1. Materials & Methods
Acknowledgments &
References
Scheme 1: Enzymatic reactions of proline metabolism in the mitochondrion,
adapted from Becker & Tanner. Handbook of Flavoproteins. 2013.5
Kinetic characterization of human ∆1-pyrroline-5-carboxylate
reductase 2 (HsPYCR2) wild-type and Arg251Cys mutant
Sagar M. Patel and Donald F. Becker
Redox Biology Center, Department of Biochemistry, University of Nebraska-Lincoln, Lincoln NE
Abstract
Proline Metabolism
Conclusions
References
1. De Ingeniis, J., Ratnikov, B., et al. (2012) Functional specialization in proline biosynthesis of
melanoma. PLoS One. 7, e45190
2. Martinelli, D., Haberle, J., et al. (2012) Understanding pyrroline-5-carboxylate synthetase
deficiency: clinical, molecular, functional, and expression studies, structure-based analysis, and
novel therapy with arginine. J Inherit Metab Dis. 35, 761-776
3. Nakayama, T., A. Al-Maawali, et al. (2015) Mutations in PYCR2, Encoding Pyrroline-5-
Carboxylate Reductase 2, Cause Microcephaly and Hypomyelination. The American Journal of
Human Genetics. 96, 709-719
4. Deutch, C. E., Klarstrom, J. L., Link, C. L., & Ricciardi, D. L. (2001). Oxidation of L-thiazolidine-4-
carboxylate by Δ1-pyrroline-5-carboxylate reductase in Escherichia coli. Current
Microbiology, 42(6), 442-446.
5. Hille, R., Susan M. Miller, and Bruce Palfey, eds. "Ch. 2. PutA and proline
metabolism." Handbook of Flavoproteins. Vol. 1. Berlin: De Gruyter, 2013. Print. p. 32
6. Yang, H., Yang, T., Baur, J. A., Perez, E., Matsui, T., Carmona, J. J.,& Sinclair, D. A. (2007).
Nutrient-sensitive mitochondrial NAD+ levels dictate cell survival. Cell, 130(6), 1095-1107.
7. Williams, I., & Frank, L. (1975). Improved chemical synthesis and enzymatic assay of Δ1-
pyrroline-5-carboxylic acid. Analytical biochemistry, 64 (1), 85-97.
8. Merrill, M. J., Yeh, G. C., & Phang, J. M. (1989). Purified human erythrocyte pyrroline-5-
carboxylate reductase. Preferential oxidation of NADPH. Journal of Biological Chemistry, 264(16),
9352-9358.
9. Lin, S. J., & Guarente, L. (2003). Nicotinamide adenine dinucleotide, a metabolic regulator of
transcription, longevity and disease. Current opinion in cell biology, 15(2), 241-246.
Special thanks to:
• Dr. Donald F. Becker
• Shelbi Christgen
• Dr. Xinwen Liang
• Dr. Julia Zhang
• Dr. Javier Seravalli
• Joshua L. Floth
• Jacob Wilkinson
• Dr. Benjamin W.
Arentson
• Dr. John J. Tanner
Proline is synthesized from glutamate by the enzymes ∆1-pyrroline-5-
carboxylate synthetase (PYCS) and P5C reductase (PYCR); the latter
enzyme is the focus of our research as shown in Scheme 1. In humans,
three isoforms of PYCR (PYCR1, PYCR2 and PYCRL) are known, with each
isoform catalyzing a reversible reaction involving the reduction of L-P5C into
L-proline with concomitant oxidation of NAD(P)H into NAD(P)+.1 PYCR1 and
PYCR2 are localized in the mitochondrion whereas PYCRL is found in the
cytosol.1 Over the past few decades, growing evidence has shown that
genetic disorders and deficiencies of human PYCR1 and PYCR2 are
associated with neurological diseases (i.e. postnatal microcephaly,
hypomyelination) as well as skin conditions (i.e. cutis laxa syndrome,
progeroid skin changes, melanoma).2,3 The goal of this project is to
characterize the biochemical penalties of PYCR2 mutation Arg251Cys found
in patients. Our hypothesis is that PYCR2 mutant R251C will demonstrate a
kinetic profile distinct from its wild-type counterpart, thus suggesting potential
impact upon proline biosynthesis. PYCR2 mutation R251C has been linked
to mental disorders and is hypothesized to interfere with dimerization of
PYCR2.3 Proline levels are normal in fibroblast samples from patients with
the above PYCR2 mutation.3 Thus, it is not clear how the PYCR2 mutation
contributes to the disease phenotype. Site-directed mutagenesis of human
PYCR2 wild-type was performed to generate mutant R251C. PYCR2 wild-
type and mutant were expressed recombinantly in E. coli, purified, and
characterized for enzymatic activity in a method modified from Deutch et al.4
by measuring loss of absorbance at 340 nm due to NAD(P)+ product
formation. The kinetic parameters (KM, kcat) will be determined for PYCR2
wild-type and mutant variant to assess the impact of the R251C mutation.
