Suk Hui Kim

Integral beta-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

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

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 genes demonstrated the biosynthesis of cihunamides through P450-mediated oxidative Trp-Trp cross-linking. Further bioinformatic analysis uncovered 252 homologous gene clusters, including

Macrocyclization of peptides is often employed to generate novel structures and biological activities in the biosynthesis of natural products and drug discovery. The enzymatic cross-linking of two side chains in a peptide via an ester or amide has a high potential for making topologically diverse cyclic peptides but is found with only a single consensus sequence in the microviridin class of natural products. Here, we report that a peptide with a new sequence pattern can be enzymatically cross-li

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

Rising antibiotic resistance urgently begs for novel targets and strategies for antibiotic discovery. Here, we report that over-activation of the periplasmic DegP protease, a member of the highly conserved HtrA family, can be a viable strategy for antibiotic development. We demonstrate that tripodal peptidyl compounds that mimic DegP-activating lipoprotein variants allosterically activate DegP and inhibit the growth of an Escherichia coli strain with a permeable outer membrane in a DegP-dependen

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

The modular biosynthetic pathway of ribosomally synthesized and post-translationally modified peptides (RiPPs) enhances their engineering potential for exploring new structures and biological functions. The ω-ester-containing peptides (OEPs), a subfamily of RiPPs, have distinct side-to-side ester or amide linkages and frequently present more than one macrocyclic domain in a "beads-on-a-string" structure. In an effort to improve the engineering potential of RiPPs, we present here the idea that th