Resolving the cofactor-binding site in the proline biosynthetic enzyme human pyrroline-5-carboxylate reductase 1

Content Provider	Scilit
Author	Christensen, Emily M. Patel, Sagar M. Korasick, David A. Campbell, Ashley C. Krause, Kurt L. Becker, Donald F. Tanner, John J.
Copyright Year	2017
Description	Journal: Journal of Biological Chemistry Pyrroline-5-carboxylate reductase (PYCR) is the final enzyme in proline biosynthesis, catalyzing the NAD(P)H-dependent reduction of $Δ^{1}$-pyrroline-5-carboxylate (P5C) to proline. Mutations in the PYCR1 gene alter mitochondrial function and cause the connective tissue disorder cutis laxa. Furthermore, PYCR1 is overexpressed in multiple cancers, and the PYCR1 knock-out suppresses tumorigenic growth, suggesting that PYCR1 is a potential cancer target. However, inhibitor development has been stymied by limited mechanistic details for the enzyme, particularly in light of a previous crystallographic study that placed the cofactor-binding site in the C-terminal domain rather than the anticipated Rossmann fold of the N-terminal domain. To fill this gap, we report crystallographic, sedimentation-velocity, and kinetics data for human PYCR1. Structures of binary complexes of PYCR1 with NADPH or proline determined at 1.9 Å resolution provide insight into cofactor and substrate recognition. We see NADPH bound to the Rossmann fold, over 25 Å from the previously proposed site. The 1.85 Å resolution structure of a ternary complex containing NADPH and a P5C/proline analog provides a model of the Michaelis complex formed during hydride transfer. Sedimentation velocity shows that PYCR1 forms a concentration-dependent decamer in solution, consistent with the pentamer-of-dimers assembly seen crystallographically. Kinetic and mutational analysis confirmed several features seen in the crystal structure, including the importance of a hydrogen bond between Thr-238 and the substrate as well as limited cofactor discrimination.
Related Links	http://www.jbc.org/content/292/17/7233.full.pdf https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5409489/pdf http://www.jbc.org/article/S0021925820429114/pdf
Ending Page	7243
Page Count	11
Starting Page	7233
ISSN	00219258
e-ISSN	1083351X
DOI	10.1074/jbc.m117.780288
Journal	Journal of Biological Chemistry
Issue Number	17
Volume Number	292
Language	English
Publisher	Elsevier BV
Publisher Date	2017-04-01
Access Restriction	Open
Subject Keyword	Journal: Journal of Biological Chemistry Biochemistry and Molecular Biology Nad(p)h-dependent Reductase Rossmann Fold X-ray Crystallography Analytical Ultracentrifugation Enzyme Kinetics Nicotinamide Adenine Dinucleotide (nadh) Site-directed Mutagenesis Substrate Specificity
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology