In Vitro Characterization of Echinomycin Biosynthesis: Formation and Hydroxylation of L-Tryptophanyl-S-Enzyme and Oxidation of (2S,3S) β-Hydroxytryptophan

Chen Zhang,Delin You,Jun Yin,Zixin Deng,Lingxin Kong,Tao Zhu,Xuan Lei,Birun Lin,Qian Liu,Shekhar C Mande

doi:10.1371/journal.pone.0056772

Abstract

Quinoxaline-2-carboxylic acid (QXC) and 3-hydroxyquinaldic acid (HQA) feature in quinomycin family and confer anticancer activity. In light of the significant potency against cancer, the biosynthetic gene clusters have been reported from many different Streptomyces strains, and the biosynthetic pathway were proposed mainly based on the in vivo feeding experiment with isotope labeled putative intermediates. Herein we report another gene cluster from Streptomyces griseovariabilis subsp. bandungensis subsp. nov responsible for the biosynthesis of echinomycin (a member of quinomycin family, also named quinomycin A) and presented in vitro evidence to corroborate the previous hypothesis on QXC biosynthesis, showing that only with the assistance of a MbtH-like protein Qui5, did the didomain NRPS protein (Qui18) perform the loading of a L-tryptophan onto its own PCP domain. Particularly, it was found that Qui5 and Qui18 subunits form a functional tetramer through size exclusion chromatography. The subsequent hydroxylation on β-carbon of the loaded L-tryptophan proved in vitro to be completed by cytochrome P450-dependent hydroxylase Qui15. Importantly, only the Qui18 loaded L-tryptophan can be hydroxylated by Qui15 and the enzyme was inactive on free L-tryptophan. Additionally, the chemically synthesized (2S,3S) β-hydroxytryptophan was detected to be converted by the tryptophan 2,3-dioxygenase Qui17 through LC-MS, which enriched our previous knowledge that tryptophan 2,3-dioxygenase nearly exclusively acted on L-tryptophan and 6-fluoro-tryptophan.

Highlights

Quinomycin antibiotics are composed of the nonribosomal peptide (NRP) backbone and a pair of identical quinoxaline or 3hydroxyquinaldic rings (Figure 1), which originates respectively from the aromatic precursor of quinoxaline-2-carboxylic acid (QXC) and 3-hydroxyquinaldic acid (HQA) [1] (Figure 1)
The biosynthetic gene clusters for echinomycin [5,6] and triostin [7] indicated that the QXC and HQA biosynthesis genes have high homology among some counterparts especially in the enzymes assigned to first several steps of QXC and HQA biosynthesis pathway
In detail for the aromatic precursor biosynthesis, at first, with the help of an MbtH-like protein, L-tryptophan is loaded onto a NRPS protein which consists of both adenylation (A) and PCP (T) domain, the assistance of the MbtH-like protein in the activity of the NRPS protein in echinomycin and triostin A biosynthesis was proposed by Kenji Watanabe, yet unsubstantiated [6,7]

Summary

Introduction

Quinomycin antibiotics are composed of the nonribosomal peptide (NRP) backbone and a pair of identical quinoxaline or 3hydroxyquinaldic rings (Figure 1), which originates respectively from the aromatic precursor of quinoxaline-2-carboxylic acid (QXC) and 3-hydroxyquinaldic acid (HQA) [1] (Figure 1). In detail for the aromatic precursor biosynthesis, at first, with the help of an MbtH-like protein, L-tryptophan is loaded onto a NRPS protein which consists of both adenylation (A) and PCP (T) domain, the assistance of the MbtH-like protein in the activity of the NRPS protein in echinomycin and triostin A biosynthesis was proposed by Kenji Watanabe, yet unsubstantiated [6,7]. The loaded tryptophan is hydroxylated at bcarbon by a cytochrome P450 superfamily member to produce (2S,3S) b-hydroxytryptophanyl-S-PCP. For the HQA pathway, the amino group on the a-carbon of (2S,3R) b-hydroxykynurenine is converted by an aminotransferase to form a carbonyl group, which is linked to the amino group steming from the nitrogen atom of L-tryptophan, resulting in the formation of another sixmembered ring neighboring the benzyl ring, it is reduced to HQA [9]

Methods

Results

Conclusion