First Nitric Oxide Regulating Enzyme Found That Is Conserved From Bacteria To Humans

Nitric oxide (NO) and a version of the molecule called S-nitrosothiol (SNO) are involved in important cellular processes including protein modification, signaling pathways and defense mechanisms. Levels of NO-related molecules are abnormal in numerous conditions, including infection, inflammation, asthma, cystic fibrosis, angina and heart disease, said principal investigator Dr. Jonathan Stamler, a Howard Hughes Medical Institute investigator at Duke University Medical Center.

"We've found the first enzyme conserved from bacteria to humans responsible for regulating an NO-related molecule," said Stamler, also a professor of medicine and biochemistry at Duke. "We've shown that removing that enzyme produces biologic effects in yeast and mice. This enzyme potentially could be a target for agents to help fight infections or to alleviate or prevent the many conditions in which NO plays a part."

Because the enzyme is present in evolutionarily distant organisms, the findings also support the idea that NO and SNO can cause cellular damage directly -- a process called "nitrosative stress." Until now, damage from NO-related molecules was thought always to involve oxidants, which produce highly reactive forms of oxygen. Instead, NO creates damage by attaching to proteins in excessive amounts, Stamler said.

The study was funded by the Howard Hughes Medical Institute (HHMI) and the National Institutes of Health. The findings are reported in the March 22 issue of the journal Nature.

The enzyme, known as glutathione-dependent formaldehyde dehydrogenase or GS-FDH, was already known as an enzyme that detoxifies formaldehyde. But Stamler and his colleagues found that a version of NO called glutathione-S-nitrosothiol (GSNO) is in fact the primary target of GS-FDH in organisms ranging from primordial bacteria to humans.

The scientists, led by HHMI investigator Limin Liu, discovered that yeast without the gene for GS-FDH died when exposed to excess SNO, while mice without GS-FDH had abnormally higher levels of SNO bound to proteins, although they appeared normal at birth. It is not yet known whether the mice may exhibit increased susceptibility to infection, cancer or heart disease, Stamler said.

Protein-bound SNO is involved in sending cellular messages, while GSNO is a reservoir of SNO. GS-FDH appears to control the balance of GSNO and protein-SNO.

"Until now the only enzyme known to be involved in nitric oxide biology was nitric oxide synthase (NOS), which produces NO and SNO," said Stamler. "That left NO with a production system but no regulatory system, particularly in mammalian systems."

Because NO levels are altered in disease states, knowing the enzyme responsible for controlling those levels may lead to improved therapies, though much more work is needed, Stamler said. The fact that GS-FDH is different in microbes and humans might potentially allow therapeutic selective inhibition, he added.

For example, infections -- which are populations of microbes such as bacteria proliferating uncontrolled -- might be effectively treated by selectively shutting down the bacteria's GS-FDH, leaving them susceptible to NO (in the form of SNO) produced by the human body, which increases in response to infection. An inhibitor for the bacteria's enzyme would not affect the human's version. Similarly, in diseases where NO levels are too low, inhibiting the human enzyme might allow levels to return to normal.

"Previously all damage was lumped together as oxidative stress, and NO molecules were thought only to act through oxidants," Stamler said. "Based on previous studies by co-author Alfred Hausladen (of Duke), we've come to understand that mechanism of injury should be separated into oxidative stresses, nitrosative stresses, and both.

"Now we have genetic proof that nitrosative stress is an entity in its own right. For those diseases that have previously been attributed to oxidative stress, like Alzheimer's disease and atherosclerosis, it is fair to ask whether nitrosative stress might play a separate and important role."

Other co-authors of the report are Ming Zeng and Loretta Que of the Department of Medicine and Joseph Heitman of the Department of Genetics, all of Duke.