Pyrroline-5-carboxylate reductase (PYCR) is the final enzyme of proline biosynthesis that reversibly reduces L-P5C into L-proline using NAD(P)H as the hydride donor (Scheme 1). In humans, three isoforms (PYCR1, PYCR2, and PYCRL) are known. Recent genome-wide association and clinical studies have revealed that homozygous mutations in PYCR2 lead to an autosomal-recessive condition of postnatal microcephaly and hypomyelination. In addition, patients possessing homozygous PYCR2 variants were observed to co-segregate with disease marked by progressive microcephaly and hypomyelination. Another study found all PYCR2 homozygous mutations were fully penetrant without observed phenocopies within each family, thus demonstrating that hypomyelinating leukodystrophy type 10 with microcephaly was due to homozygous variants of PYCR2. In this study, we seek to characterize PYCR2 mutants R119C and R251C and to determine their biochemical penalties. Since HsPYCR1 and HsPYCR2 share 84.4% amino acid sequence identity, structural insights for HsPYCR2 are inferred from homology with an experimentally determined crystal structure of HsPYCR1 (Figure 1). From this model, Arg119 and Arg251 are predicted to be important for the catalytic activity and dimerization of PYCR2, respectively. Here, we establish the substrate binding order mechanism for wild-type PYCR2 by analysis of the products’ inhibition kinetics profiles. Also, we performed the purification of both PYCR2 mutants and characterized their enzyme activities and relative protein thermal stabilities. In particular, our data show a tremendous loss in catalytic activity of the R119C mutant with respect to P5C substrate ((kcat app/ KM app) = (0.0025 ± 0.0008) x 105 M-1 s-1)) compared to wild-type PYCR2 enzyme ((kcat app/ KM app) = (0.41 ± 0.02) x 105 M-1 s-1)) consistent with the location of Arg119 at the posterior wall of the enzyme active site.

Characterization and enzyme mechanism of human pyrroline-5-carboxylate reductase 2 (HsPYCR2) wild-type and disease variants

256th National Meeting (2018)

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