title: 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 thePYCR1gene alter mitochondrial function and cause the connective tissue disorder cutis laxa. Furthermore, PYCR1 is overexpressed in multiple cancers, and thePYCR1knock-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.
authors.affiliation: From the Departments of Chemistry and.
authors.forename: Emily M
authors.initials: EM
authors.lastname: Christensen
authors.affiliation: the Department of Biochemistry and Redox Biology Center, University of Nebraska, Lincoln, Nebraska 68588.
authors.forename: Sagar M
authors.initials: SM
authors.lastname: Patel
authors.affiliation: Biochemistry University of Missouri, Columbia, Missouri 65211.
authors.forename: David A
authors.initials: DA
authors.lastname: Korasick
authors.affiliation: Biochemistry University of Missouri, Columbia, Missouri 65211.
authors.forename: Ashley C
authors.initials: AC
authors.lastname: Campbell
authors.affiliation: the Department of Biochemistry, University of Otago, Dunedin 9054, New Zealand, and.
authors.forename: Kurt L
authors.initials: KL
authors.lastname: Krause
authors.affiliation: the Department of Biochemistry and Redox Biology Center, University of Nebraska, Lincoln, Nebraska 68588.
authors.forename: Donald F
authors.initials: DF
authors.lastname: Becker
authors.affiliation: From the Departments of Chemistry and tannerjj@missouri.edu.|Biochemistry University of Missouri, Columbia, Missouri 65211.
authors.forename: John J
authors.initials: JJ
authors.lastname: Tanner
mesh_terms: D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006801:Humans; D007700:Kinetics; D008024:Ligands; D009154:Mutation; D009249:NADP / Q000737:chemistry; D011392:Proline / Q000737:chemistry*; D011485:Protein Binding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011762:Pyrroline Carboxylate Reductases / Q000737:chemistry*; D013379:Substrate Specificity; D014461:Ultracentrifugation; D000097502:delta-1-Pyrroline-5-Carboxylate Reductase
chemical_list: D008024:Ligands; D009249:NADP; D011392:Proline; D011762:Pyrroline Carboxylate Reductases; D000097502:delta-1-Pyrroline-5-Carboxylate Reductase
keywords: NAD(P)H-dependent reductase; Rossmann fold; X-ray crystallography; analytical ultracentrifugation; enzyme kinetics; nicotinamide adenine dinucleotide (NADH); reductase; site-directed mutagenesis; substrate specificity