Large Pocket Research Articles

DEPACT and PACMatch: A Workflow of Designing De Novo Protein Pockets to Bind Small Molecules.

Engineering of new functional proteins such as enzymes and biosensors involves the design of new protein pockets for the specific binding of small molecules. Here, we report a workflow composed of two new computational methods to execute this task. The DEPACT (Design Pocket as a Cluster based on Templates) method is a data-driven approach to design and evaluate small-molecule-binding pockets as isolated clusters, while the PACMatch method is a computational approach to match pocket residues in a cluster model to positions on given protein scaffolds. Using DEPACT and its scoring function, pocket clusters of natural-pocket-like chemical compositions and protein-ligand interaction strength can be designed. DEPACT can design pocket clusters containing water- or metal-ion-mediated protein-ligand interactions. While being able to efficiently treat relatively large pocket cluster models (e.g., of around 10 pocket residues), PACMatch outperforms previous methods in test cases of recovering the native positions of pocket residues in natural enzyme-substrate complexes.

Molecular and functional characterization of a SCD 1b from European sea bass (Dicentrarchus labrax L.)

Fatty acid desaturation is a highly complex and regulated process involving different molecular and genetic actors. Ultimally, the fatty acid desaturase enzymes are responsible for the introduction of double bonds at different positions of specific substrates, resulting in a wide variety of mono- and poly-unsaturated fatty acids. This substrate-specificity makes it possible to meet all the functional needs of the different tissues against a wide variety of internal and external conditions, giving rise to a varied profile of expression and functionality of the different desaturases in the body. Being our main interest to study and characterize at the molecular level the fatty acid desaturation process in fishes, we have focused our effort on characterizing SCD 1b from European sea bass (Dicentrarchus labrax, L.). In this work, we have characterized a tearoyl-CoA Desaturase cDNA that codes a protein of 334 amino acids, which shares the greatest homology to marine fish SCD 1b. Northern blot analysis showed two transcripts of 3.5kb and 1.4kb. Two putative cis-acting conserved motifs are localized in the cDNA 5'-end: a polypyrimidine CT dinucleotide repeat tract and two non-palindromic putative NRL-response elements (NREs). The deduced protein presents two Δ9 FADs like domain, three His-rich motifs, a total of nine His residues acting as di‑iron coordination ligands. The SCD 1b 3D protein modelling shows a structure made up primarily of α-helices, four of which could be transmembrane helices. The catalytic region is oriented to the cytosolic side of the Endoplasmic Reticulum membrane, where the 9-histidine residues are arranged coordinated to two non-heme Fe2+ ions. A new His-containing motif NX3H-like includes an Asn residue that participates in the coordination of Fe2+1 through a water molecule. The protein has a large pocket with a large opening to the outside. It includes a tunnel in which the substrate-binding site is located. The external shape is reminiscent of a boathook. It shows group specificity, although a greater preference for 18C substrates. The length of the tunnel, delimited by seven amino acids that forms a pocket at the end of the tunnel, the possibility that the substrates adopt different conformations inside the tunnel as well as and the movement of acyl chain inside the tunnel, could explain the high preference for 18C fatty acids and the group specificity of the enzyme. The cDNA encodes a functional SCD enzyme, whose subcellular localization is the Endoplasmic Reticulum, which complements the ole1Δ gene-disrupted gene in DTY-11A Saccharomyces cerevisiae strain and produces an increment of palmitoleic and oleic acids. The scd 1b gene is expressed in all tested tissues, showing the liver and adipose tissue a higher level of expression against the brain, heart, gonad and intestine. Scd 1b expression was always bigger than those of the Δ6 fad gene, being especially significant in adipose tissue and liver. From our data, we conclude that, in contrast to the functional significance of SCD 1b in adipose tissue, liver and heart, Δ6 FAD seems to play a more determining role in the biosynthesis of unsaturated fatty acids in the intestine, brain and gonad in fish.

