Modular structure of neuronal nitric oxide synthase: Localization of the arginine binding site and modulation by pterin

A putative dihydrofolate reductase (DHFR) module has been identified in neuronal nitric oxide synthase, consisting of amino acids 558-721, and is proposed to be the site of tetrahydrobiopterin (BH₄) binding. This polypeptide has been expressed in E. coli as a fusion protein with glutathione S-transferase (GST), using the plasmid pGEX-4T1. The protein binds N^ω-nitro-L-arginine (NNA) tightly, but this binding is not stimulated by BH₄. cDNAs for Module II (residues 220-557) and Module III (residues 220-721) have been expressed as fusion proteins with GST. Module II does not bind NNA. However, Module III does bind NNA and binding is significantly stimulated by BH₄. These observations are taken as strong evidence that the DHFR module contains the L-arginine binding site and, presumably, the BH₄ binding site by analogy to its homology with DHFR, but that tight binding of BH₄ requires amino acids 220-577.