摘要
S-腺苷甲硫氨酸自由基(rSAM)酶家族是目前已知的最大酶超家族之一,由22000多个成员组成.这些酶在大自然中广泛存在,被认为是地球上最早的生物催化剂之一.随着大量的微生物基因组信息被解析,分析显示,微生物中大量核糖体肽类天然产物的生物合成基因簇中含有rSAM酶;其中Xenorhabdus、Yersinia和 Erwinia三个属的基因组中均含有一个高度保守的rSAM酶负责其相邻核糖体肽的前体修饰,此类前体肽和rSAM酶构成的XYE系统所合成的化合物鲜有报道.本研究合成一个来源于Xenorhabdus sp.KJ12.1的XYE系统的前体肽和rSAM修饰酶基因,在大肠杆菌中进行共表达,得到新型核糖体肽Xenopeptide,通过结构解析发现,rSAM酶XenB负责分子内2个碳碳键的形成.本研究为微生物中此类化合物的深度挖掘和合成生物学改造提供了理论支撑.
Abstract
Ribosomally-synthesized and post-translationally-modified peptides(RiPPs)are a major class of natural products.It originates from a corresponding biosynthetic gene cluster where the genes for a precursor peptide and several post-translational modification(PTM)enzymes are located.The precursor peptide is translated by a ribosome,recognized on the N-terminal leader peptide and modified on the C-terminal core peptide by PTM enzymes,to finally give a structurally unique mature compound.So far,the RiPPs found by researchers only account for a small part of the family,and there are a large number of compounds remaining to be discovered.The S-adenosylmethionine radical(rSAM)enzyme family is one of the largest enzyme superfamilies,containing more than 22000 members.These enzymes are widely distributed in eukarya,bacteria and archaea and are considered to be one the earliest biocatalysts on earth which perform essential and inseparable functions in cells.As more substantial microbial genomic information becomes available,a large number of RiPPs are excavated as natural products by high-throughput analyses.Genome mining in microorganisms reveals great abundance of biosynthetic gene clusters consisting of co-occurring rSAM enzyme and RiPPs'peptide genes,mostly leading to complex bioactive compounds whether reported or not.Recently,three genera,Xenorhabdus,Yersinia,and Erwinia,are unified with the definition of XYE system,for they all employ a highly conserved rSAM enzyme responsible for modifying its adjacent peptide to create a distinctive C-C or C-O bond in the final RiPPs product.Yet complex compounds synthesized through the rSAM enzyme modifying a precursor peptide in the XYE system have rarely been reported.Through algorithm analysis,we monitored a biosynthetic gene cluster in the Xenorhabdus sp.KJ12.1 comforming a similar construction of that in an XYE system.We fully synthesized the precursor peptide and the rSAM enzyme genes from Xenorhabdus sp.KJ12.1,heterologously expressed the modifying system in Escherichia coli and successfully obtained a novel ribopeptide Xenopeptide.Structure analysis showed that the rSAM enzyme XenB is involved in the formation of two C-C bonds on the side groups of the precursor,one between tryptophan 14(W 14)and asparagine 16(N16),the other between tryptophan21(W21)and lysine23(K23).Although bioactivity tests proved the compound without a confirmed antimicrobial activity,we can still expect the utilization of the product elsewhere with its chemical complexity.These results indicate a developmental potential of combining computer technology and biological engineering.Our work provides a fundamentally theoretical basis for in-depth genome mining and a practically technical flow for finding modified compounds in microorganisms.The combination of genetic manipulation,biosynthetic characterization and structure determination is expected to further decipher the mechanistic enzymology involved in C-C crosslink formation.This strategy will shed light on the structure-activity relationships involved in the maturation of this subclade of RiPPs.Moreover,thorough biosynthetic engineering of Xenopeptide's precursor peptide and relevant enzymes through rational design and directed mutation will facilitate the development of a variety of Xenopeptide analogs with enhanced bioactivities.This paper will not only disclose the enzymatic underpinnings underlying the biosynthesis of C-C crosslinks,but also point out new directions for structural derivatization of bioactive peptides.
维普
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