Functional Impact of a Cancer-Related Variant in Human Δ¹‑Pyrroline-5-Carboxylate Reductase 1

Authors

Oseeyi I. Daudu, University of Nebraska-LincolnFollow
Kaylen R. Meeks, University of Missouri
Lu Zhang, University of Nebraska - Lincoln
Javier Seravalli, University of Nebraska-LincolnFollow
John J. Tanner, University of Missouri
Donald F. Becker, University of Nebraska-LincolnFollow

Document Type

Article

Date of this Version

1-10-2023

Citation

ACS Omega 2023, 8, 3509−3519

Comments

Used by permission.

Abstract

Pyrroline-5-carboxylate reductase (PYCR) is a proline biosynthetic enzyme that catalyzes the NAD(P)H-dependent reduction of Δ¹-pyrroline-5-carboxylate (P5C) to proline. Humans have three PYCR isoforms, with PYCR1 often upregulated in different types of cancers. Here, we studied the biochemical and structural properties of the Thr171Met variant of PYCR1, which is found in patients with malignant melanoma and lung adenocarcinoma. Although PYCR1 is strongly associated with cancer progression, characterization of a PYCR1 variant in cancer patients has not yet been reported. Thr171 is conserved in all three PYCR isozymes and is located near the P5C substrate binding site. We found that the amino acid replacement does not affect thermostability but has a profound effect on PYCR1 catalytic activity. The k_cat of the PYCR1 variant T171M is 100- to 200-fold lower than wild-type PYCR1 when P5C is the variable substrate, and 10- to 25-fold lower when NAD(P)H is varied. A 1.84 Å resolution X-ray crystal structure of T171M reveals that the Met side chain invades the P5C substrate binding site, suggesting that the catalytic defect is due to steric clash preventing P5C from achieving the optimal pose for hydride transfer from NAD(P)H. These results suggest that any impact on PYCR1 function associated with T171M in cancer does not derive from increased catalytic activity.