9D7L

Minimal PutA proline dehydrogenase domain (design #2) covalently inactivated by (S)-but-3-yn-2-ylglycine

This is a non-PDB format compatible entry.

Summary for 9D7L

• Entry DOI: 10.2210/pdb9d7l/pdb
• Descriptor: Bifunctional protein PutA, {[(3E)-4-{10-[(2S,3S,4R)-5-{[(R)-{[(R)-{[(2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl]oxy}(hydroxy)phosphoryl]oxy}-2,3,4-trihydroxypentyl]-7,8-dimethyl-2,4-dioxo-2,3,4,10-tetrahydrobenzo[g]pteridin-5(1H)-yl}but-3-en-2-ylidene]amino}acetic acid (non-preferred name), FORMIC ACID, ... (4 entities in total)
• Functional Keywords: beta/alpha barrel, flavoenzyme, proline catabolism, oxidoreductase
• Biological source: Sinorhizobium meliloti; More
• Total number of polymer chains: 2
• Total formula weight: 89465.41

Authors

Tanner, J.J.,Meeks, K.R.,Ji, J. (deposition date: 2024-08-16, release date: 2025-02-05, Last modification date: 2025-02-19)

Primary citation

Meeks, K.R.,Ji, J.,Scott, G.K.,Campbell, A.C.,Nix, J.C.,Tadeo, A.,Ellerby, L.M.,Benz, C.C.,Tanner, J.J.
Biochemical, structural, and cellular characterization of S-but-3-yn-2-ylglycine as a mechanism-based covalent inactivator of the flavoenzyme proline dehydrogenase.
Arch.Biochem.Biophys., 765:110319-110319, 2025

PubMed Abstract: The mitochondrial flavoenzymes proline dehydrogenase (PRODH) and hydroxyproline dehydrogenase (PRODH2) catalyze the first steps of proline and hydroxyproline catabolism, respectively. The enzymes are targets for chemical probe development because of their roles in cancer cell metabolism (PRODH) and primary hyperoxaluria (PRODH2). Mechanism-based inactivators of PRODH target the FAD by covalently modifying the N5 atom, with N-propargylglycine (NPPG) being the current best-in-class of this type of probe. Here we investigated a close analog of NPPG, but-3-yn-2-ylglycine (B32G), distinguished by having a methyl group adjacent to the ethynyl group of the propargyl warhead. UV-visible spectroscopy shows that a bacterial PRODH catalyzes the oxidation of the S-enantiomer of B32G, a necessary first step in mechanism-based inactivation. In contrast, the enzyme does not react with the R-enantiomer. Enzyme activity assays show that S-B32G inhibits bacterial PRODH in a time-dependent manner consistent with covalent inactivation; however, the inactivation efficiency is ∼600-times lower than NPPG. We generated the crystal structure of PRODH inactivated by S-B32G at 1.68 Å resolution and found that inactivation induces a covalent link between the FAD N5 and the ε-nitrogen of an active site lysine, confirming that S-B32G follows the same mechanism as NPPG. Despite its lower inactivation efficiency at the purified bacterial enzyme, S-B32G exhibited comparable activity to NPPG against PRODH and PRODH2 in human cells and mouse livers. Molecular modeling is used to rationalize the stereospecificity of B32G.

PubMed: 39870289
DOI: 10.1016/j.abb.2025.110319

Experimental method

X-RAY DIFFRACTION (1.68 Å)