Guanidine N-methylation by BlsL Is Dependent on Acylation of Beta-amine Arginine in the Biosynthesis of Blasticidin S.

Xiankun Wang,Xinyi He,Guiyang Yu,Aiqin Du,Zixin Deng

doi:10.3389/fmicb.2017.01565

Abstract

The peptidyl nucleoside blasticidin S (BS) produced by Streptomyces griseochromogenes was the first non-mercurial fungicide used to prevent rice blast and increasingly used as a selection reagent in transgenic study. Acylation by addition of a leucine residue at the beta amine group of arginine side chain of demethylblasticidin S (DBS) has been proposed as a novel self-resistance to the cytotoxic biosynthetic intermediate. But the resultant product leucyldemethylblasticidin S (LDBS) has not been isolated as a metabolite, and LDBS synthetase activity remained to be demonstrated in S. griseochromogenes. In this study, we isolated LDBS in a BS heterologous producer S. lividans WJ2 upon the deletion of blsL, which encodes a S-Adenosyl methionine-dependent methyltransferase. Purified BlsL efficiently methylated LDBS at the delta N of beta-arginine to generate the ultimate intermediate LBS, but nearly didn’t methylate DBS to final product BS. Above experiments demonstrated that LDBS is indeed an intermediate in BS biosynthetic pathway, and acylation of beta-amino group of arginine side chain is prerequisite for efficient guanidine N-methylation in addition to being a self-resistance mechanism.

Highlights

Blasticidin S (BS) was first isolated from the broth of Streptomyces griseochromogenes as an effective non-mercurial fungicide (Takeuchi et al, 1958)
Sequence analysis of BlsL revealed that low similarity to guanidine acetate methyltransferase (GAMT, 33% identity, 85% similarity) and protein arginine methyltransferase (PRMT, 35% identity, 74% similarity)
Protein arginine methyltransferase (PRMT) both catalyzes the formation of monomethylarginine (MMA) and symmetrical dimethylarginine (SDMA) from arginine residues of versatile proteins, and is involved in regulation of many cellular processes (Poulard et al, 2016)

Summary

Introduction

Blasticidin S (BS) was first isolated from the broth of Streptomyces griseochromogenes as an effective non-mercurial fungicide (Takeuchi et al, 1958). BS displayed wide-range antimicrobial spectrum, it exerts its biological activities by inhibiting protein translation through a unique mechanism by which it traps the deformed P-site tRNA and strongly suppresses peptidyl-tRNA hydrolysis by release factors and peptide bond formation (Svidritskiy et al, 2013). As it’s friendly to environment and not harmful to fish, blasticidin S was soon used on a large scale by foliar application in Asia to control rice blast caused by Pyricularia oryzae (Misato et al, 1958). It has been increasingly used as the selection reagent in mammalian transgenic studies (Kamakura et al, 1990; Freitas et al, 2002). Blasticidin S is consisted of a N-methyl-guanidine tail attached to a pyranose ring and bonded to a cytosine (Otake et al, 1966), its core moiety cytosylglucuronic acid (CGA) is shared by mildiomycin, gougerotin, arginomycin (Fox et al, 1968; Harada et al, 1978; Argoudelis et al, 1987)

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Results

Conclusion