Charge density waves and Fermi surface reconstruction in the clean overdoped cuprate superconductor Tl2Ba2CuO6+\u03b4

Hall effect and quantum oscillation measurements on high temperature cuprate superconductors show that underdoped compositions have small Fermi surface pockets whereas when heavily overdoped, a single much larger pocket is found. The origin of this change in electronic structure has been unclear, but may be related to the high temperature superconductivity. Here we show that the clean overdoped single-layer cuprate Tl2Ba2CuO6+δ (Tl2201) displays CDW order with a remarkably long correlation length ξ ≈ 200 Å which disappears above a hole doping of pCDW ≈ 0.265. We show that the evolution of the electronic properties of Tl2201 as the doping is lowered may be explained by a Fermi surface reconstruction which accompanies the emergence of the CDW below pCDW. Our results demonstrate importance of CDW correlations in understanding the electronic properties of overdoped cuprates.

Recognition of quinolone antibiotics by the multidrug efflux transporter MexB of Pseudomonas aeruginosa.

The drug/proton antiporter MexB is the engine of the major efflux pump MexAB-OprM in Pseudomonas aeruginosa. This protein is known to transport a large variety of compounds, including antibiotics, thus conferring a multi-drug resistance phenotype. Due to the difficulty of producing co-crystals, only two X-ray structures of MexB in a complex with ligands are available to date, and mechanistic aspects are largely hypothesized based on the body of data collected for the homologous protein AcrB of Escherichia coli. In particular, a recent study (Ornik-Cha, Wilhelm, Kobylka et al., Nat. Commun., 2021, 12, 6919) reported a co-crystal structure of AcrB in a complex with levofloxacin, an antibiotic belonging to the important class of (fluoro)-quinolones. In this work, we performed a systematic ensemble docking campaign coupled to the cluster analysis and molecular-mechanics optimization of docking poses to study the interaction between 36 quinolone antibiotics and MexB. We additionally investigated surface complementarity between each molecule and the transporter and thoroughly assessed the computational protocol adopted against the known experimental data. Our study reveals different binding preferences of the investigated compounds towards the sub-sites of the large deep binding pocket of MexB, supporting the hypothesis that MexB substrates oscillate between different binding modes with similar affinity. Interestingly, small changes in the molecular structure translate into significant differences in MexB–quinolone interactions. All the predicted binding modes are available for download and visualization at the following link: https://www.dsf.unica.it/dock/mexb/quinolones.

Status of the mastoid antrum and the eustachian tube function in cases of chronic otitis media

Introduction: Chronic otitis media (COM) is now synonymous with chronic suppurative otitis media, which is not actively used since COM is seldom associated with “the collection of pus.” Acute suppuration of the ear which transcends 6 weeks is considered to be COM which suggests an abnormality of the ear drum. With a prevalence incidence of 1%, Eustachian tube (ET) dysfunction is among the most important factors in the development of COM. Recently, mastoid air cells have been acknowledged as an essential compounding factor for the pathogenesis of middle ear disease. Since ET, mastoid air cell system, and nasopharyngeal mass like adenoids can play a pivotal role in the causation and persistence of COM, the current study will be taken to observe the status of ET, mastoid air cells system, nasopharynx in general, and adenoids in particular in patients of COM. Aim: This study aims to study the status of the mastoid antrum on surgical exploration, the status of ET, and nasopharynx for adenoids in patients of large, subtotal perforation, and posterosuperior retraction pockets (PSRPs). Study Design: This study was an observational, cross-sectional study. Study Setting: All the patients with large, subtotal perforation, and PSRPs visiting the Outpatient Department and Inpatient Department of ENT in Acharya Vinoba Bhave Rural Hospital (AVBRH) between 2022 and 2023 will be studied. Methods: All the selected patients of large, subtotal perforation, and posterosuperior retraction pocket (PSRP) in the age bracket of 15–65 years and satisfying the inclusion criteria will be considered and accrued in this study. We plan to look at a sample size of 50 patients (50 ears). Diagnostic nasal video endoscopy to get insight into ET function, size, and grade of adenoids by grading system given by Cassano et al. and pure-tone audiometry by ALPS AD2000 to know the type and amount of hearing loss will be carried out. Status of the mastoid antrum, the extent of the disease, status of ossicles, and associated abnormalities of temporal bone along with any other incidental findings will be noted intraoperatively. Expected Results: The data will be tabulated, analyzed statistically, and will be discussed in the context of existing research. Conclusion: The purpose of the present study is to help the surgeon have a better insight into the status of the mastoid antrum, ET in cases of large and subtotal perforation, and PSRP and nasopharynx in general with significance to adenoids in patients of COM in rural area.

