2 - 10.1074/jbc.RA119.009321
DO - 10.1074/jbc.RA119.009321
M3 - Article
C2 - 31239351
AN - SCOPUS:85070772879
VL - 294
SP - 12405
EP - 12414
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 33
ER -