5L1N

Pyrococcus horikoshii CoA Disulfide Reductase Quadruple Mutant

5L1N の概要

• エントリーDOI: 10.2210/pdb5l1n/pdb
• 分子名称: Coenzyme A disulfide reductase, FLAVIN-ADENINE DINUCLEOTIDE, COENZYME A (3 entities in total)
• 機能のキーワード: sulfur metabolism half-sites reactivity, oxidoreductase
• 由来する生物種: Pyrococcus horikoshii
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 102740.85

構造登録者

Sea, K.,Chen, B.,Crane III, E.J.,Sazinsky, M.H. (登録日: 2016-07-29, 公開日: 2017-08-09, 最終更新日: 2024-10-09)

主引用文献

Sea, K.,Lee, J.,To, D.,Chen, B.,Sazinsky, M.H.,Crane, E.J.
A broader active site inPyrococcus horikoshiiCoA disulfide reductase accommodates larger substrates and reveals evidence of subunit asymmetry.
FEBS Open Bio, 8:1083-1092, 2018

PubMed Abstract: Within the family of pyridine nucleotide disulfide oxidoreductase (PNDOR), enzymes are a group of single-cysteine containing FAD-dependent reductases that utilize a tightly bound coenzyme A to assist in the NAD(P)H-dependent reduction of di-, per-, and polysulfide substrates in bacteria and archaea. For many of these homodimeric enzymes, it has proved difficult to determine the substrate specificity and metabolic function based on sequence and genome analysis alone. Coenzyme A-disulfide reductase (CoADR) isolated from (CoADR) reduces Co-A per- and polysulfides, but, unlike other highly homologous members of this group, is a poor CoA disulfide reductase. The CoADR structure has a narrower access channel for CoA substrates, which suggested that this restriction might be responsible for the enzyme's poor activity toward the bulky CoA disulfide substrate. To test this hypothesis, the substrate channel was widened by making four mutations along the channel wall (Y65A, Y66A, P67G, and H367G). The structure of the quadruple mutant shows a widened substrate channel, which is supported by a fourfold increase in for the NAD(P)H-dependent reduction of CoA disulfide and enhanced activity toward the substrate at lower temperatures. Anaerobic titrations of the enzyme with NADH revealed a half-site reactivity not observed with the wild-type enzyme in which one subunit of the enzyme could be fully reduced to an EH state, while the other remained in an EH or EH·NADH state. These results suggest that for these closely related enzymes, substrate channel morphology is an important determinant of substrate specificity, and homology modeling will be the preferred technique for predicting function among PNDORs.

PubMed: 29988575
DOI: 10.1002/2211-5463.12439

実験手法

X-RAY DIFFRACTION (3.6 Å)