A Novel Mechanism for Nitrosative Stress Tolerance Dependent on GTP Cyclohydrolase II Activity Involved in Riboflavin Synthesis of Yeast

Khairul Anam,Hiroshi Takagi,Ryo Nasuno

doi:10.1038/s41598-020-62890-3

Abstract

The biological functions of nitric oxide (NO) depend on its concentration, and excessive levels of NO induce various harmful situations known as nitrosative stress. Therefore, organisms possess many kinds of strategies to regulate the intracellular NO concentration and/or to detoxify excess NO. Here, we used genetic screening to identify a novel nitrosative stress tolerance gene, RIB1, encoding GTP cyclohydrolase II (GTPCH2), which catalyzes the first step in riboflavin biosynthesis. Our further analyses demonstrated that the GTPCH2 enzymatic activity of Rib1 is essential for RIB1-dependent nitrosative stress tolerance, but that riboflavin itself is not required for this tolerance. Furthermore, the reaction mixture of a recombinant purified Rib1 was shown to quench NO or its derivatives, even though formate or pyrophosphate, which are byproducts of the Rib1 reaction, did not, suggesting that the reaction product of Rib1, 2,5-diamino-6-(5-phospo-d-ribosylamino)-pyrimidin-4(3 H)-one (DARP), scavenges NO or its derivatives. Finally, it was revealed that 2,4,5-triamino-1H-pyrimidin-6-one, which is identical to a pyrimidine moiety of DARP, scavenged NO or its derivatives, suggesting that DARP reacts with N2O3 generated via its pyrimidine moiety.

Highlights

Nitric oxide (NO) is a small signaling molecule that plays various roles in a number of biological processes[1,2]
In order to isolate genes involved in nitrosative stress tolerance, the screening was performed in acidified medium containing NaNO2, as previous studies reported[26,27]
DAF-FM was reacted with NOC-5 in the presence of TAPO with varied concentrations and the fluorescence was monitored (Fig. 5D). These results showed that TAPO inhibited the increase of fluorescence in a dose-dependent manner, which let us conclude that DARP scavenges nitric oxide (NO) or its derivatives through its pyrimidine moiety to reduce the toxicity of nitrosative stress

Summary

Introduction

Nitric oxide (NO) is a small signaling molecule that plays various roles in a number of biological processes[1,2]. A low level of NO plays a role in oxidative stress tolerance in bacteria[15]. An excessive level of RNS including NO could cause nitrosative stress leading to cellular damage or death. RNS, including NO and peroxynitrite, function as anti-microbial agents against pathogenic bacteria, fungi, and yeasts. Macrophages produce NO and superoxide by inducible NOS and NADPH oxidase, respectively, and these two molecules react with each other to generate peroxynitrite with higher reactivity and the ability to kill pathogens. NT traps NO with its cysteine residues and S-nitrosylated residues of NT are regenerated through the reduction system by Trx/Trr. The discovery of NT suggests the presence of further anti-nitrosative stress systems in various microbes, such as pathogenic and model bacteria, fungi, and yeasts, including S. cerevisiae

Methods

Results

Conclusion