Identifying FAAH Inhibitors as New Therapeutic Options for the Treatment of Chronic Pain through Drug Repurposing.

Chronic pain determines a substantial burden on individuals, employers, healthcare systems, and society. Most of the affected patients report dissatisfaction with currently available treatments. There are only a few and poor therapeutic options—some therapeutic agents are an outgrowth of drugs targeting acute pain, while others have several serious side effects. One of the primary degradative enzymes for endocannabinoids, fatty acid amide hydrolase (FAAH) attracted attention as a significant molecular target for developing new therapies for neuropsychiatric and neurological diseases, including chronic pain. Using chemical graph mining, quantitative structure–activity relationship (QSAR) modeling, and molecular docking techniques we developed a multi-step screening protocol to identify repurposable drugs as FAAH inhibitors. After screening the DrugBank database using our protocol, 273 structures were selected, with five already approved drugs, montelukast, repaglinide, revefenacin, raloxifene, and buclizine emerging as the most promising repurposable agents for treating chronic pain. Molecular docking studies indicated that the selected compounds interact with the enzyme mostly non-covalently (except for revefenacin) through shape complementarity to the large substrate-binding pocket in the active site. A molecular dynamics simulation was employed for montelukast and revealed stable interactions with the enzyme. The biological activity of the selected compounds should be further confirmed by employing in vitro and in vivo studies.

Precise druggability of the PTH type 1 receptor.

Class B G protein-coupled receptors (GPCRs) are notoriously difficult to target by small molecules because their large orthosteric peptide-binding pocket embedded deep within the transmembrane domain limits the identification and development of non-peptide small molecule ligands. Using the parathyroid hormone type 1 receptor (PTHR) as a prototypic class B GPCR target, and a combination of molecular dynamics simulations and elastic network model-based methods, we demonstrate that PTHR druggability can be effectively addressed. Here we found a key mechanical site that modulates the collective dynamics of the receptor and used this ensemble of PTHR conformers to identify selective small molecules with strong negative allosteric and biased properties for PTHR signaling in cell and PTH actions in vivo. This study provides a computational pipeline to detect precise druggable sites and identify allosteric modulators of PTHR signaling that could be extended to GPCRs to expedite discoveries of small molecules as novel therapeutic candidates.

The Dichotomy of Intimate Garments: Women’s Stays and Pockets in Georgian Britain

In John Collet’s 1777 print, Tight Lacing, or Fashion Before Ease a woman is depicted holding onto a bed poster as her maid and husband strain to tighten the lacing on her stays. Meanwhile, a large tie-on pocket can be seen hanging from the woman’s waist. Although they were worn closely together on the female body stays and pockets have been studied independently from each other. This is understandable, as at first glance, they appear incomparable. Stays were the precursor to nineteenth century corsets and they moulded the body into the desired and fashionable shape. Pockets on the other hand were utilitarian bags meant for carrying necessary items; separate from women’s clothing they were tied around the waist with string and were hidden under the skirts. Despite the initial differences, these garments did not function independently from each other. Using caricatures, etiquette books, and physical examples found in museum collections, this study demonstrates that when examined in tandem it becomes apparent that stays and pockets served distinct, yet overlapping functions in women’s lives. They informed women’s relationships with their surrounding environments, strengthened women’s connection to the domestic realm, yet also gave women freedom to move outside the home and participate within the public sphere. Furthermore, both items also acted as private spaces, which allowed women to actively partake in romantic courtship and sexual flirtations in a gender appropriate way. By juxtaposing stays and pockets this study contributes to the growing historical discussion surrounding these items, but also begins to piece together a larger picture of the role undergarments, as a whole, played in women’s lives.

