TY - JOUR
T1 - Structural basis for the substrate inhibition of proline utilization A by proline
AU - Korasick, David A.
AU - Pemberton, Travis A.
AU - Arentson, Benjamin W.
AU - Becker, Donald F.
AU - Tanner, John J.
N1 - Funding Information:
Acknowledgments: Research reported in this publication was supported by the NIGMS of the National Institutes of Health under award numbers R01GM065546 and R01GM061068. We thank Jay Nix for help with X-ray diffraction data collection. This research used resources of the Advanced Light Source, which is a DOE Office of Science User Facility under contract no. DE-AC02-05CH11231.
PY - 2018
Y1 - 2018
N2 - Proline utilization A (PutA) is a bifunctional flavoenzyme that catalyzes the two-step oxidation of L-proline to L-glutamate using spatially separated proline dehydrogenase (PRODH) and L-glutamate-γ-semialdehyde dehydrogenase (GSALDH) active sites. Substrate inhibition of the coupled PRODH-GSALDH reaction by proline is a common kinetic feature of PutAs, yet the structural basis for this phenomenon remains unknown. To understand the mechanism of substrate inhibition, we determined the 2.15 resolution crystal structure of Bradyrhizobium japonicum PutA complexed with proline. Proline was discovered in five locations remote from the PRODH active site. Most notably, strong electron density indicated that proline bound tightly to the GSAL binding site of the GSALDH active site. The pose and interactions of proline bound in this site are remarkably similar to those of the natural aldehyde substrate, GSAL, implying that proline inhibits the GSALDH reaction of PutA. Kinetic measurements show that proline is a competitive inhibitor of the PutA GSALDH reaction. Together, the structural and kinetic data show that substrate inhibition of the PutA coupled reaction is due to proline binding in the GSAL site.
AB - Proline utilization A (PutA) is a bifunctional flavoenzyme that catalyzes the two-step oxidation of L-proline to L-glutamate using spatially separated proline dehydrogenase (PRODH) and L-glutamate-γ-semialdehyde dehydrogenase (GSALDH) active sites. Substrate inhibition of the coupled PRODH-GSALDH reaction by proline is a common kinetic feature of PutAs, yet the structural basis for this phenomenon remains unknown. To understand the mechanism of substrate inhibition, we determined the 2.15 resolution crystal structure of Bradyrhizobium japonicum PutA complexed with proline. Proline was discovered in five locations remote from the PRODH active site. Most notably, strong electron density indicated that proline bound tightly to the GSAL binding site of the GSALDH active site. The pose and interactions of proline bound in this site are remarkably similar to those of the natural aldehyde substrate, GSAL, implying that proline inhibits the GSALDH reaction of PutA. Kinetic measurements show that proline is a competitive inhibitor of the PutA GSALDH reaction. Together, the structural and kinetic data show that substrate inhibition of the PutA coupled reaction is due to proline binding in the GSAL site.
KW - Flavoenzyme
KW - L-glutamate-γ-semialdehyde dehydrogenase
KW - Proline dehydrogenase
KW - Substrate inhibition
KW - X-ray crystallography
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U2 - 10.3390/molecules23010032
DO - 10.3390/molecules23010032
M3 - Article
C2 - 29295473
AN - SCOPUS:85039708083
VL - 23
JO - Molecules
JF - Molecules
SN - 1420-3049
IS - 1
M1 - 32
ER -