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

Abstract

Pyrroline-5-carboxylate reductase (PYCR) is the final enzyme in proline biosynthesis, catalyzing the NAD(P)H-dependent reduction of Δ¹-pyrroline-5-carboxylate (P5C) to proline. Mutations in the <i>PYCR1</i> gene alter mitochondrial function and cause the connective tissue disorder cutis laxa. Furthermore, PYCR1 is overexpressed in multiple cancers, and the <i>PYCR1</i> 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.