4NME

Crystal structure of proline utilization A (PutA) from Geobacter sulfurreducens PCA inactivated by N-propargylglycine

Summary for 4NME

• Entry DOI: 10.2210/pdb4nme/pdb
• Related: 4NM9 4NMA 4NMB 4NMC 4NMD 4NMF
• Descriptor: Proline dehydrogenase and Delta-1-pyrroline-5-carboxylate dehydrogenase, N-propargylglycine-modified flavin adenine dinucleotide, 1,2-ETHANEDIOL, ... (4 entities in total)
• Functional Keywords: flavoenzyme, rossmann fold, aldehyde dehydrogenase, flavin adenine dinucleotide, nicotinamide adenine dinucleotide, proline catabolism, substrate channeling, bifunctional enzyme, mechanism-based inactivation, oxidoreductase
• Biological source: Geobacter sulfurreducens
• Total number of polymer chains: 2
• Total formula weight: 227439.76

Authors

Singh, H.,Tanner, J.J. (deposition date: 2013-11-14, release date: 2014-02-19, Last modification date: 2024-11-06)

Primary citation

Singh, H.,Arentson, B.W.,Becker, D.F.,Tanner, J.J.
Structures of the PutA peripheral membrane flavoenzyme reveal a dynamic substrate-channeling tunnel and the quinone-binding site.
Proc.Natl.Acad.Sci.USA, 111:3389-3394, 2014

PubMed Abstract: Proline utilization A (PutA) proteins are bifunctional peripheral membrane flavoenzymes that catalyze the oxidation of L-proline to L-glutamate by the sequential activities of proline dehydrogenase and aldehyde dehydrogenase domains. Located at the inner membrane of Gram-negative bacteria, PutAs play a major role in energy metabolism by coupling the oxidation of proline imported from the environment to the reduction of membrane-associated quinones. Here, we report seven crystal structures of the 1,004-residue PutA from Geobacter sulfurreducens, along with determination of the protein oligomeric state by small-angle X-ray scattering and kinetic characterization of substrate channeling and quinone reduction. The structures reveal an elaborate and dynamic tunnel system featuring a 75-Å-long tunnel that links the two active sites and six smaller tunnels that connect the main tunnel to the bulk medium. The locations of these tunnels and their responses to ligand binding and flavin reduction suggest hypotheses about how proline, water, and quinones enter the tunnel system and where L-glutamate exits. Kinetic measurements show that glutamate production from proline occurs without a lag phase, consistent with substrate channeling and implying that the observed tunnel is functionally relevant. Furthermore, the structure of reduced PutA complexed with menadione bisulfite reveals the elusive quinone-binding site. The benzoquinone binds within 4.0 Å of the flavin si face, consistent with direct electron transfer. The location of the quinone site implies that the concave surface of the PutA dimer approaches the membrane. Altogether, these results provide insight into how PutAs couple proline oxidation to quinone reduction.

PubMed: 24550478
DOI: 10.1073/pnas.1321621111

Experimental method

X-RAY DIFFRACTION (2.088 Å)