Nitric Oxide Synthase Active Site Research Articles

Inhibitor Bound Crystal Structures of Bacterial Nitric Oxide Synthase.

Nitric oxide generated by bacterial nitric oxide synthase (NOS) increases the susceptibility of Gram-positive pathogens Staphylococcus aureus and Bacillus anthracis to oxidative stress, including antibiotic-induced oxidative stress. Not surprisingly, NOS inhibitors also improve the effectiveness of antimicrobials. Development of potent and selective bacterial NOS inhibitors is complicated by the high active site sequence and structural conservation shared with the mammalian NOS isoforms. To exploit bacterial NOS for the development of new therapeutics, recognition of alternative NOS surfaces and pharmacophores suitable for drug binding is required. Here, we report on a wide number of inhibitor-bound bacterial NOS crystal structures to identify several compounds that interact with surfaces unique to the bacterial NOS. Although binding studies indicate that these inhibitors weakly interact with the NOS active site, many of the inhibitors reported here provide a revised structural framework for the development of new antimicrobials that target bacterial NOS. In addition, mutagenesis studies reveal several key residues that unlock access to bacterial NOS surfaces that could provide the selectivity required to develop potent bacterial NOS inhibitors.

Quantum chemical calculations of the NHA bound nitric oxide synthase active site: O2 binding and implications for the catalytic mechanism.

Density functional theory has been employed to model the binding of the intermediate substrate NHA, by nitric oxide synthases. In particular, the orientation and interactions of possibly catalytically important substrate hydrogens, with and without molecular oxygen bound to the active site heme group, are considered. Without O(2), three possible conformers have been found, with the energetically most favored structure being that in which both protons of the -NHOH moiety of NHA are directed toward the heme group. With oxygen bound, four different structures were found. The energetically lowest structure is again found to have both hydrogens of the -NHOH group pointing toward the heme group, thus forming hydrogen bonds between -NH- and the terminal oxygen, and between -OH and the inner oxygen of the heme-O(2) group. In addition, unprotonated structures of the substrate bound to the active site are considered and the proton affinity calculated.

Mapping the active site polarity in structures of endothelial nitric oxide synthase heme domain complexed with isothioureas

Analyzing the active site topology and plasticity of nitric oxide synthase (NOS) and understanding enzyme-drug interactions are crucial for the development of potent, isoform-selective NOS inhibitors. A small hydrophobic pocket in the active site is identified in the bovine eNOS heme domain structures complexed with potent isothiourea inhibitors: seleno analogue of S-ethyl-isothiourea, S-isopropyl-isothiourea, and 2-aminothiazoline, respectively. These structures reveal the importance of nonpolar van der Waals contacts in addition to the well-known hydrogen bonding interactions between inhibitor and enzyme. The scaffold of a potent NOS inhibitor should be capable of donating hydrogen bonds to as well as making nonpolar contacts with amino acids in the NOS active site.

L-Arginine Binding to Nitric-oxide Synthase: THE ROLE OF H-BONDS TO THE NONREACTIVE GUANIDINIUM NITROGENS

Nitric-oxide synthase (NOS) catalyzes the oxidation of L-arginine to nitric oxide and L-citrulline. Because overproduction of nitric oxide causes tissue damage in neurological, inflammatory, and autoimmune disorders, design of NOS inhibitors has received much attention. Most inhibitors described to date include a guanidine-like structural motif and interact with the guanidinium region of the L-arginine-binding site. We report here studies with L-arginine analogs having one or both terminal guanidinium nitrogens replaced by functionalities that preserve some, but not all, of the molecular interactions possible for the -NH(2), =NH, or =NH(2)(+) groups of L-arginine. Replacement groups include -NH-alkyl, -alkyl, =O, and =S. Binding of L-canavanine, an analog unable to form hydrogen bonds involving a N(5)-proton, was also examined. From our results and previous work, we infer the orientation of these compounds in the L-arginine-binding site and use IC(50) or K(i) values and optical difference spectra to quantitate their affinity relative to L-arginine. We find that the non-reactive guanidinium nitrogen of L-arginine binds in a pocket that is relatively intolerant of changes in the size or hydrogen bonding properties of the group bound. The individual H-bonds involved are, however, weaker than expected (<2 versus 3-6 kcal). These findings elucidate substrate binding forces in the NOS active site and identify an important constraint on NOS inhibitor design.