Biochemistry, Department of


First Advisor

Donald F. Becker

Date of this Version

Summer 8-2020


Patel S.M. (2020). Characterization of Human Pyrroline-5-Carboxylate Reductase Enzymes Responsible for L-Proline Biosynthesis [Ph.D. dissertation, Department of Biochemistry, University of Nebraska-Lincoln].


A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy, Major: Biochemistry, Under the Supervision of Professor Donald F. Becker. Lincoln, Nebraska: August, 2020

Copyright 2020 Sagar Mahendrakumar Patel


Pyrroline-5-carboxylate reductases (EC are important housekeeping enzymes of L-proline biosynthesis, which generate L-proline and influence redox cycling of NAD(P)H/NAD(P)+ to support cellular growth in all domains of life. Structural evidence from X-ray crystal structures of HsPYCR1 (PDB codes 5UAT, 5UAU, and 5UAV) shows both NADPH bound in the N-terminal Rao-Rossmann fold motif and an important hydrogen bond or proton donor role for Thr238 with L-P5C. The Thr238Ala mutation results in 10-fold loss in catalytic efficiency with varied L-P5C relative to the wild-type enzyme, thus indicating Thr238’s potential hydrogen bond and proton donation to L-P5C is critical for catalysis. For HsPYCR2, Henri-Michaelis-Menten kinetic analysis reveals 164-fold loss in catalytic efficiency for the Arg119Cys mutant, with varied L-P5C and fixed NADH, relative to wild-type HsPYCR2. Profound effects on thermostability and secondary structure characteristics of the Arg251Cys mutant were determined by thermal shift assays and circular dichroism spectroscopy, respectively. Product(s) inhibition kinetics collectively indicate NADP+ and NAD+ are mixed noncompetitive inhibitors against NADPH and NADH, respectively, whereas L-proline is a competitive inhibitor against L-P5C. Taken together, these findings support a sequential-ordered binding enzyme mechanism of L-P5C binding first followed by NAD(P)H-binding.

The ability of HsPYCR1 and 2 for reverse enzyme activity was observed with L-T4C as the reducing substrate. Structural evidence of a HsPYCR1─L-T4C binary complex, reverse direction saturation kinetics for both isozymes, and ligand inhibition kinetics evidence of L-proline as a competitive inhibitor with varied L-T4C, all indicate L-T4C shares the same active site as L-proline. Upon further evaluation of reverse direction reactions, we discovered association reactions between tris(alkyl)phosphine compounds and NAD+. Stopped-flow absorbance kinetics demonstrated rapid and reversible NAD+─tris(alkyl)phosphine nonenzymatic reactions with optimal absorbance at 334 nm for the reaction product. NMR spectroscopy identified a covalent adduct between the phosphorus of tris(2-carboxyethyl)phosphine or tris(3-hydroxypropyl)phosphine interacting at C4 of dihydronicotinamide ring of NAD+. Ultimately, this thesis dissertation provides strong structural and kinetic insights into human pyrroline-5-carboxylate reductase enzymes responsible for L-proline biosynthesis.

Advisor: Donald F. Becker