Achieving S2- in aqueous Solution: An evaluation using First-Principle molecular dynamics simulations

First principles molecular dynamics simulations were carried out in several systems built to simulate solutions of S2- in NaOH 20 M. The simulations were performed in an NVT-ensemble at 300 K for eleven picoseconds. The obtained results confirm previous experimental results, that is the S2- dianion does not exist in aqueous solution, not even in a hyper-alkaline solution. However, this circumstance can change if a large enough pocket containing the S2- dianion without counterions is created in the system.

Role of substrate recognition in modulating strigolactone receptor selectivity in witchweed

Witchweed, or Striga hermonthica, is a parasitic weed that destroys billions of dollars' worth of crops globally every year. Its germination is stimulated by strigolactones exuded by its host plants. Despite high sequence, structure, and ligand-binding site conservation across different plant species, one strigolactone receptor in witchweed, ShHTL7, uniquely exhibits a picomolar EC50 for downstream signaling. Previous biochemical and structural analyses have hypothesized that this unique ligand sensitivity can be attributed to a large binding pocket volume in ShHTL7 resulting in enhanced ability to bind substrates, but additional structural details of the substrate-binding process would help explain its role in modulating the ligand selectivity. Using long-timescale molecular dynamics simulations, we demonstrate that mutations at the entrance of the binding pocket facilitate a more direct ligand-binding pathway to ShHTL7, whereas hydrophobicity at the binding pocket entrance results in a stable “anchored” state. We also demonstrate that several residues on the D-loop of AtD14 stabilize catalytically inactive conformations. Finally, we show that strigolactone selectivity is not modulated by binding pocket volume. Our results indicate that while ligand binding is not the sole modulator of strigolactone receptor selectivity, it is a significant contributing factor. These results can be used to inform the design of selective antagonists for strigolactone receptors in witchweed.

Clinical pregnancy resulting from intracytoplasmic sperm injection of prematurely ovulated oocytes retrieved from the posterior cul-de-sac

ObjectiveTo report a clinical pregnancy resulting from intracytoplasmic sperm injection of prematurely ovulated oocytes retrieved from the posterior cul-de-sac.DesignCase report.SettingAcademic center.Patient(s)A 40-year-old nulligravid woman underwent ovarian stimulation for in vitro fertilization (IVF). Daily injections of gonadotropin-releasing hormone antagonist were initiated on cycle day 8. A 10,000 IU dose of human chorionic gonadotropin was administered on cycle day 15 to trigger follicular maturation. The estradiol and luteinizing hormone levels on the trigger day were 1528 pg/mL and 2.4 mIU/mL, respectively. The patient underwent oocyte retrieval 35 hours after the trigger. Transvaginal sonography at the time of the retrieval revealed a large pocket of free fluid in the posterior cul-de-sac. Only 3 follicles measuring 10–12 mm were noted in both ovaries. No lead follicles were visualized.Intervention(s)Aspiration of free fluid from the posterior cul-de-sac.Main Outcome Measure(s)Clinical pregnancy.Result(s)The fluid in the posterior cul-de-sac was aspirated, and 3 mature oocytes were retrieved. Aspiration of the smaller ovarian follicles measuring 10–12 mm did not yield oocytes. All mature oocytes retrieved from the posterior cul-de-sac were fertilized with intracytoplasmic sperm injection. Three cleavage-stage embryos were transferred 3 days later. A single intrauterine pregnancy with cardiac activity was confirmed at a gestational age of 7 weeks.Conclusion(s)In the setting of premature ovulation, aspiration of free fluid from the posterior cul-de-sac can result in the retrieval of mature oocytes, which may result in clinical pregnancies.

