6X9A

Structure of proline utilization A with trans-4-hydroxy-D-proline bound in the L-glutamate-gamma-semialdehyde dehydrogenase active site

Summary for 6X9A

• Entry DOI: 10.2210/pdb6x9a/pdb
• Descriptor: Bifunctional protein PutA, FLAVIN-ADENINE DINUCLEOTIDE, FORMIC ACID, ... (8 entities in total)
• Functional Keywords: flavoenzyme, rossmann fold, proline dehydrognease, aldehyde dehydrogenase, proline catabolism, substrate channeling, bifunctional enzyme, oxidoreductase
• Biological source: Sinorhizobium meliloti (strain SM11)
• Total number of polymer chains: 2
• Total formula weight: 267266.93

Authors

Tanner, J.J.,Campbell, A.C. (deposition date: 2020-06-02, release date: 2020-12-30, Last modification date: 2023-10-18)

Primary citation

Campbell, A.C.,Bogner, A.N.,Mao, Y.,Becker, D.F.,Tanner, J.J.
Structural analysis of prolines and hydroxyprolines binding to the l-glutamate-gamma-semialdehyde dehydrogenase active site of bifunctional proline utilization A.
Arch.Biochem.Biophys., 698:108727-108727, 2020

PubMed Abstract: Proline utilization A (PutA) proteins are bifunctional proline catabolic enzymes that catalyze the 4-electron oxidation of l-proline to l-glutamate using spatially-separated proline dehydrogenase and l-glutamate-γ-semialdehyde dehydrogenase (GSALDH, a.k.a. ALDH4A1) active sites. The observation that l-proline inhibits both the GSALDH activity of PutA and monofunctional GSALDHs motivated us to study the inhibition of PutA by proline stereoisomers and analogs. Here we report five high-resolution crystal structures of PutA with the following ligands bound in the GSALDH active site: d-proline, trans-4-hydroxy-d-proline, cis-4-hydroxy-d-proline, l-proline, and trans-4-hydroxy-l-proline. Three of the structures are of ternary complexes of the enzyme with an inhibitor and either NAD or NADH. To our knowledge, the NADH complex is the first for any GSALDH. The structures reveal a conserved mode of recognition of the inhibitor carboxylate, which results in the pyrrolidine rings of the d- and l-isomers having different orientations and different hydrogen bonding environments. Activity assays show that the compounds are weak inhibitors with millimolar inhibition constants. Curiously, although the inhibitors occupy the aldehyde binding site, kinetic measurements show the inhibition is uncompetitive. Uncompetitive inhibition may involve proline binding to a remote site or to the enzyme-NADH complex. Together, the structural and kinetic data expand our understanding of how proline-like molecules interact with GSALDH, reveal insight into the relationship between stereochemistry and inhibitor affinity, and demonstrate the pitfalls of inferring the mechanism of inhibition from crystal structures alone.

PubMed: 33333077
DOI: 10.1016/j.abb.2020.108727

Experimental method

X-RAY DIFFRACTION (1.41 Å)