9FCN

OPR3 loop swap variant L6(AchrOYE4) in complex with NADPH4

Summary for 9FCN

• Entry DOI: 10.2210/pdb9fcn/pdb
• Descriptor: 12-oxophytodienoate reductase 3,Old yellow enzyme OYE4, FLAVIN MONONUCLEOTIDE, 1,4,5,6-TETRAHYDRONICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE, ... (4 entities in total)
• Functional Keywords: flavoenzymes, ene-reductases, old yellow enzymes, coenzymes, flavoprotein
• Biological source: Solanum lycopersicum (tomato); More
• Total number of polymer chains: 2
• Total formula weight: 90743.33

Authors

Bijelic, A.,Kerschbaumer, B.,Macheroux, P. (deposition date: 2024-05-15, release date: 2025-05-28, Last modification date: 2026-03-25)

Primary citation

Kerschbaumer, B.,Friesser, E.M.,Wallner, S.,Oberdorfer, G.,Friess, M.,Breinbauer, R.,Macheroux, P.,Bijelic, A.
Structural and evolutionary dissection of NADPH-binding motifs in NADPH-preferring ene-reductases.
Protein Sci., 35:e70521-e70521, 2026

PubMed Abstract: Ene-reductases (ERs) catalyze nicotinamide-dependent, stereoselective reductions of activated CC bonds. While their catalytic chemistry and applications are well-explored, cosubstrate (NAD(P)H) binding remains poorly understood. Most ERs strongly prefer NADPH despite lacking canonical dinucleotide-binding folds and instead employ flexible loop motifs. We recently elucidated the NADPH-binding mode of the NADPH-preferring ER Solanum lycopersicum OPR3 (SlOPR3), identifying four key residues (R283/R343/Y364/R366) that form two motifs: a 2'-phosphate (2'-P)-binding site (R343/Y364/R366) and a loop 6 (L6)-mediated adenine clamp (R283/R343). Guided by this model, we analyzed the conservation of these motifs across 51 NADPH-preferring ERs from different Old Yellow Enzyme (OYE) classes by multi-sequence alignment and homology modeling. Analyses revealed a class-dependent distribution: class-II ERs predominantly conserve the OPR3-like motifs, whereas other classes employ alternative mechanisms, including dimerization-induced modes. Functional dissection of SlOPR3 through mutagenesis, kinetics, and crystallography established a functional hierarchy of the motif elements, indicating that R343 and R366 are indispensable for NADPH binding in OPR3-like ERs, while the adenine clamp acts as a conformation-sensitive affinity tuner. Ancestral sequence reconstruction revealed the stepwise and convergent assembly of motif elements, culminating in the complete motif set in plant, fungal, and cyanobacterial lineages. Our findings delineate (i) a strict functional hierarchy of NADPH-binding residues in OPR3-like ERs, (ii) alternative binding solutions in other OYE classes, and (iii) a convergent evolutionary trajectory, advancing the fundamental understanding of NADPH binding in NADPH-preferring ERs and offering a modular framework to predict NADPH preference in ERs.

PubMed: 41848427
DOI: 10.1002/pro.70521

Experimental method

X-RAY DIFFRACTION (2.25 Å)