TY - JOUR
T1 - Disease variants of human Δ1-pyrroline-5-carboxylate reductase 2 (PYCR2)
AU - Patel, Sagar M.
AU - Seravalli, Javier
AU - Liang, Xinwen
AU - Tanner, John J.
AU - Becker, Donald F.
N1 - Funding Information:
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 (JJT) and R01GM132640 (JJT and DFB) .
Publisher Copyright:
© 2021 Elsevier Inc.
PY - 2021/5/30
Y1 - 2021/5/30
N2 - Pyrroline-5-carboxylate reductase (PYCR in humans) catalyzes the final step of L-proline biosynthesis by catalyzing the reduction of L-Δ1-pyrroline-5-carboxylate (L-P5C) to L-proline using NAD(P)H as the hydride donor. In humans, three isoforms PYCR1, PYCR2, and PYCR3 are known. Recent genome-wide association and clinical studies have revealed that homozygous mutations in human PYCR2 lead to postnatal microcephaly and hypomyelination, including hypomyelinating leukodystrophy type 10. To uncover biochemical and structural insights into human PYCR2, we characterized the steady-state kinetics of the wild-type enzyme along with two protein variants, Arg119Cys and Arg251Cys, that were previously identified in patients with microcephaly and hypomyelination. Kinetic measurements with PYCR2 suggest a sequential binding mechanism with L-P5C binding before NAD(P)H and NAD(P)+ releasing before L-Pro. Both disease-related variants are catalytically impaired. Depending on whether NADPH or NADH was used, the catalytic efficiency of the R119C protein variant was 40 or 366 times lower than that of the wild-type enzyme, while the catalytic efficiency of the R251C protein variant was 7 or 26 times lower than that of the wild-type enzyme. In addition, thermostability and circular dichroism measurements suggest that the R251C protein variant has a pronounced folding defect. These results are consistent with the involvement of Arg119Cys and Arg251Cys in disease pathology.
AB - Pyrroline-5-carboxylate reductase (PYCR in humans) catalyzes the final step of L-proline biosynthesis by catalyzing the reduction of L-Δ1-pyrroline-5-carboxylate (L-P5C) to L-proline using NAD(P)H as the hydride donor. In humans, three isoforms PYCR1, PYCR2, and PYCR3 are known. Recent genome-wide association and clinical studies have revealed that homozygous mutations in human PYCR2 lead to postnatal microcephaly and hypomyelination, including hypomyelinating leukodystrophy type 10. To uncover biochemical and structural insights into human PYCR2, we characterized the steady-state kinetics of the wild-type enzyme along with two protein variants, Arg119Cys and Arg251Cys, that were previously identified in patients with microcephaly and hypomyelination. Kinetic measurements with PYCR2 suggest a sequential binding mechanism with L-P5C binding before NAD(P)H and NAD(P)+ releasing before L-Pro. Both disease-related variants are catalytically impaired. Depending on whether NADPH or NADH was used, the catalytic efficiency of the R119C protein variant was 40 or 366 times lower than that of the wild-type enzyme, while the catalytic efficiency of the R251C protein variant was 7 or 26 times lower than that of the wild-type enzyme. In addition, thermostability and circular dichroism measurements suggest that the R251C protein variant has a pronounced folding defect. These results are consistent with the involvement of Arg119Cys and Arg251Cys in disease pathology.
KW - Circular dichroism
KW - Product inhibition kinetics
KW - Proline biosynthesis
KW - Protein thermostability
KW - Steady-state kinetics
KW - Substrate-binding order
KW - Δ-pyrroline-5-carboxylate reductase
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U2 - 10.1016/j.abb.2021.108852
DO - 10.1016/j.abb.2021.108852
M3 - Article
C2 - 33771508
AN - SCOPUS:85104969793
SN - 0003-9861
VL - 703
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
M1 - 108852
ER -