Computational Redesign of an ω-Transaminase from Pseudomonas jessenii for Asymmetric Synthesis of Enantiopure Bulky Amines.

ω-Transaminases (ω-TA) are attractive biocatalysts for the production of chiral amines from prochiral ketones via asymmetric synthesis. However, the substrate scope of ω-TAs is usually limited due to steric hindrance at the active site pockets. We explored a protein engineering strategy using computational design to expand the substrate scope of an (S)-selective ω-TA from Pseudomonas jessenii (PjTA-R6) toward the production of bulky amines. PjTA-R6 is attractive for use in applied biocatalysis due to its thermostability, tolerance to organic solvents, and acceptance of high concentrations of isopropylamine as amino donor. PjTA-R6 showed no detectable activity for the synthesis of six bicyclic or bulky amines targeted in this study. Six small libraries composed of 7–18 variants each were separately designed via computational methods and tested in the laboratory for ketone to amine conversion. In each library, the vast majority of the variants displayed the desired activity, and of the 40 different designs, 38 produced the target amine in good yield with >99% enantiomeric excess. This shows that the substrate scope and enantioselectivity of PjTA mutants could be predicted in silico with high accuracy. The single mutant W58G showed the best performance in the synthesis of five structurally similar bulky amines containing the indan and tetralin moieties. The best variant for the other bulky amine, 1-phenylbutylamine, was the triple mutant W58M + F86L + R417L, indicating that Trp58 is a key residue in the large binding pocket for PjTA-R6 redesign. Crystal structures of the two best variants confirmed the computationally predicted structures. The results show that computational design can be an efficient approach to rapidly expand the substrate scope of ω-TAs to produce enantiopure bulky amines.

Mechanistic Insights into the P450 TleB-Catalyzed Unusual Intramolecular C-N Bond Formation Involved in the Biosynthesis of Indolactam V.

Indolactam V, a known biosynthetic precursor of indolactam alkaloids, is the main pharmacophore of this family and exhibits potential protein kinase C activation. A key step in the biosynthesis of indolactam V is the formation of an indole-fused nine-membered lactam core by intramolecular C-N bond formation. In this work, we report a computational study of the unique cytochrome P450 TleB enzyme-catalyzed direct and selective C-H bond amination reaction that can generate indolactam V from the dipeptide N-methylvalyl-tryptophanol. By performing molecular dynamics simulations and quantum-mechanical/molecular-mechanical calculations, we revealed that the C-H bond amination involves one step of proton transfer from N1-H of the indole ring to the FeIV═O unit, one step of hydrogen abstraction of N13-H in the side chain of the substrate by the FeIV-OH unit, and diradical coupling, in which two conformational changes of the side chain of the substrate are necessary. In the enzyme-substrate complex of TleB, the N-H bond of the indole ring of the substrate forms a strong hydrogen bond with the FeIV═O unit in compound I, and the porphyrin radical cation accepts an electron from the substrate to form the closed-shell electronic configuration. Thus, compound I in the enzyme-substrate complex cannot be described as FeIV═O coupled to a porphyrin radical cation, which is different from those of other P450 enzymes. Besides, two stages of conformational changes of the side chains of the substrate may increase the relative energies of reaction intermediates by 10-12 kcal/mol. From the structure point of view, it is the rotatable long side chain of the substrate and the large flexible active pocket of TleB that make the intramolecular diradical coupling feasible. Our findings may provide useful information to further understand the Tleb-catalyzed intramolecular C-H bond amination and the other bio-catalyzed intramolecular diradical coupling.

Electronic properties of single-layer CoO2/Au(111)

We report direct measurements via angle-resolved photoemission spectroscopy (ARPES) of the electronic dispersion of single-layer (SL) CoO2. The Fermi contour consists of a large hole pocket centered at the point. To interpret the ARPES results, we use density functional theory (DFT) in combination with the multi-orbital Gutzwiller Approximation (DFT+GA), basing our calculations on crystalline structure parameters derived from x-ray photoelectron diffraction and low-energy electron diffraction. Our calculations are in good agreement with the measured dispersion. We conclude that the material is a moderately correlated metal. We also discuss substrate effects, and the influence of hydroxylation on the CoO2 SL electronic structure.

