Abstract
l-Thioproline (l-thiazolidine-4-carboxylate, l-T4C) is a cyclic sulfur-containing analog of l-proline found in multiple kingdoms of life. The oxidation of l-T4C leads to l-cysteine formation in bacteria, plants, mammals, and protozoa. The conversion of l-T4C to l-Cys in bacterial cell lysates has been attributed to proline dehydrogenase and l-Δ1-pyrroline-5-carboxylate (P5C) reductase (PYCR) enzymes but detailed kinetic studies have not been conducted. Here, we characterize the dehydrogenase activity of human PYCR isozymes 1 and 2 with l-T4C using NAD(P)+ as the hydride acceptor. Both PYCRs exhibit significant l-T4C dehydrogenase activity; however, PYCR2 displays nearly tenfold higher catalytic efficiency (136 M−1 s−1) than PYCR1 (13.7 M−1 s−1). Interestingly, no activity was observed with either l-Pro or the analog dl-thiazolidine-2-carboxylate, indicating that the sulfur at the 4-position is critical for PYCRs to utilize l-T4C as a substrate. Inhibition kinetics show that l-Pro is a competitive inhibitor of PYCR1 \(\left({K_{IC}^{app}}=15.7 \,{\rm mM} \right)\) with respect to l-T4C, consistent with these ligands occupying the same binding site. We also confirm by mass spectrometry that l-T4C oxidation by PYCRs leads to cysteine product formation. Our results suggest a new enzyme function for human PYCRs in the metabolism of l-T4C.
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The datasets generated and analyzed during the current study for the figures and tables are available from the corresponding author on reasonable request.
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Abbreviations
- l-T4C:
-
l-Thiazolidine-4-carboxylate
- l-Pro:
-
l-Proline
- l-Cys:
-
l-Cysteine
- dl--T2C:
-
dl--Thiazolidine-2-carboxylate
- l-GSAL:
-
l-Glutamate γ-semialdehyde
- GSALDH:
-
l-GSAL dehydrogenase
- IAM:
-
Iodoacetamide
- NADH:
-
Reduced nicotinamide adenine dinucleotide
- NAD+ :
-
Oxidized nicotinamide adenine dinucleotide
- NAD(P)H:
-
Reduced nicotinamide adenine dinucleotide (phosphate)
- NAD(P)+ :
-
Oxidized nicotinamide adenine dinucleotide (phosphate)
- l-P5C:
-
l-∆1-Pyrroline-5-carboxylate
- PYCR:
-
∆1-Pyrroline-5-carboxylate reductase
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Acknowledgements
We thank Drs. Thomas Clemente, Daniel Schachtman, Jaekwon Lee, Paul Black, and Concetta C. DiRusso (all from University of Nebraska-Lincoln) for providing access equipment in their laboratories for this study.
Funding
Research reported in this publication was supported in part by the National Institute of General Medical Sciences of the National Institutes of Health under award numbers R01GM065546 (J.J.T.) and R01GM132640 (J.J.T. and D.F. B.).
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SMP and DFB designed the experiments. SMP conducted the experiments and analyzed the data. JS performed and analyzed the data of the LC-ESI tandem mass spectrometry of l-T4C reaction mixtures with and without PYCR1 and PYCR2 enzymes. KMS purified PYCR1. All authors contributed to data analysis, interpretation, and writing of the manuscript; all have approved the final version of the manuscript.
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Patel, S.M., Seravalli, J., Stiers, K.M. et al. Kinetics of human pyrroline-5-carboxylate reductase in l-thioproline metabolism. Amino Acids 53, 1863–1874 (2021). https://doi.org/10.1007/s00726-021-03095-4
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DOI: https://doi.org/10.1007/s00726-021-03095-4