Identification of 8-Azaguanine Biosynthesis-Related Genes Provides Insight Into the Enzymatic and Non-enzymatic Biosynthetic Pathway for 1,2,3-Triazole.

Feifei Hou,Mo Xian,Qi Gan,Wei Huang,Yupeng Wan

doi:10.3389/fbioe.2020.603514

Abstract

8-Azaguanine (1) is a special 1,2,3-triazole containing natural product that possesses potent antibacterial and antitumor activities. In the present study, the entire 8-azaguanine biosynthetic gene cluster was located from Streptomyces CGMCC4.1633. Targeted gene disruption, heterologous expression analysis, and feeding experiments identified crucial genes for 8-azaguanine production. Moreover, we characterized the structure of two novel metabolites, analyzed NO (or reactive nitrogen species) related genes 8-azgA/B and radical SAM enzyme homologous 8-AzgG, and verified the non-enzymatic ring formation reaction of 8-azaguanine 1,2,3-triazole. All of the data and presumptions provide insight into the timing and mechanism of the enzymatic and non-enzymatic pathway that produce 8-azaguanine-type 1,2,3-triazole.

Highlights

Introduction8-Azaguanine (8-AZG, 1, known as pathocidin) is a triazole analog of guanine (Figure 1A), which was first found in Streptomyces albus var. pathocidicus culture medium as a secondary metabolite with activities including antibacterial and antitumor (Anzai et al, 1961), such as polyploid-specific effects to high-ploidy breast cancer (Choudhary et al, 2016); potential activity against Mycobacterium tuberculosis (Sao Emani et al, 2018); modulating HIF levels in neoplastic cells (Xia et al, 2009); stabilizing mutated von Hippel–Lindau protein (Ding et al, 2012); and direct potentiation of natural killer cell cytotoxicity (Kim et al, 2019)
Genome mining for 8-AZG biosynthetic gene cluster (BGC) candidates was performed using a local BLASTP analysis on the genome with a GTP cyclohydrolase (GCH) probe, which might be involved in the first step hydrolytic cleavage of guanine moiety C-8 (Hirasawa and Isono, 1978)
On the basis of the in vivo and in vitro analysis, we propose a complete 8-AZG biosynthetic pathway as follows: imidazole ring of the starting unit GTP was hydrolytic opened by 8-AzgF to produce 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5 -triphosphate

Summary

Introduction

8-Azaguanine (8-AZG, 1, known as pathocidin) is a triazole analog of guanine (Figure 1A), which was first found in Streptomyces albus var. pathocidicus culture medium as a secondary metabolite with activities including antibacterial and antitumor (Anzai et al, 1961), such as polyploid-specific effects to high-ploidy breast cancer (Choudhary et al, 2016); potential activity against Mycobacterium tuberculosis (Sao Emani et al, 2018); modulating HIF levels in neoplastic cells (Xia et al, 2009); stabilizing mutated von Hippel–Lindau protein (Ding et al, 2012); and direct potentiation of natural killer cell cytotoxicity (Kim et al, 2019). Earlier works in the feeding experiments with 14C-labeled compounds demonstrate [2-14C] guanine could be efficiently incorporated into 8-AZG, [8-14C] guanine was not incorporated (Hirasawa and Isono, 1978) According to these results, we presume that the skeleton of 8-AZG is formed from guanine or its derivatives by the replacement of C-8 with unknown source of nitrogen (Figure 1A). We noticed the study of another group (Zhao et al, 2020), completed at the same time as our work They have reported the biosynthetic gene cluster mining for 8-AZG and carried out a series of in vivo and thorough in vitro work to reveal the biosynthetic route to the natural product 8-AZG. Mutant intermediates structural elucidation, and in vitro tests explore the biosynthetic pathway for 8-AZG These two independent studies drew similar conclusions and complemented the results of each other’s works

Methods

Results

Conclusion