Structural characterization of nitric oxide synthase isoforms reveals striking active-site conservation

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Nitric oxide synthases (NOS) are crucial enzymes involved in various physiological processes.
• Understanding the structural basis of NOS activity is key for therapeutic intervention.
• Selective inhibition of NOS isozymes (e.g., endothelial NOS and inducible NOS) is a significant therapeutic goal.

Purpose of the Study:

• To determine the high-resolution crystal structures of human endothelial nitric oxide synthase (eNOS) and human inducible nitric oxide synthase (iNOS) catalytic domains.
• To elucidate the binding modes of the arginine substrate and an inhibitor.
• To provide a structural basis for the design of isozyme-selective NOS inhibitors.

Main Methods:

• X-ray crystallography was employed to solve the crystal structures of eNOS and iNOS catalytic domains.
• Structures were determined in complex with arginine substrate (for eNOS) and S-ethylisothiourea (SEITU) inhibitor (for iNOS).
• High-resolution (2.4 Å for eNOS, 2.25 Å for iNOS) structural data were refined.

Main Results:

• The small molecules (arginine and SEITU) bind within a narrow cleft in the active site, interacting with heme and tetrahydrobiopterin.
• Both eNOS and iNOS active sites exhibit high similarity, with conserved residues including a key glutamate.
• An unexpected structural zinc ion was identified at the intermolecular interface, coordinated by four cysteine residues from adjacent monomers.

Conclusions:

• The high structural similarity between eNOS and iNOS active sites presents a challenge for selective inhibitor design.
• The identified conserved binding interactions and the novel zinc-binding site offer potential targets for developing isozyme-specific inhibitors.
• These structural findings provide critical insights for future drug discovery efforts targeting nitric oxide synthase pathways.