
メソスコピック酵素濃度におけるミカエリス・メンテン動力学では、酵素と複合体の間の詳細釣り合いが成り立たない非古典的な過渡的な挙動が観察される。この過渡的な挙動は、触媒回転数が臨界値以下の場合に、酵素速度の基質依存性が双曲線的ではなくなることを示す。一方、触媒回転数が臨界値を大きく超えると、詳細釣り合いの条件の下で双曲線的なミカエリス・メンテン式が漸近的に回復される。


coremsg

メソスコピック-ミカエリス-メンテン動力学における統計的平衡への緩和


メソスコピック・ミカエリス・メンテン動力学における統計的平衡への緩和


title_rewrite


组蛋白电离性残基的质子化状态变化可以显著影响核小体表面的静电势,从而调节核小体与染色质结合因子的相互作用以及核小体之间的自组装。


The nucleosome serves as the fundamental unit of chromatin organization, with electrostatic interactions acting as the driving forces in the folding of nucleosomes into chromatin. Perturbations in cellular pH conditions can lead to changes in the protonation states of titratable histone residues, impacting nucleosome surface electrostatic potentials and interactions. However, the effects of proton uptake or release of histone ionizable groups on nucleosome-partner protein interactions and higher-order chromatin structures remain largely unexplored. Here, we conducted comprehensive analyses of histone titratable residue pKa values in various nucleosome contexts, utilizing 96 experimentally determined structures. We revealed that pH-induced changes in histone residue protonation states modulated nucleosome surface electrostatic potentials and significantly influenced nucleosome-partner protein interactions. Furthermore, we observed that proton uptake or release often accompanied nucleosome-partner protein interactions, facilitating their binding processes. Additionally, using a dataset of 1266 recurrent histone cancer mutations, we systematically characterized their impact on nucleosome surface electrostatics, demonstrating their profound effects on electrostatic interactions between nucleosomes and partner proteins. Finally, our findings suggest that alterations in histone protonation or cancer mutations can also regulate nucleosome self-association, thereby modulating the organization and dynamics of higher-order chromatin structure.

### Competing Interest Statement

The authors have declared no competing interest.

Electrostatic interactions in nucleosome and higher-order structures are regulated by protonation state of histone ionizable residue

核小体和高级结构中的静电相互作用受到组蛋白电离性残基质子化状态的调节


His-Me ヌクレアーゼは単一の二価金属イオンと保存されたヒスチジン残基を用いて、DNAの加水分解を触媒する。


Metal-ion-dependent nucleases play crucial roles in cellular defense and biotechnological applications. Time-resolved crystallography has resolved catalytic details of metal-ion-dependent DNA hydrolysis and synthesis, uncovering the essential roles of multiple metal ions during catalysis. The superfamily of His-Me nucleases is renowned for binding one divalent metal ion and requiring a conserved histidine to promote catalysis. Many His-Me family nucleases, including homing endonucleases and Cas9 nuclease, have been adapted for biotechnological and biomedical applications. However, it remains unclear how this single metal ion in His-Me nucleases, together with the histidine, promotes water deprotonation, nucleophilic attack, and phosphodiester bond breakage. By observing DNA hydrolysis in crystallo with His-Me I-PpoI nuclease as a model system, we proved that only one divalent metal ion is required during its catalysis. Moreover, we uncovered several possible deprotonation pathways for the nucleophilic water. Interestingly, binding of the single metal ion and water deprotonation are concerted during catalysis. Our results reveal catalytic details of His-Me nucleases, which is distinct from multi-metal-ion-dependent DNA polymerases and nucleases.

Teaser Soaking crystals of the HNH Cas9 family I-PpoI nuclease reveals how one metal ion and a histidine residue are sufficient for cleaving DNA

### Competing Interest Statement

The authors have declared no competing interest.

Observing one-divalent-metal-ion dependent and histidine-promoted His-Me family I-PpoI nuclease catalysis in crystallo

単一二価金属イオン依存性およびヒスチジン促進型his-me-i-ppoiヌクレアーゼ触媒反応のin-crystallo観察


単一二価金属イオン依存性およびヒスチジン促進型His-Me I-PpoIヌクレアーゼ触媒反応のin crystallo観察



LptB2FGCは、ATP結合と加水分解を介して、リポ多糖の膜間輸送を効率的かつ一方向的に制御する。


Lipopolysaccharides (LPS) confer resistance against harsh conditions, including antibiotics, in Gram-negative bacteria. The lipopolysaccharide transport (Lpt) complex, consisting of seven proteins (A-G), exports LPS across the cellular envelope. LptB2FG forms an ATP-binding cassette transporter that transfers LPS to LptC. How LptB2FG couples ATP binding and hydrolysis with LPS transport to LptC remains unclear. We observed the conformational heterogeneity of LptB2FG and LptB2FGC in micelles and/or proteoliposomes using pulsed dipolar electron spin resonance spectroscopy. Additionally, we monitored LPS binding and release using laser-induced liquid bead ion desorption mass spectrometry. The β-jellyroll domain of LptF stably interacts with the LptG and LptC β-jellyrolls in both the apo and vanadate-trapped states. ATP binding at the cytoplasmic side is allosterically coupled to the selective opening of the periplasmic LptF β-jellyroll domain. In LptB2FG, ATP binding closes the nucleotide binding domains, causing a collapse of the first lateral gate as observed in structures. However, the second lateral gate, which forms the putative en try site for LPS, exhibits a heterogeneous conformation. LptC binding limits the flexibility of this gate to two conformations, likely representing the helix of LptC as either released from or inserted into the transmembrane domains. Our results reveal the regulation of the LPS entry gate through the dynamic behavior of the LptC transmembrane helix, while its β-jellyroll domain is anchored in the periplasm. This, combined with long-range ATP-dependent allosteric gating of the LptF β-jellyroll domain, may ensure efficient and unidirectional transport of LPS across the periplasm.

