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

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 n 2 o 3 generated via its pyrimidine moiety. Nitric oxide (NO) is a small signaling molecule that plays various roles in a number of biological processes 1,2. In mammalian cells, NO is generally produced by three different isoforms of NO synthase (NOS) from l-arginine and NADPH 3. NOS consists of the oxygenase domain responsible for oxidation of l-arginine and the reductase domain which transfers electrons from NADPH to the oxygenase domain 4. Some Gram-positive bacteria have enzymes called bacterial NOS (bNOS), which is homologous to the oxygenase domain of NOS, and which exerts its NOS activity via interaction with uncertain reductase proteins 5. On the other hand, nitrate and nitrite serve as NO sources in plants, in which nitrate and nitrite are reduced to NO by nitrate reductase and nitrite reductase, respectively 6. Nitrite is also reduced to NO by heme-containing proteins including hemoglobin or molybdopterin proteins such as xanthine oxidase via their nitrite reductase activity 7,8. Furthermore, reduction of nitrite to NO is also catalyzed by the mitochondrial respiratory activity complex III and/or IV 9. NO activates soluble guanylyl cyclase (sGC) by binding to the heme in sGC. Activated sGC releases cyclic GMP, which is a second messenger leading to the relaxation of smooth muscle cells in mammals 10. Additionally, NO induces the posttranslational modification of proteins, such as S-nitrosation. S-nitrosation is formed via the reaction of NO with thiyl radical or between NO + , which is one electron oxidized form of NO, and the thiol group of cysteine residues in the target protein 11. Thiol group also undergoes S-nitrosation via transnitrosation, in which NO + group is transferred from a sulfur atom in nitrosothiol compound to that in a target thiol compound. The NO-responsive transcription factor OxyR is a...

Nitrite Triggers Reprogramming of the Oral Polymicrobial Metabolome by a Commensal Streptococcus

“…In our study, aromatic compounds from the Shikimate pathway, particularly 3-dehydroshikimate, were significantly increased in S. parasanguinis cultures that contained nitrite. Many aromatic compounds have inhibitory activity against nitrosative stress due to their ability to scavenge NO x ( Sirimulla et al., 2012 ; Anam et al., 2020 ). Moreover, 5-enolpyruvylshikimate-3-phosphate synthase, an enzyme in the Shikimate pathway that participates in the synthesis of aromatic amino acids, is critical for growth on nitrate in P. aeruginosa ( Filiatrault et al., 2006 ).…”

Identification and Functional Analysis of GTP Cyclohydrolase II in Candida glabrata in Response to Nitrosative Stress

“…The plasmid pCU-PDC1-CAGL0F04279g was also used to overexpress CAGL0F04279g in the WT strain. In order to express RIB1 from S. cerevisiae , the previously constructed plasmid pET53-RIB1 ( Anam et al, 2020 ) was used as a template, followed by the same manipulation as the case of CAGL0F04279g.…”

“…Recently, we discovered a novel nitrosative stress resistance system dependent on GTP cyclohydrolase II (GCH2) in the yeast S. cerevisiae ( Anam et al, 2020 ). GCH2, the first-step and rate-limiting enzyme of the riboflavin (RF) biosynthesis pathway, converts its substrate GTP into 2,5-diamino-6-(5-phospo-d-ribosylamino)-pyrimidin-4(3H)-one (DARP); then DARP scavenges NO, leading to nitrosative stress resistance.…”

Production of vitamin B2 (riboflavin) by Bacillus subtilis

“…However, the major disadvantage in the use of yeast is iron inhibition which is required to be overcome for the synthesis of riboflavin 38 . Therefore, the focus is laid on genetically engineered yeast by developing iron‐resistant genes for the production of riboflavin (20 g L –1 ) 39 …”