Bacterial Nitric-oxide Synthases Operate without a Dedicated Redox Partner

Ivan Gusarov,Marina Starodubtseva,Zhi-Qiang Wang,Lindsey Mcquade,Stephen J Lippard,Dennis J Stuehr,Evgeny Nudler

doi:10.1074/jbc.m710178200

Abstract

Bacterial nitric-oxide (NO) synthases (bNOSs) are smaller than their mammalian counterparts. They lack an essential reductase domain that supplies electrons during NO biosynthesis. This and other structural peculiarities have raised doubts about whether bNOSs were capable of producing NO in vivo. Here we demonstrate that bNOS enzymes from Bacillus subtilis and Bacillus anthracis do indeed produce NO in living cells and accomplish this task by hijacking available cellular redox partners that are not normally committed to NO production. These "promiscuous" bacterial reductases also support NO synthesis by the oxygenase domain of mammalian NOS expressed in Escherichia coli. Our results suggest that bNOS is an early precursor of eukaryotic NOS and that it acquired its dedicated reductase domain later in evolution. This work also suggests that alternatively spliced forms of mammalian NOSs lacking their reductase domains could still be functional in vivo. On a practical side, bNOS-containing probiotic bacteria offer a unique advantage over conventional chemical NO donors in generating continuous, readily controllable physiological levels of NO, suggesting a possibility of utilizing such live NO donors for research and clinical needs.

Highlights

Nitric oxide (NO)2 signaling is essential in organisms ranging from bacteria to humans [1,2,3,4,5,6]
Our results suggest that bacterial NOS-like proteins (bNOS) is an early precursor of eukaryotic NOS and that it acquired its dedicated reductase domain later in evolution
To demonstrate bNOS-dependent NO production under physiological condition in vivo, we first relied on a quantitative assessment of the NO end products nitrate and nitrite

Summary

Introduction

Nitric oxide (NO)2 signaling is essential in organisms ranging from bacteria to humans [1,2,3,4,5,6]. These “promiscuous” bacterial reductases support NO synthesis by the oxygenase domain of mammalian NOS expressed in Escherichia coli. To construct pUCND6, a PCR fragment containing the araA promoter and regulatory elements from B. subtilis was cloned in front of the nos gene in the pBNOSB.su(I/V) plasmid.

Results

Conclusion