The structure of Medicago truncatula δ(1)-pyrroline-5-carboxylate reductase provides new insights into regulation of proline biosynthesis in plants

Abstract

The two pathways for proline biosynthesis in higher plants share the last step, the conversion of δ(1)-pyrroline-5-carboxylate (P5C) to L-proline, which is catalyzed by P5C reductase (P5CR, EC 1.5.1.2) with the use of NAD(P)H as a coenzyme. There is increasing amount of evidence to suggest a complex regulation of P5CR activity at the post-translational level, yet the molecular basis of these mechanisms is unknown. Here we report the three-dimensional structure of the P5CR enzyme from the model legume Medicago truncatula (Mt). The crystal structures of unliganded MtP5CR decamer, and its complexes with the products NAD(+), NADP(+), and L-proline were refined using x-ray diffraction data (at 1.7, 1.85, 1.95, and 2.1 Å resolution, respectively). Based on the presented structural data, the coenzyme preference for NADPH over NADH was explained, and NADPH is suggested to be the only coenzyme used by MtP5CR in vivo. Furthermore, the insensitivity of MtP5CR to feed-back inhibition by proline, revealed by enzymatic analysis, was correlated with structural features. Additionally, a mechanism for the modulation of enzyme activity by chloride anions is discussed, as well as the rationale for the possible development of effective enzyme inhibitors.

Keywords: P5C reductase; P5CR; abiotic stress; coenzyme preference; decamer; protein structure; salt stress.

Figure 1

Scheme of the enzymatic reaction catalyzed by P5CR.

Figure 2

Sequence alignment of P5CRs from various sources. The following organisms are listed and square brackets indicate UniProt accession numbers: Mt, Medicago truncatula [G7KRM5], Mt_2 indicates the second isoform of MtP5CR [A2Q2Y7]; At, Arabidopsis thaliana [P54904]; Os, Oryza sativa [Q8GT01]; Ps, Pisum sativum [Q04708]; Gm, Glycine max [K7KEQ2]; So, Spinacia oleracea, only partial sequence is available (Murahama et al., 2001); Hs, Homo sapiens [P32322]; Sp, Streptococcus pyogenes [Q9A1S9]; Nm, Neisseria meningitides [Q9K1N1]. Amino acid residues are colored according to the type of residue. Numbering on the top reflects the MtP5CR sequence, while that on the right of each row is protein-specific. Residues interacting with NAD(P)⁺ and L-proline in MtP5CR structure and conserved among other species are highlighted in gray and light blue, respectively. α-Helices, 3₁₀ helices and β-strands corresponding to MtP5CR structure are depicted on top as red, blue and yellow bars, respectively. L1, L3, and L19 indicate the location of loops 1, 3, and 19, which were discussed in the paper. Seven C-terminal residues from the human P5CR sequence were omitted. The human and the two bacterial sequences were chosen for alignment as their crystal structures had already been determined.

Figure 3

Effect of NADP⁺ on the activity of MtP5CR. The standard reaction mixture, containing 1 mM NADH or 0.5 mM NADPH, was added with increasing concentrations of the oxidized dinucleotide. The resulting activity was expressed as a percent of mean value in untreated controls. Three replications were carried out for each treatment. Non- linear regression analysis of data [log(inhibitor) vs. normalized response-Variable slope] was computed using Prism 6 for Windows, version 6.03.

Figure 4

Differential effect of Cl⁻ anions and Na⁺ cations on MtP5CR activity. To reduce the carryover of ions in the standard reaction mixture, the enzyme was assayed in the presence of 0.2 mM L-P5C (in 20 mM Tris-HCl buffer pH 7.5) with either 1 mM NADH or 0.5 mM NADPH as the electron donor. To avoid unspecific effects due to pH variations, after fixing the desired concentration of Cl⁻ and Na⁺ ions, the pH was brought to pH 7.75 with Tris base or tricine, respectively. Three replications were carried out for each treatment. Non-linear regression analysis of data [log(inhibitor) vs. normalized response-Variable slope for enzyme inhibition; log(agonist) vs. normalized response-Variable slope for enzyme stimulation] was computed using Prism 6 for Windows, version 6.03.

Figure 5

Overall structure of MtP5CR. (A) The top view. The black pentagon indicates the five-fold NCS axis. (B) The side view. Dimensions are given in Å. (C) The dimer of MtP5CR. The structures with NADP⁺ and with L-proline are superimposed (RMSD 0.34 Å) to show the binding sites of the coenzyme and product. The protein chain from the L-proline complex as well as MOPS and Cl⁻ from the NADP⁺ structure are omitted for clarity. Chains A and B are shown.

Figure 6

Comparison of known P5CR crystal structures. The presented enzymes originate from: MtP5CR/NADP⁺ complex (this work, light blue); HsP5CR/NAD⁺ (PDB ID: 2izz, orange); NmP5CR/NADP⁺ (2ag8, green); and SpP5CR/NADP⁺ (2ahr, purple). The protein molecules are shown as pipe-and-plank models, while the coenzymes are presented as ball-and-stick models. Secondary structure elements are denoted as: α, α-helices; η, 3₁₀ helices; β, β-strands.

Figure 7

L-Proline binding. Green mesh represents omit difference F_o-F_c electron density map contoured at the 4 σ level. Nicotinamide originates from the superposed chain B of the MtP5CR/NADP⁺ complex. Distances are given in Å.

Figure 8

Coenzyme (ball-and-stick model) binding by MtP5CR. (A) The MtP5CR/NAD⁺ complex. (B) The close-up view of the ribose 2′O-bound phosphate in NADP⁺. Both figures are oriented as in Figure 1C. Green mesh areas represent omit difference F_o–F_c electron density maps contoured at the 4 σ level. Water molecules that take part in binding interactions are depicted as small red balls. Chain B is represented as a yellow semitransparent surface. Note that Thr129 belongs to chain A and is only covered by a part of the C-terminal domain of chain B. The surface of interacting amino acids is semitransparent in (A) and clipped in (B) so it does not obscure the coenzymes. Black dashed lines represent bonding protein-coenzyme interactions. Intramolecular interaction between the N atom of nicotinamide and O from pyrophosphate group of NAD⁺ is depicted as a black solid line at the bottom of (A). Distances are given in Å.

Figure 9

Binding of activity modulators. (A) Binding of chloride in the active site. Note multiple hydrogen bonds between Cl⁻ and peptide amides. Yellow mesh represents anomalous difference electron density map contoured at 4 σ. Superposed L-proline (from the MtP5CR/L-proline complex, wires) is shown to visualize the Cl⁻ overlapping the carboxyl O. (B) Superposition of MtP5CR/L-proline and MtP5CR/NAD(P)⁺ complexes reveals that the MOPS molecule (originating from NAD(P)⁺ complexes) partially overlaps with L-proline (wires).

Figure 10

Kinetic analysis of the inhibition of MtP5CR by HEPES. Enzyme activity was measured at varying substrate concentrations in the presence of various inhibitor levels. Invariable substrates were fixed as in the standard mixture. Three replications were carried out for each treatment. Lines intersecting at the x-axis in the Lineweaver-Burk plots accounted for non-competitive inhibition types in all cases. Non-linear regression analysis of data [Michaelis-Menten fit, Lineweaver-Burk (double-reciprocal) transform, linear regression over Michaelis-Menten data, and non-competitive inhibition] was computed using Prism 6 for Windows, version 6.03.