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Updated: Apr 10, 2026
Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO
Published on: December 28, 2019
Inhibitor Bound Crystal Structures of Bacterial Nitric Oxide Synthase
Jeffrey K Holden1,2, Dillon Dejam1,2, Matthew C Lewis1,2
1Departments of †Molecular Biology and Biochemistry, ‡Pharmaceutical Sciences, and §Chemistry, University of California, Irvine, California 92697-3900, United States.
Abstract:
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.
Insights
Bacterial nitric oxide synthase (NOS) contributes to pathogen survival and antibiotic resistance. New inhibitors targeting unique bacterial NOS surfaces offer a promising strategy for developing novel antimicrobial drugs.
Area of Science:
- Microbiology and Biochemistry
- Drug Discovery and Development
- Structural Biology
Background:
- Bacterial nitric oxide synthase (NOS) enhances pathogen survival and antibiotic resistance in Gram-positive bacteria like Staphylococcus aureus and Bacillus anthracis.
- Inhibiting bacterial NOS can improve antimicrobial efficacy, but developing selective inhibitors is challenging due to conserved active sites shared with mammalian NOS isoforms.
Purpose of the Study:
- To identify novel drug-binding surfaces and pharmacophores on bacterial NOS distinct from mammalian isoforms.
- To provide a structural basis for developing new therapeutics targeting bacterial NOS.
Main Methods:
- Determined crystal structures of bacterial NOS bound to various inhibitors.
- Performed binding studies to assess inhibitor interactions.
- Conducted mutagenesis studies to identify key residues influencing inhibitor binding and selectivity.
Main Results:
- Identified several compounds that interact with unique surfaces on bacterial NOS, distinct from the active site.
- Observed weak binding of these inhibitors to the NOS active site, suggesting alternative binding modes.
- Mutagenesis revealed key residues that can be targeted to enhance selectivity for bacterial NOS.
Conclusions:
- The identified unique bacterial NOS surfaces and inhibitor interactions provide a revised structural framework for developing novel antimicrobials.
- Targeting these alternative surfaces and utilizing mutagenesis insights can lead to potent and selective bacterial NOS inhibitors, overcoming challenges posed by conserved active sites.
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