Covalent Modification of the Flavin in Proline Dehydrogenase by Thiazolidine-2-Carboxylate

Abstract

Proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the FAD-dependent 2-electron oxidation of l-proline to Δ¹-pyrroline-5-carboxylate. PRODH has emerged as a possible cancer therapy target, and thus the inhibition of PRODH is of interest. Here we show that the proline analogue thiazolidine-2-carboxylate (T2C) is a mechanism-based inactivator of PRODH. Structures of the bifunctional proline catabolic enzyme proline utilization A (PutA) determined from crystals grown in the presence of T2C feature strong electron density for a 5-membered ring species resembling l-T2C covalently bound to the N5 of the FAD in the PRODH domain. The modified FAD exhibits a large butterfly bend angle, indicating that the FAD is locked into the 2-electron reduced state. Reduction of the FAD is consistent with the crystals lacking the distinctive yellow color of the oxidized enzyme and stopped-flow kinetic data showing that T2C is a substrate for the PRODH domain of PutA. A mechanism is proposed in which PRODH catalyzes the oxidation of T2C at the C atom adjacent to the S atom of the thiazolidine ring (C5). Then, the N5 atom of the reduced FAD attacks the C5 of the oxidized T2C species, resulting in the covalent adduct observed in the crystal structure. To our knowledge, this is the first report of T2C inactivating (or inhibiting) PRODH or any other flavoenzyme. These results may inform the design of new mechanism-based inactivators of PRODH for use as chemical probes to study the roles of proline metabolism in cancer.

Figure 1.

Electron density evidence (1.74 Å resolution) for covalent modification of the FAD in PRODH by T2C. (A) Modified FAD of SmPutA covered by a polder omit map (4σ). (B) Polder omit map contoured at 12σ, showing a peak indicating the S atom. The FAD and T2C are colored gray and pink, respectively.

Figure 2.

Interactions of (A) the T2C covalent adduct with the active site, compared with (B) the noncovalent THFA complex (PDB 5KF6).

Figure 3.

Conformation of the modified FAD is consistent with 2-electron reduction. (A) Comparison of PutA structures highlighting the bend of the isoalloxazine. From left to right: oxidized SmPutA complexed with THFA (PDB 5KF6); T2C-modified SmPutA; Geobacter sulfurreducens PutA (GsPutA) reduced by dithionite (PDB 4NMD); NPPG-inactivated GsPutA (PDB 4NME). (B) Comparison of the ribityl chain conformations in PutA structures. From left to right: oxidized SmPutA complexed with THFA (PDB 5KF6); T2C-modified SmPutA; GsPutA reduced by dithionite (PDB 4NMD); NPPG-inactivated GsPutA (PDB 4NME).

Figure 4.

Electron density evidence for covalent modification of cysteines in the crystal harvested on day 30. (A) Location of the modified cysteine residues in SmPutA. SmPutA is colored by domain, and the modified cysteines are shown as spheres. (B) Modified Cys46, Cys470, and Cys638 of SmPutA observed in the structure determined from a crystal harvested on day 30. The cage represents a refined 2F_o−F_c map (1σ). (C) Equivalent cysteine residues are not modified in the structure of T2C-inactivated SmPutA determined from a crystal harvested on day 14 (2F_o−F_c map, 1σ). (D) Depiction of the disorder in helix 7 caused by the modification of Cys470. The native (PDB 5KF6) and T2C-modified enzymes are colored yellow and gray, respectively. THFA in the unmodified structures is colored cyan.

Figure 5.

Stopped-flow kinetics of SmPutA with proline and T2C. Oxidized SmPutA (20.3 μM after mixing) was mixed with (A) 20 mM l-proline (after mixing) and (B) 40 mM _D,l-T2C (after mixing) and was monitored by stopped-flow multiwavelength absorption. The spectra shown were recorded at 0.005−197 s after mixing. (C) Single wavelength traces with proline at 452 nm (blue) and 340 nm (orange) and with T2C at 452 nm (red) and 340 nm (gray). Observed rate constants (k_obs) for the reduction of the FAD by proline and T2C, and formation of NADH in the reaction with proline, were estimated by fitting the traces to a single exponential equation. No formation of NADH was observed in the reaction of SmPutA with T2C.

Figure 6.

Kinetics of inactivation of SmPutA by T2C. The points represent the PRODH activity measured after preincubating SmPutA with 2 mM T2C for various time periods and removing the excess T2C. The error bars represent the standard deviation of three technical replicates. The curve represents the fit to an exponential decay function, which yields an apparent pseudo-first order rate constant for enzyme inactivation of 0.44 ± 0.08 days⁻¹.

Scheme 1.. Reactions of Proline Catabolism

Scheme 2.. Chemical Structures of THFA and

l-T2C

Scheme 3.. Proposed Pathways for the Oxidation of T2C by the PRODH Active Site of PutA^a

^a(A) PRODH-catalyzed oxidation of T2C at the C4 atom, which is analogous to the oxidation of proline to P5C. Species 4 is proposed to modify surface cysteine residues of PutA to produce 5 (see Figure 4). (B) PRODH-catalyzed oxidation of T2C at the C5 atom, resulting in the covalent modification of the FAD and inactivation of the PRODH site (see Figure 1).