Precision druggability of a class B GPCR, the PTH type 1 receptor

Class B G protein-coupled receptors (GPCRs) are notoriously difficult to target by small molecules because their large orthosteric peptide-binding pocket embedded deep within the transmembrane domain limits the identification and development of nonpeptide small molecule ligands. Using the parathyroid hormone type 1 receptor (PTHR) as a prototypic class B GPCR target, and a combination of molecular dynamics simulations and elastic network model-based methods, we demonstrate that PTHR druggability can be effectively addressed. Here we found a key mechanical site that modulates the collective dynamics of the receptor and used this ensemble of PTHR conformers to identify selective small molecules with strong negative allosteric and biased properties for PTHR signaling in cell and PTH actions in vivo. This study provides a computational pipeline to detect precise druggable sites and identify allosteric modulators in PTHR signaling that could be extended broadly to GPCRs, in particular to class B peptide hormone receptors to expedite discoveries of small molecules toward the treatment of diverse hormonal and metabolic diseases.

Antipsychotic clozapine binding to alpha-2-macroglobulin protects interacting partners against oxidation and preserves the anti-proteinase activity of the protein

In this study, the interaction between clozapine, an atypical antipsychotic drug, and alpha-2-macroglobulin (α2M), a multipurpose anti-proteinase, was investigated under simulated (patho) physiological conditions using multiple spectroscopic techniques and molecular modeling. It was found that α2M binds clozapine with a moderate affinity (the binding constant of 0.9 × 105 M−1 at 37 °C). The preferable binding site for both clozapine's atropisomers was revealed to be a large pocket at the interface of C and D monomer subunits of the protein. Hydrogen bonds and the hydrophobic effect were proposed as dominant forces in complex formation. The binding of clozapine did not induce significant conformational change of the protein, as confirmed by virtually unaltered α2M secondary structure and anti-proteinase activity. However, both clozapine and α2M shielded each other from the deleterious influence of strong oxidants: sodium hypochlorite and 2,2′-azobis-2-methyl-propanimidamide dihydrochloride (AAPH). Moreover, clozapine in a concentration range that is usually targeted in the plasma during patients' treatment effectively protected the anti-proteinase activity of α2M under AAPH-induced free radical overproduction. Our results suggest that the cooperation between α2M and clozapine may be a path by which these two molecules synergistically protect neural tissue against injury caused by disturbed proteostasis or oxidative stress.

Arabidopsis Carboxylesterase 20 Binds Strigolactone and Increases Branches and Tillers When Ectopically Expressed in Arabidopsis and Maize.

Severe drought stress can delay maize silk emergence relative to the pollen shedding period, resulting in poor fertilization and reduced grain yield. Methods to minimize the delay in silking could thus improve yield stability. An Arabidopsis enhancer-tagged carboxylesterase 20 (AtCXE20) line was identified in a drought tolerance screen. Ectopic expression of AtCXE20 in Arabidopsis and maize resulted in phenotypes characteristic of strigolactone (SL)-deficient mutants, including increased branching and tillering, decreased plant height, delayed senescence, hyposensitivity to ethylene, and reduced flavonols. Maize silk growth was increased by AtCXE20 overexpression, and this phenotype was partially complemented by exogenous SL treatments. In drought conditions, the transgenic maize plants silked earlier than controls and had decreased anthesis-silking intervals. The purified recombinant AtCXE20 protein bound SL in vitro, as indicated by SL inhibiting AtCXE20 esterase activity and altering AtCXE20 intrinsic fluorescence. Homology modeling of the AtCXE20 three-dimensional (3D) protein structure revealed a large hydrophobic binding pocket capable of accommodating, but not hydrolyzing SLs. The AtCXE20 protein concentration in transgenic maize tissues was determined by mass spectrometry to be in the micromolar range, well-above known endogenous SL concentrations. These results best support a mechanism where ectopic expression of AtCXE20 with a strong promoter effectively lowers the concentration of free SL by sequestration. This study revealed an agriculturally important role for SL in maize silk growth and provided a new approach for altering SL levels in plants.

