PDBsum entry 6pyp

Oxidoreductase

PDB id: 6pyp

Contents

Protein chains

348 a.a.

Ligands

NAD

PO4

3SY

POP

Metals

__K

Waters ×383

PDB id:

6pyp

Name:

Oxidoreductase

Title:

Binary complex of human glycerol 3-phosphate dehydrogenase, r269a mutant

Structure:

Glycerol-3-phosphate dehydrogenase [nad(+)], cytoplasmic. Chain: a, b. Synonym: gpdh-c. Engineered: yes. Mutation: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: gpd1. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

1.95Å R-factor: 0.167 R-free: 0.190

Authors:

A.M.Gulick

Key ref:

A.R.Mhashal et al. (2020). Modeling the Role of a Flexible Loop and Active Site Side Chains in Hydride Transfer Catalyzed by Glycerol-3-phosphate Dehydrogenase. ACS Catal, 10, 11253-11267. PubMed id: 33042609 DOI: 10.1021/acscatal.0c02757

Date:

30-Jul-19 Release date: 08-Jul-20

Protein chains

P21695  (GPDA_HUMAN) -  Glycerol-3-phosphate dehydrogenase [NAD(+)], cytoplasmic from Homo sapiens

Seq: 349 a.a.

Struc: 348 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.1.1.1.8 - glycerol-3-phosphate dehydrogenase (NAD(+)).
• Reaction: sn-glycerol 3-phosphate + NAD⁺ = dihydroxyacetone phosphate + NADH + H⁺

sn-glycerol 3-phosphate (Bound ligand (Het Group name = NAD) corresponds exactly) + NAD(+) (Bound ligand (Het Group name = PO4) matches with 50.00% similarity) = dihydroxyacetone phosphate + NADH + H(+)

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

reference

DOI no: 10.1021/acscatal.0c02757

ACS Catal 10:11253-11267 (2020)

PubMed id: 33042609

Modeling the Role of a Flexible Loop and Active Site Side Chains in Hydride Transfer Catalyzed by Glycerol-3-phosphate Dehydrogenase.

A.R.Mhashal, A.Romero-Rivera, L.S.Mydy, J.R.Cristobal, A.M.Gulick, J.P.Richard, S.C.L.Kamerlin.

ABSTRACT

Glycerol-3-phosphate dehydrogenase is a biomedically important enzyme that plays a crucial role in lipid biosynthesis. It is activated by a ligand-gated conformational change that is necessary for the enzyme to reach a catalytically competent conformation capable of efficient transition-state stabilization. While the human form (hlGPDH) has been the subject of extensive structural and biochemical studies, corresponding computational studies to support and extend experimental observations have been lacking. We perform here detailed empirical valence bond and Hamiltonian replica exchange molecular dynamics simulations of wild-type hlGPDH and its variants, as well as providing a crystal structure of the binary hlGPDH·NAD R269A variant where the enzyme is present in the open conformation. We estimated the activation free energies for the hydride transfer reaction in wild-type and substituted hlGPDH and investigated the effect of mutations on catalysis from a detailed structural study. In particular, the K120A and R269A variants increase both the volume and solvent exposure of the active site, with concomitant loss of catalytic activity. In addition, the R269 side chain interacts with both the Q295 side chain on the catalytic loop, and the substrate phosphodianion. Our structural data and simulations illustrate the critical role of this side chain in facilitating the closure of hlGPDH into a catalytically competent conformation, through modulating the flexibility of a key catalytic loop (292-LNGQKL-297). This, in turn, rationalizes a tremendous 41,000 fold decrease experimentally in the turnover number, k_cat, upon truncating this residue, as loop closure is essential for both correct positioning of key catalytic residues in the active site, as well as sequestering the active site from the solvent. Taken together, our data highlight the importance of this ligand-gated conformational change in catalysis, a feature that can be exploited both for protein engineering and for the design of allosteric inhibitors targeting this biomedically important enzyme.