Seokhee Kim

Integral β-barrel proteins are found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. The machine that assembles these proteins contains an integral membrane protein, called YaeT in Escherichia coli , which has one or more polypeptide transport–associated (POTRA) domains. The crystal structure of a periplasmic fragment of YaeT reveals the POTRA domain fold and suggests a model for how POTRA domains can bind different peptide sequences, as required for a machine t

Various in vivo mutagenesis methods have been developed to facilitate fast and efficient continuous evolution of proteins in cells. However, they either modify the DNA region that does not match the target gene, or suffer from low mutation rates. Here, we report a mutator, eMutaT7 (enhanced MutaT7), with very fast in vivo mutation rate and high gene-specificity in Escherichia coli. eMutaT7, a cytidine deaminase fused to an orthogonal RNA polymerase, can introduce up to ∼4 mutations per 1 kb per

Cytochrome P450 enzymes (P450s) catalyze diverse oxidative cross-coupling reactions between aromatic substrates in the natural product biosynthesis. Specifically, P450s install distinct biaryl macrocyclic linkages in three families of ribosomally synthesized and post-translationally modified peptides (RiPPs). However, the chemical diversity of biaryl-containing macrocyclic RiPPs remains largely unexplored. Here, we demonstrate that P450s have the capability to generate diverse biaryl linkages on

DegP, a member of the highly conserved HtrA family, performs quality-control degradation of misfolded proteins in the periplasm of gram-negative bacteria and is required for high-temperature survival of Escherichia coli. Substrate binding transforms DegP from an inactive oligomer containing two trimers into active polyhedral cages, typically containing four or eight trimers. Although these observations suggest a causal connection, we show that cage assembly and proteolytic activation can be unco

ω-Ester-containing peptides (OEPs) are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) containing intramolecular ω-ester or ω-amide bonds. Although their distinct side-to-side connections may create considerable topological diversity of multicyclic peptides, it is largely unknown how diverse ring patterns have been developed in nature. Here, using genome mining of biosynthetic enzymes of OEPs, we identified genes encoding nine new groups of putative OEPs wi

Cihunamides A-D (1-4), novel antibacterial RiPPs, were isolated from volcanic-island-derived Streptomyces sp. The structures of 1-4 were elucidated by ¹ H, ¹³ C, and ¹⁵ N NMR, MS, and chemical derivatization; they contain a tetrapeptide core composed of WNIW, cyclized by a unique C-N linkage between two Trp units. Genome mining of the producer strain revealed two biosynthetic genes encoding a cytochrome P450 enzyme and a precursor peptide. Heterologous co-express

Microviridins are ribosomally synthesized and post-translationally modified peptides (RiPPs) that contain multiple intramolecular ω-ester or ω-amide crosslinks between two side chains in peptides. This type of the side-to-side macrocyclization may generate diverse structures with distinct topology and ring sizes, but the majority of the microviridin-like RiPPs present only a single consensus sequence with a tricyclic architecture. Here, we expanded the natural diversity of the microviridin-like

Intracellular proteases combat proteotoxic stress by degrading damaged proteins, but their activity must be carefully controlled to maintain cellular fitness. The activity of Escherichia coli DegP, a highly conserved periplasmic protease, is regulated by substrate-dependent allosteric transformations between inactive and active trimer conformations and by the formation of polyhedral cages that confine the active sites within a proteolytic chamber. Here, we investigate how these distinct control

Targeted in vivo hypermutation accelerates directed evolution of proteins through concurrent DNA diversification and selection. Although systems employing a fusion protein of a nucleobase deaminase and T7 RNA polymerase present gene-specific targeting, their mutational spectra have been limited to exclusive or dominant C:G→T:A mutations. Here we describe eMutaT7transition, a new gene-specific hypermutation system, that installs all transition mutations (C:G→T:A and A:T→G:C) at comparable frequen

Methods that can randomly introduce mutations in the microbial genome have been used for classical genetic screening and, more recently, the evolutionary engineering of microbial cells. However, most methods rely on either cell-damaging agents or disruptive mutations of genes that are involved in accurate DNA replication, of which the latter requires prior knowledge of gene functions, and thus, is not easily transferable to other species. In this study, we developed a new mutator for in vivo mut

Backbone N-methylation is one of the prominent peptide modifications that can greatly enhance the pharmacological properties of a peptide. Naturally occurring backbone N-methylated peptides are produced via nonribosomal or ribosomal pathways, the latter of which was only recently identified in the borosin family of ribosomally synthesized and post-translationally modified peptides. Although previous bioinformatic analyses have revealed new putative genes for borosin biosynthesis, the natural sco

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a structurally diverse class of natural products with a distinct biosynthetic logic, the enzymatic modification of genetically encoded precursor peptides. Although their structural and biosynthetic diversity remains largely underexplored, the identification of novel subclasses with unique structural motifs and biosynthetic pathways is challenging. Here, it is reported that peptide/protein L-aspartyl O-methyltransferas

Abstract Cihunamides A–D ( 1 – 4 ), novel antibacterial RiPPs, were isolated from volcanic‐island‐derived Streptomyces sp. The structures of 1 – 4 were elucidated by 1 H, 13 C, and 15 N NMR, MS, and chemical derivatization; they contain a tetrapeptide core composed of WNIW, cyclized by a unique C−N linkage between two Trp units. Genome mining of the producer strain revealed two biosynthetic genes encoding a cytochrome P450 enzyme and a precursor peptide. Heterologous co‐expression of the core ge