PDBsum entry 6x9c

Oxidoreductase

PDB id: 6x9c

Contents

Protein chains

1209 a.a.

Ligands

NAI ×2

PEG

FMT

FDA ×2

SO4 ×5

PRO ×3

PGE

Metals

_MG ×2

Waters ×1992

PDB id:

6x9c

Name:

Oxidoreductase

Title:

Structure of proline utilization a with l-proline bound in the l- glutamate-gamma-semialdehyde dehydrogenase active site

Structure:

Bifunctional protein puta. Chain: a, b. Engineered: yes

Source:

Sinorhizobium meliloti (strain sm11). Organism_taxid: 707241. Strain: sm11. Gene: puta, sm11_chr0102. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

1.44Å R-factor: 0.181 R-free: 0.205

Authors:

J.J.Tanner,A.C.Campbell

Key ref:

A.C.Campbell et al. (2020). Structural analysis of prolines and hydroxyprolines binding to the l-glutamate-γ-semialdehyde dehydrogenase active site of bifunctional proline utilization A. Arch Biochem Biophys, 698, 108727. PubMed id: 33333077 DOI: 10.1016/j.abb.2020.108727

Date:

02-Jun-20 Release date: 30-Dec-20

Protein chains

F7X6I3  (F7X6I3_SINMM) -  Bifunctional protein PutA from Sinorhizobium meliloti (strain SM11)

Seq: 1233 a.a.

Struc: 1209 a.a.

Enzyme reactions

• Enzyme class 2: E.C.1.2.1.88 - L-glutamate gamma-semialdehyde dehydrogenase.
• Reaction: L-glutamate 5-semialdehyde + NAD⁺ + H2O = L-glutamate + NADH + 2 H⁺

L-glutamate 5-semialdehyde + NAD(+) (Bound ligand (Het Group name = PRO) matches with 88.89% similarity) + H2O (Bound ligand (Het Group name = NAI) corresponds exactly) = L-glutamate + NADH + 2 × H(+)

• Enzyme class 3: E.C.1.5.5.2 - proline dehydrogenase.
• Reaction: L-proline + a quinone = (S)-1-pyrroline-5-carboxylate + a quinol + H⁺

L-proline + quinone (Bound ligand (Het Group name = PRO) corresponds exactly) = (S)-1-pyrroline-5-carboxylate + quinol + H(+)

• Cofactor: FAD

FAD (Bound ligand (Het Group name = FDA) corresponds exactly)

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1016/j.abb.2020.108727

Arch Biochem Biophys 698:108727 (2020)

PubMed id: 33333077

Structural analysis of prolines and hydroxyprolines binding to the l-glutamate-γ-semialdehyde dehydrogenase active site of bifunctional proline utilization A.

A.C.Campbell, A.N.Bogner, Y.Mao, D.F.Becker, J.J.Tanner.

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.