Effects of new micro-pocketed bore surface topographies on the performance behaviours of aerodynamic journal bearing

This paper presents the exploration for improving the static and dynamic performance behaviours of a self-acting rigid gas journal bearing employing some new conceived micro-depth pocketed surface topographies at the bore. The conceived micro-pockets comprise of relatively large size rectangular pocket having micro-depth at bearing bore in the converging zone followed by placing of different designs of sub-pockets at the trailing edge of the previous one in the direction of journal rotation. The form of equation achieved from Patir and Cheng’s model for the case of hydrodynamic lubrication regime and the related dynamic pressure equations have been solved using the finite volume method discretisation scheme followed by the solution of the algebraic equations using the Gauss-Seidel iterative method. The minimum film thickness, frictional force, side leakage, bearing dynamic coefficients, effective stiffness, effective damping, and critical mass parameters have been investigated with each new bore surface topography and compared with the performances of conventional aerodynamic journal bearing. Substantial improvements in both static and dynamic performances have been found with the new micro-pocketed bore surface topographies as compared to conventional one. Moreover, the established best bore design has produced significant increase (21%) in minimum film thickness, substantial reduction (12%) in coefficient of friction, and excellent improvement (170%) in the stability parameter (critical mass) as compared to the conventional case.

Gas-liquid two-phase flow patterns in the wake of a submerged nozzle under co-flow condition

The present study aims to reveal gas-liquid flow patterns as gas is injected into co-flowing water. An experimental work was conducted in a loop in which pure water was circulated. The gas-injection nozzle was submerged in the flowing water. Effects of the water flow velocity and gas flow rate were jointly considered. Two-phase flow patterns were visualized using the high-speed photography and then analyzed statistically. The evolution of the cavity-water interface and collapse of cavity were explained. The results show that a sequential arrangement of a long attached cavity and dispersed bubbles in the streamwise direction constitutes a unique flow pattern. The cavity length is dependent of the contest between the gas velocity and water flow velocity. A single-valley distribution of pinch-off frequency as the gas/water velocity ratio varies is insensitive to the water flow velocity. At high water flow velocities, collapse of cavity follows the pattern of wake vortices. Distinct coalescence is evidenced at high gas/water velocity ratio as a large gas pocket is separated from the attached cavity. It is ascribed to the retarded motion of the gas pocket and high inertia of the subsequently detached small bubble.

Functional and structural characterization of a flavoprotein monooxygenase essential for biogenesis of tryptophylquinone cofactor

Bioconversion of peptidyl amino acids into enzyme cofactors is an important post-translational modification. Here, we report a flavoprotein, essential for biosynthesis of a protein-derived quinone cofactor, cysteine tryptophylquinone, contained in a widely distributed bacterial enzyme, quinohemoprotein amine dehydrogenase. The purified flavoprotein catalyzes the single-turnover dihydroxylation of the tryptophylquinone-precursor, tryptophan, in the protein substrate containing triple intra-peptidyl crosslinks that are pre-formed by a radical S-adenosylmethionine enzyme within the ternary complex of these proteins. Crystal structure of the peptidyl tryptophan dihydroxylase reveals a large pocket that may dock the protein substrate with the bound flavin adenine dinucleotide situated close to the precursor tryptophan. Based on the enzyme-protein substrate docking model, we propose a chemical reaction mechanism of peptidyl tryptophan dihydroxylation catalyzed by the flavoprotein monooxygenase. The diversity of the tryptophylquinone-generating systems suggests convergent evolution of the peptidyl tryptophan-derived cofactors in different proteins.