### Competing Interest Statement

The authors have declared no competing interest.

Dynamic basis of lipopolysaccharide export by LptB2FGC

リポ多糖輸送複合体lptb2fgcの動的基盤


リポ多糖輸送複合体LptB2FGCの動的基盤



マウス酸性マンマリーキチナーゼ(mAMCase)は、酸性条件と中性条件の両方で機能するために、pH に応じて異なるメカニズムを使用する。


Chitin is an abundant biopolymer and pathogen-associated molecular pattern that stimulates a host innate immune response. Mammals express chitin-binding and chitin-degrading proteins to remove chitin from the body. One of these proteins, Acidic Mammalian Chitinase (AMCase), is an enzyme known for its ability to function under acidic conditions in the stomach but is also active in tissues with more neutral pHs, such as the lung. Here, we used a combination of biochemical, structural, and computational modeling approaches to examine how the mouse homolog (mAMCase) can act in both acidic and neutral environments. We measured kinetic properties of mAMCase activity across a broad pH range, quantifying its unusual dual activity optima at pH 2 and 7. We also solved high resolution crystal structures of mAMCase in complex with oligomeric GlcNAcn, the building block of chitin, where we identified extensive conformational ligand heterogeneity. Leveraging these data, we conducted molecular dynamics simulations that suggest how a key catalytic residue could be protonated via distinct mechanisms in each of the two environmental pH ranges. These results integrate structural, biochemical, and computational approaches to deliver a more complete understanding of the catalytic mechanism governing mAMCase activity at different pH. Engineering proteins with tunable pH optima may provide new opportunities to develop improved enzyme variants, including AMCase, for therapeutic purposes in chitin degradation.

### Competing Interest Statement

S.J.V.D. and R.M.L. are listed as inventors on a patent for the use of chitinases to treat fibrotic lung disease. S.J.V.D., R.M.L., and J.S.F. are listed as inventors on a patent for mutant chitinases with enhanced expression and activity.

Structural characterization of ligand binding and pH-specific enzymatic activity of mouse Acidic Mammalian Chitinase

マウスの酸性マンマリーキチナーゼの配位子結合と-ph-特異的な酵素活性の構造的特徴付け


マウスの酸性マンマリーキチナーゼの配位子結合と pH 特異的な酵素活性の構造的特徴付け



Pseudomonas sp. strain JS3066は1-ナフチルアミンを唯一の炭素・窒素源として利用できる細菌株であり、その分解経路と初発反応を触媒する酵素NpaA1の構造と機能が明らかになった。


1-Naphthylamine (1NA), which is harmful to human and aquatic animals, has been used widely in the manufacturing of dyes, pesticides, and rubber antioxidants. Nevertheless, little is known about its environmental behavior and no bacteria have been reported to use it as the growth substrate. Herein, we describe a pathway for 1NA degradation in isolate Pseudomonas sp. strain JS3066, determine the structure and mechanism of the enzyme NpaA1 that catalyzes the initial reaction, and reveal how the pathway evolved. From genetic and enzymatic analysis, a cluster of 5 genes encoding a dioxygenase system was determined to be responsible for the initial steps in 1NA degradation through glutamylation of 1NA. The γ-glutamylated 1NA was subsequently oxidized to 1,2-dihydroxynaphthalene which was further degraded by the well-established pathway of naphthalene degradation via catechol. Enzymatic analysis showed that NpaA1 catalyzed conversion of various anilines and naphthylamine derivatives. Structural and biochemical studies of NpaA1 revealed that the broad substrate specificity of NpaA1 is due to a large hydrophobic pocket, which is different from type I glutamine synthetase (GSI). The findings enhance understanding of degrading polycyclic aromatic amines, and will also enable the application of bioremediation at naphthylamine contaminated sites.

### Competing Interest Statement

The authors have declared no competing interest.

Discovery of the 1-naphthylamine biodegradation pathway reveals an enzyme that catalyzes 1-naphthylamine glutamylation

1-ナフチルアミンの生分解経路の発見により-1-ナフチルアミンのグルタミン化を触媒する酵素が明らかになった


1-ナフチルアミンの生分解経路の発見により、1-ナフチルアミンのグルタミン化を触媒する酵素が明らかになった



光生物触媒を利用することで、従来の小分子触媒では困難だった不斉ラジカル反応を実現し、様々な非天然アミノ酸を立体選択的に合成できる。


We report on the oxidative&nbsp;cross-coupling of organoboron reagents and amino acids via pyridoxal biocatalysis to produce non-canonical amino acids, uncovering stereoselective, intermolecular free-radical transformations.

Stereoselective amino acid synthesis by photobiocatalytic oxidative coupling - Nature

光生物触媒的酸化カップリングによるステレオ選択的アミノ酸合成