6UFP

Structure of proline utilization A with the FAD covalently modified by L-thiazolidine-2-carboxylate and three cysteines (Cys46, Cys470, Cys638) modified to S,S-(2-HYDROXYETHYL)THIOCYSTEINE

Summary for 6UFP

• Entry DOI: 10.2210/pdb6ufp/pdb
• Descriptor: Bifunctional protein PutA, DIHYDROFLAVINE-ADENINE DINUCLEOTIDE, (2S)-1,3-thiazolidine-2-carboxylic acid, ... (9 entities in total)
• Functional Keywords: flavoenzyme, rossmann fold, proline catabolism, substrate channeling, bifunctional enzyme, oxidoreductase
• Biological source: Sinorhizobium meliloti (strain SM11); More
• Total number of polymer chains: 2
• Total formula weight: 268352.88

Authors

Campbell, A.C.,Tanner, J.J. (deposition date: 2019-09-24, release date: 2020-03-18, Last modification date: 2024-11-13)

Primary citation

Campbell, A.C.,Becker, D.F.,Gates, K.S.,Tanner, J.J.
Covalent Modification of the Flavin in Proline Dehydrogenase by Thiazolidine-2-Carboxylate.
Acs Chem.Biol., 15:936-944, 2020

PubMed Abstract: Proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the FAD-dependent 2-electron oxidation of l-proline to Δ-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.

PubMed: 32159324
DOI: 10.1021/acschembio.9b00935

Experimental method

X-RAY DIFFRACTION (1.737 Å)