Crystal structure of multi-functional enzyme FadB from Cupriavidus necator: Non-formation of FadAB complex.

Cupriavidus necator H16 is a gram-negative chemolithoautotrophic bacterium that has been extensively studied for biosynthesis and biodegradation of polyhydroxyalkanoate (PHA) plastics. To improve our understanding of fatty acid metabolism for PHA production, we determined the crystal structure of multi-functional enoyl-CoA hydratase from Cupriavidus necator H16 (CnFadB). The predicted model of CnFadB created by AlphaFold was used to solve the phase problem during determination of the crystal structure of the protein. The CnFadB structure consists of two distinctive domains, an N-terminal enol-CoA hydratase (ECH) domain and a C-terminal 3-hydroxyacyl-CoA dehydrogenase (HAD) domain, and the substrate- and cofactor-binding modes of these two functional domains were identified. Unlike other known FadB enzymes that exist as dimers complexed with FadA, CnFadB functions as a monomer without forming a complex with CnFadA. Small angle X-ray scattering (SAXS) measurement further proved that CnFadB exists as a monomer in solution. The non-sequential action of FadA and FadB in C. necator appears to affect ß-oxidation and PHA synthesis/degradation.

Structural insights into a maleylpyruvate hydrolase from sphingobium sp. SYK-6, a bacterium degrading lignin-derived aryls.

Sphingobium sp. strain SYK-6, an aerobic gram-negative bacillus found in soil, is known for utilizing lignin-derived monoaryls and biaryls as carbon sources and degrading aromatic compounds. The Sphingobium sp. strain SYK-6 genome contains three genes involved in salicylate catabolism: SLG_11260, SLG_11270, and SLG_11280. Here, we report that the gene product of SLG_11280 functions as a maleylpyruvate hydrolase (SsMPH) with Km and Kcat values of 166.2 µM and 3.76 min-1, respectively. This study also reveals the crystal structures of both the apo and pyruvate-manganese ion-bound SsMPH, which revealed that like other fumarylacetoacetate hydrolases, SsMPH dimerizes and has nine unique 310-helices. Molecular docking studies of maleylpyruvate also revealed the likely binding mode of SsMPH and its substrate.

Production of extracellular PETase from Ideonella sakaiensis using sec-dependent signal peptides in E. coli.

Poly(ethylene terephthalate) (PET) is the most commonly used polyester polymer resin in fabrics and storage materials, and its accumulation in the environment is a global problem. The ability of PET hydrolase from Ideonella sakaiensis 201-F6 (IsPETase) to degrade PET at moderate temperatures has been studied extensively. However, due to its low structural stability and solubility, it is difficult to apply standard laboratory-level IsPETase expression and purification procedures in industry. To overcome this difficulty, the expression of IsPETase can be improved by using a secretion system. This is the first report on the production of an extracellular IsPETase, active against PET film, using Sec-dependent translocation signal peptides from E. coli. In this work, we tested the effects of fusions of the Sec-dependent and SRP-dependent signal peptides from E. coli secretory proteins into IsPETase, and successfully produced the extracellular enzyme using pET22b-SPMalE:IsPETase and pET22b-SPLamB:IsPETase expression systems. We also confirmed that the secreted IsPETase has PET-degradation activity. The work will be used for development of a new E. coli strain capable of degrading and assimilating PET in its culture medium.

Effect of Process Parameters on Formation and Aggregation of Nanoparticles Prepared with a Shirasu Porous Glass Membrane.

The objectives of this study were to prepare itraconazole (ITZ) nanoparticles using a Shirasu porous glass (SPG) membrane and to characterize the effects of diverse preparation parameters on the physical stability of nanoparticles. SPG membrane technology was used for the antisolvent precipitation method. The preparation of nanoparticles was carried out over a wide range of continuous-phase factors (type of surfactant, surfactant concentration), dispersed-phase factors (solvent type, solvent volume used to dissolve ITZ), and technical factors (pressure, membrane pore size, stirring speed in the continuous phase, temperature). Improved physical stability of nanoparticles was observed when surfactant with a lower molecular weight and higher hydrophilic segment ratio was used. The water miscibility of the solvent also had an effect on the physical stability. N,N-Dimethylacetamide contributed to creating a well-rounded shape and narrow size distribution due to high miscibility. Concentration of the surfactant and solvent volume used for dissolving ITZ were related to instability of nanoparticles, resulting from depletion attraction and Ostwald ripening. In addition to these factors, technical factors changed the environment surrounding ITZ nanoparticles, such as the physicochemical equilibrium between surfactant and ITZ nanoparticles. Therefore, the appropriate continuous-phase factors, dispersed-phase factors, and technical factors should be maintained for stabilizing ITZ nanoparticles.

