6GI8

Crystal structure of pentaerythritol tetranitrate reductase (PETNR) mutant L25A

6GI8 の概要

• エントリーDOI: 10.2210/pdb6gi8/pdb
• 分子名称: Pentaerythritol tetranitrate reductase, FLAVIN MONONUCLEOTIDE, ACETATE ION, ... (4 entities in total)
• 機能のキーワード: mutant l25a, flavoprotein
• 由来する生物種: Enterobacter cloacae
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 41113.67

構造登録者

Levy, C.W. (登録日: 2018-05-10, 公開日: 2019-03-20, 最終更新日: 2024-01-17)

主引用文献

Iorgu, A.I.,Baxter, N.J.,Cliff, M.J.,Levy, C.,Waltho, J.P.,Hay, S.,Scrutton, N.S.
Nonequivalence of Second Sphere "Noncatalytic" Residues in Pentaerythritol Tetranitrate Reductase in Relation to Local Dynamics Linked to H-Transfer in Reactions with NADH and NADPH Coenzymes.
Acs Catalysis, 8:11589-11599, 2018

PubMed Abstract: Many enzymes that catalyze hydride transfer reactions work via a mechanism dominated by quantum mechanical tunneling. The involvement of fast vibrational modes of the reactive complex is often inferred in these reactions, as in the case of the NAD(P)H-dependent pentaerythritol tetranitrate reductase (PETNR). Herein, we interrogated the H-transfer mechanism in PETNR by designing conservative (L25I and I107L) and side chain shortening (L25A and I107A) PETNR variants and using a combination of experimental approaches (stopped-flow rapid kinetics, X-ray crystallography, isotope/temperature dependence studies of H-transfer and NMR spectroscopy). X-ray data show subtle changes in the local environment of the targeted side chains but no major structural perturbation caused by mutagenesis of these two second sphere active site residues. However, temperature dependence studies of H-transfer revealed a coenzyme-specific and complex thermodynamic equilibrium between different reactive configurations in PETNR-coenzyme complexes. We find that mutagenesis of these second sphere "noncatalytic" residues affects differently the reactivity of PETNR with NADPH and NADH coenzymes. We attribute this to subtle, dynamic structural changes in the PETNR active site, the effects of which impact differently in the nonequivalent reactive geometries of PETNR-NADH and PETNR-NADPH complexes. This inference is confirmed through changes observed in the NMR chemical shift data for PETNR complexes with unreactive 1,4,5,6-tetrahydro-NAD(P) analogues. We show that H-transfer rates can (to some extent) be buffered through entropy-enthalpy compensation, but that use of integrated experimental tools reveals hidden complexities that implicate a role for dynamics in this relatively simple H-transfer reaction. Similar approaches are likely to be informative in other enzymes to understand the relative importance of (distal) hydrophobic side chains and dynamics in controlling the rates of enzymatic H-transfer.

PubMed: 31119061
DOI: 10.1021/acscatal.8b02810

実験手法

X-RAY DIFFRACTION (1.42 Å)