Nitric oxide (NO) has emerged as an important new gaseous messenger molecule that can mediate physiological and pathological responses in humans. NO regulates blood pressure in healthy human subjects and is also constantly synthesized by a healthy beating heart for its proper function. While controlled production of NO is essential for an individual's well being, an overproduction can lead to severe problems. In mammals NO is produced by a family of three enzymes collectively known as nitric oxide synthases (NOS). NOS is a bidomain protein made up of a heme-containing catalytic domain covalently fused to a reductase domain via a linker that binds caldmodulin. Physiological actions of NO are mediated by its heme protein receptor, soluble guanylyl cyclase, which gets activated by NO and catalyzes the biosynthesis of second messenger, cGMP. NO can mediate its actions via both cGMP-dependent and -independent mechanisms. The latter includes the ability of NO to kill bacterial and viral pathogens. The toxicity of NO to bacteria has given rise to the assumption that NO signaling pathway is indigenous to eukaryotes. We have discovered that some gram-positive bacterial pathogens encode a NOS-like gene whose product bears strong sequence and structural resemblance to the catalytic heme domain of NOS found in mammals. The major long-term goal of this proposal is to understand the structural bases of catalysis and molecular recognition by bacterial NOS. We will particularly focus our efforts on NOS from Bacillus anthracis, the etiological agent of anthrax, and Staphylococcus aureus, an organism that exhibits remarkable resistance to antibiotics. A secondary long-term goal is to utilize bacterial NOS as a model system to gain molecular insights into the workings of mammalian NOS. A tertiary long-range goal is to understand the function of NOS in bacterial pathogens and to discover novel pathways in which it might participate. To achieve these goals, we plan to utilize a battery of X-ray crystallographic, biochemical, spectroscopic, molecular biological, and genetic methods. Knowledge gained from the proposed research will not only generate key information regarding how bacteria generate and deal with nitrogen oxides, but also provide us with critical leads for the development of novel strategies to combat biological warfare.
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