Research supported by
grants:
• Redox Biology Center
(P30 GM103335)
• National Institutes of
Health
(R01 GM061068)
Data as (mean ± SD) of n=3.
• WT enzyme has substrate specificity value kcat /KM for NADH ~4-
fold higher than R251C mutant enzyme, which suggests R251C
mutation has a slightly lower catalytic efficiency for NADH than
WT enzyme.
• WT enzyme demonstrates fairly similar substrate specificity
values kcat /KM for NADH and NADPH cofactors, however in
context of NADPH cofactor, WT enzyme demonstrates ~5-fold
higher kcat /KM than R251C mutant, which suggests R251C
mutation has slightly lower catalytic efficiency for NAPDH than
WT enzyme, owing to R251C mutant’s large KM value
• Previous kinetic observations may be relevant for NADH or NAD+
in physiological context of mammalian cells, because Yang H et
al.’s MALDI-MS method estimated [NAD+] in mitochondria of
HEK293 cells is 245.6 μM, corresponding to 2053 pmol NAD+/mg
mitochondrial protein6 where that physiological [NAD+] is nearly
2-fold greater than the observed NADH KM value for the R251C
mutant enzyme. Considering that NADH may be lower than NAD+
levels (e.g., physiologic NAD+/NADH ratio ~ 0.1-10 in mammalian
cells9), it is possible that R251C mutant enzyme has lower
activity at physiological levels of NADH relative to WT enzyme.
• WT enzyme has fairly similar kcat /KM substrate specificity value
for L-P5C substrate as that of R251C mutant enzyme whether
NADH or NADPH is varied, which suggests R251C mutation
does not have large kinetic effect when varying [D,L-P5C].
• Further characterizations of X-ray crystal structures and of
oligomeric states of WT and R251C mutant enzymes will be done
to better understand how mutation in enzyme may contribute to
disease state.
Table 1. Kinetic Properties of HsPYCR2 Enzyme Samples (n=3)
Data and calculations are presented as (mean ± standard error) based on model fit for Equation: Ligand Binding, one site saturation by SigmaPlot 12.0 (Systat Software, Inc).
† Since measurements based on use of concentration of RACEMIC mixture of D- and L-P5C & hPYCR2 enzyme is specific for the substrate L isomer of P5C only, KM calculation corrected as ½-values assuming
½ of RACEMIC substrate (L-isomer) responsible for kinetics.
‡ These results were obtained in lower TOTAL IONIC STRENGTH reaction mixtures.
Protein purification: First, full-length wild-type {HsPYCR2} was generated using PCR-based cloning strategy for recombinant
overexpression in a pKA8H vector with a TEV protease cleavable N-terminal (8x)His-tag in E. coli strain BL21(DE3) pLysS cells.
Cells were cultured in LB broth with ampicillin & chloramphenicol and 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG,
ThermoScientific) was added at OD600~ 0.8-1.0 for 3 hours at 37°C to induce expression of PYCR2. Next, cells were harvested,
spun down, and resuspended in cell lysis buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-base or NaH2PO4, 500 µM THP
reducing agent, 0.1% TritonX-100 detergent & 10% glycerol) containing 5 protease inhibitors with 0.1% TritonX-100 detergent
and 500 µM THP. Next, cells were further lysed by large probe sonication and centrifuged to collect the supernatant. HsPYCR2
was purified by Ni2+-NTA-resin (QIAGEN) affinity column chromatography using column buffers of increasing [imidazole] from 5
mM  60 mM  500 mM. PCR-based site directed mutagenesis strategy using primers designed by New England BioLabs
NEBaseChanger online tool generated R251C mutant enzyme which was purified similarly to wild-type enzyme.
Racemic D,L-P5C substrate chemical synthesis: Utilized Williams I et al.7 protocol except using only ½ of each synthesis
component (D,L-5-HydroxyLysine MonoHydrochloride (SIGMA ALDRICH), Na-MetaPeriodate (SIGMA ALDRICH), glycerol
(Fisher), and HCl (Omnitrace EMD)) with reaction mixture loaded onto 2 cm (diam.) x 78 cm (height) column Dowex 50WX4,
hydrogen form, 200-400 mesh, 4% crosslinked CATION EXCHANGE resin on polystyrene containing sulfonic groups (SIGMA
ALDRICH) column washed with 1 M HCl to collect eluted fractions. Then, concentrations of racemic D,L-P5C substrate fractions
determined by using O(2)-AminoBenzaldehyde (ACROS ORGANICS) dissolved in 200-proof ethanol and Glycine-HCl Buffer, pH
2.0 for colorimetric assay using 96-well plate passed through Biotek plate-reader spectrophotometer set λ = 443 nm and
analyzed with KC4 computer software.
