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Biosynthetic Studies on a D-tryptophan-containing Lasso Peptide, MS-271
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| Title: | Biosynthetic Studies on a D-tryptophan-containing Lasso Peptide, MS-271 |
| Other Titles: | D-トリプトファン含有ラッソペプチド天然物MS-271の生合成研究 |
| Authors: | 馮, 智1 Browse this author |
| Authors(alt): | Feng, Zhi1 |
| Issue Date: | 25-Mar-2021 |
| Publisher: | Hokkaido University |
| Abstract: | MS-271, originally isolated from Streptomyces sp. M-271 as a potent inhibitor of
calmodulin-activated myosin light-chain kinase, is a lasso peptide natural product
comprising 21 amino acids (aa) with a D-tryptophan (Trp) at its C-terminus. Lasso
peptides are peptide natural products that have a characteristic isopeptide-bonded
slipknot structure. In terms of their biosynthesis, lasso peptides belong to a group of
ribosomally synthesized and post-translationally modified peptides (RiPPs). Thus, the
biosynthesis of MS-271, especially the mechanism of D-Trp introduction, is of great
interest. In this study, I investigated the biosynthesis of MS-271.
In chapter 2, I first identified MS-271 biosynthetic gene cluster (msl) spanning a ca.
11-kbp region from mslR1 to mslH by draft genome sequencing followed by searching
for DNA region encoding the amino acid sequence of MS-271. Sequence analysis
revealed that precursor peptide gene (mslA) encoded 42-residue peptide with a leader
peptide at its N-terminus, and most importantly, the C-terminal core region contained
all 21 amino acid residues of MS-271 including the C-terminal Trp. This suggested that
the D-Trp residue is introduced via epimerization into a ribosomal peptide as a post translational modification. The cluster also contained genes for modification enzymes
such as a macrolactam synthetase (mslC), precursor peptide recognition element
(mslB1), cysteine protease (mslB2), disulfide oxidoreductases (mslE, mslF). Although
obvious epimerase genes were absent in the cluster, the cluster encoded a protein of
unknown function (mslH). Hence, I next carried out heterologous expression of the msl
cluster in Streptomyces lividans. As the results, the production of MS-271 was
confirmed by LC-MS and chiral amino acid analysis, indicating that the cluster contains
all the necessary genes for MS-271 production including a novel peptide epimerase
gene. I also showed that MslB1, B2, C and H were indispensable for MS-271
production by gene knockout experiments. Overall, these results suggested that MslH
is responsible for the epimerization of the C-terminal Trp.
In chapter 3, I characterized the function of MslH in vivo and in vitro. Considering
that many modification enzymes involved in RiPP biosynthesis require leader peptides
for their substrate recognition, I speculated that the epimerization occurs on the nascent
full-length MslA in MS-271 biosynthesis. As expected, in vivo experiments revealed
the formation of epi-MslA when mslA was expressed with mslH in Escherichia coli.
Additional coexpression of precursor peptide recognition element (mslB1) enhanced the formation of MslA. Furthermore, in vitro experiments revealed that MslH catalyzed
epimerization of C-terminal Trp of MslA in metal- and cofactor-independent manner
and that the leader peptide in MslA is indispensable for the substrate recognition by
MslH. I also examine substrate specificity of MslH by heterologous expression of the
msl cluster to produce MS-271 derivatives by altering the core peptide sequences of the
mslA gene, and demonstrated that MslH exhibited broad substrate specificities toward
the N-terminal region of core peptides while the C-terminal “CFW” sequence is
important for substrate recognition. Overall, I fully characterized MslH as a novel
peptide epimerase. This is the first example epimerase that catalyzes epimerization at
the C⍺ center adjacent to a carboxylic acid in a cofactor-independent manner.
Taken together, I identified MslH, previously annotated as a hypothetical protein, as
a novel epimerase involved in the post-translational epimerization of the C-terminal
Trp residue of the precursor peptide MslA. I also demonstrated that MslH exhibited
broad substrate specificity toward the N-terminal region of the core peptide, showing
that MslH-type epimerases offer opportunities in peptide bioengineering. |
| Conffering University: | 北海道大学 |
| Degree Report Number: | 甲第14473号 |
| Degree Level: | 博士 |
| Degree Discipline: | 工学 |
| Examination Committee Members: | (主査) 教授 松本 謙一郎, 特任教授 髙木 睦, 教授 大利 徹, 准教授 南 篤志, 准教授 小笠原 泰志 |
| Degree Affiliation: | 総合化学院（総合化学専攻） |
| Type: | theses (doctoral) |
| URI: | http://hdl.handle.net/2115/81306 |
| Appears in Collections: | 課程博士 (Doctorate by way of Advanced Course) > 総合化学院(Graduate School of Chemical Sciences and Engineering)
学位論文 (Theses) > 博士 （工学）
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