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 Δ-pyrroline-5-carboxylate (P5C) to proline. Mutations in the gene alter mitochondrial function and cause the connective tissue disorder cutis laxa. Furthermore, PYCR1 is overexpressed in multiple cancers, and the 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.

Disorders affecting vitamin B6 metabolism

“…The contribution of each pathway to lysine catabolism depends on the age of the individual and cell-type studied; studies in mice suggest that the saccharopine pathway is primarly reponsible for AASA/P6C prodcution. 80,81 POX, L-pipecolate oxidase; PYRC, pyrroline-5-carboxylate reductase (three homologous genes exist; PYCR1 and PYCR2 localize to mitochondria and possibly with the outer mitochondrial membrane and PYCRL (aka PYCR3) is cytosolic 122,123 deficiency. An overview of the clinical features of patients with ALDH7A1 deficiency is as detailed in Figure 5 (adapted from Karnebeek et al 69 ).…”

A universal mammalian vaccine cell line substrate

“…Phospholipase A2, group 1B (PLA2G1B) is an enzyme that catalyzes the release of fatty acids from glycero-3-phosphocholines [ 34 ]. Pyrroline-5-carboxylate reductase (PYCR1) is a mitochondrial enzyme that catalyzes proline biosynthesis reducing pyrroline-5-carboxylate to L-proline [ 35 ]. Protein transport protein Sec61 subunit gamma (SEC61G) may function to aid protein translocation in the endoplasmic reticulum [ 36 ].…”

Peer Review #1 of "Investigation of microbial community interactions between Lake Washington methanotrophs using ­­­­­­­genome-scale metabolic modeling (v0.1)"

“…Reactions whose fluxes reached the defined lower and upper bounds were determined to be unbounded reactions, group together based on stoichiometry, and systematically corrected. These cycles were eliminated by removing duplicate reactions, turning off lumped reactions, fixing reaction directionality, or selectively turning reactions on or off based on cofactor specificities found from literature (Ishida et al, 1969;Dekker, Lane & Shapley, 1971;Chen, Lee & Chang, 1991;Schomburg & Stephan, 1995;Achterholt, Priefert & Steinbuchel, 1998;Hadfield et al, 1999;Hutter & Singh, 1999;Kai, Matsumura & Izui, 2003;Feist et al, 2007;Dean, 2012;Flamholz et al, 2012;Lin et al, 2014;Christensen et al, 2017). The FVA optimization algorithm is shown below.…”