HsPYCR2 Enzyme Activity Assay: Utilizing methods modified from Deutch et al.4, Merrill MJ et al.8, & De Ingeniis et al.1,
we used Cary Kinetics computer software with Varian Cary Bio 100 UV-VIS spectrophotometer set λ = 340 nm and T = 37.0°C of
cuvette holder block set by Varian Cary (1x1) double-beam Peltier & maintained by pumped flow of 70% ethanol cooling water
bath, to measure loss of absorbance due to NAD(P)+ product formation performed in triplicate each over 2.5 min.-time course
with slope measured of reaction curves within range of 1.5 min. using Ruler-Least Squares option. Prepared 600 µl-reaction
mixtures in tall-window Cary quartz cuvette w/ following materials at FINAL concentrations after mixing:
• 0.1 M Tris buffer (pH 7.5)
• 1% Brij 35 detergent
• 1 mM EDTA chelator (pH 7.5)
remaining volume filled by 10 mM Tris-HCl buffer, pH 8.0 (FOR [NAD(P)H] varied series) & TOTAL IONIC STRENGTH =
450 to 480 mM Tris+Cl- by using IONIC STRENGTH BALANCING mixture of 1M HCl + added 1M Tris-HCl buffer, pH 9.0
to achieve pH 7.5) (FOR [D,L-P5C] varied series)
• fixed concentration of pH-neutralized D,L-P5C substrate at 1000 µM OR NAD(P)H cofactor substrate at 200 µM or 500 µM
• (1/10) dil. (8x)His-tag-HsPYCR2_WT enz. & (8x)His-tag-HsPYCR2_R251C MUTANT enz. with [FINAL enzyme after
mixing] = 0.0581 µM, so 0.00125 mg-worth
Ensuring [pH-neutralized D,L-P5C substrate] or [NAD(P)H cofactor substrate] >> [total protein enzyme] in a steady-state
kinetics situation for each enzyme activity assay, proceeded to use following Beer-Lambert Law rearranged equation for
calculation of enzyme activity as initial rate of product NAD(P)+ formation (in units µM s-1)
[NAD(P)+, in µM]
time, in s
=
A340 min
∈NAD(P)H, 6.22 mM−1cm−1 × cuvette path length of 1 cm or 0.25 cm
×
1 min
60 s
×
1000 µM
1 mM
Also, we employed 3-spot pH test strips (Ricca Chemical Co.) to determine pH of combined post-reaction samples ≈ 8.0 for each
enzyme activity assay.
37°Cdry
heatbath
Kepton
ice
wild-type R251C mutant
substrate KM (µM) Vlimit (µM s-1
) kcat (s-1
) kcat /KM (M-1
s-1
) KM (µM) Vlimit (µM s-1
) kcat (s-1
) kcat /KM (M-1
s-1
)
NADH 11.3 ± 3 0.35 ± 0.02 6.1 ± 0.3 (5.37 x 105
) ± 0.24 151 ± 24 1.14 ± 0.05 19.7 ± 0.9 (1.30 x 105
) ± 0.16
NADPH 80.0 ± 13 1.4 ± 0.09 24.7 ± 1.6 (3.09 x 105
) ± 0.17 586 ± 137 1.9 ± 0.19 33.0 ± 3.3 (5.63 x 104
) ± 0.25
D,L-P5C (with
varied NADH)
192 ± 30†
0.80 ± 0.04 13.8 ± 0.7 (7.17 x 104
) ± 0.16 192 ± 38†,‡
0.80 ± 0.05‡
13.8 ± 0.9‡
(7.16 x 104
) ± 0.21‡
D,L-P5C (with
varied NADPH)
121 ± 23†,‡
0.98 ± 0.06‡
16.9 ± 1.0‡
(1.39 x 105
) ± 0.62‡
375 ± 78†
2.36 ± 0.22 40.6 ± 3.7 (1.08 x 105
) ± 0.23
Data as (mean ± SD) of n=3. Data as (mean ± SD) of n=3.Data as (mean ± SD) of n=3.
Data as (mean ± SD) of n=3. Data as (mean ± SD) of n=3. Data as (mean ± SD) of n=3.
Data as (mean ± SD) of n=3. Data as (mean ± SD) of n=3.