Biodegradable Polymer Versus Polymer-Free Ultrathin Sirolimus-Eluting Stents: Analysis of the Stent Arm Registry From the HOST-IDEA Randomized Trial.

BACKGROUND: The efficacy and safety of each third-generation drug-eluting stent with ultrathin struts and advanced polymer technology remain unclear. We investigated the clinical outcomes of percutaneous coronary intervention using the Coroflex ISAR polymer-free sirolimus-eluting stent (SES) or Orsiro biodegradable polymer SES. METHODS: The HOST-IDEA trial (Harmonizing Optimal Strategy for Treatment of Coronary Artery Stenosis-Coronary Intervention With Next-Generation Drug-Eluting Stent Platforms and Abbreviated Dual Antiplatelet Therapy), initially designed with a 2×2 factorial approach, sought to randomize patients undergoing percutaneous coronary intervention based on dual antiplatelet therapy duration (3 versus 12 months) and stent type (Coroflex ISAR versus Orsiro). Despite randomizing 2013 patients for dual antiplatelet therapy duration, the stent arm transitioned to a registry format during the trial. Among these, 328 individuals (16.3%) were randomized for Coroflex ISAR or Orsiro SES, while 1685 (83.7%) underwent percutaneous coronary intervention without stent-type randomization. In this study, the Coroflex ISAR (n=559) and Orsiro groups (n=1449) were matched using a propensity score. The prespecified primary end point was target lesion failure, a composite of cardiac death, target vessel myocardial infarction, and clinically driven target lesion revascularization at 12 months. RESULTS: The baseline patient and procedural characteristics were well balanced between the Coroflex ISAR and Orsiro groups after propensity score matching (n=559, each group). The Coroflex ISAR group was significantly associated with a higher rate of target lesion failure, mainly driven by clinically driven target lesion revascularization, compared with the Orsiro group (3.4% versus 1.1%; hazard ratio, 3.21 [95% CI, 1.28-8.05]; P=0.01). A higher risk of target lesion failure in the Coroflex ISAR group was consistently observed across various subgroups. The rates of any bleeding (hazard ratio, 0.85 [95% CI, 0.51-1.40]; P=0.52) and major bleeding (hazard ratio, 1.58 [95% CI, 0.61-4.08]; P=0.34) were comparable between the 2 groups. CONCLUSIONS: In this propensity score-matched analysis of the stent arm registry from the HOST-IDEA trial, the Orsiro SES was associated with significantly better outcomes in terms of 1-year target lesion failure, mainly driven by clinically driven target lesion revascularization, than the Coroflex ISAR SES. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02601157.

Discovery and rational engineering of PET hydrolase with both mesophilic and thermophilic PET hydrolase properties.

Excessive polyethylene terephthalate (PET) waste causes a variety of problems. Extensive research focused on the development of superior PET hydrolases for PET biorecycling has been conducted. However, template enzymes employed in enzyme engineering mainly focused on IsPETase and leaf-branch compost cutinase, which exhibit mesophilic and thermophilic hydrolytic properties, respectively. Herein, we report a PET hydrolase from Cryptosporangium aurantiacum (CaPETase) that exhibits high thermostability and remarkable PET degradation activity at ambient temperatures. We uncover the crystal structure of CaPETase, which displays a distinct backbone conformation at the active site and residues forming the substrate binding cleft, compared with other PET hydrolases. We further develop a CaPETaseM9 variant that exhibits robust thermostability with a Tm of 83.2 °C and 41.7-fold enhanced PET hydrolytic activity at 60 °C compared with CaPETaseWT. CaPETaseM9 almost completely decompose both transparent and colored post-consumer PET powder at 55 °C within half a day in a pH-stat bioreactor.

Three-directional engineering of IsPETase with enhanced protein yield, activity, and durability.

The mesophilic PETase from Ideonella sakaiensis (IsPETase) has been shown to exhibit high PET hydrolysis activity, but its low stability limits its industrial applications. Here, we developed a variant, Z1-PETase, with enhanced soluble protein yield and durability while maintaining or improving activity at lower temperatures. The selected Z1-PETase not only exhibited a 20-fold improvement in soluble protein yield compared to the previously engineered IsPETaseS121E/D186H/S242T/N246D (4p) variant, but also demonstrated a 30% increase in low-temperature activity at 40 °C, along with an 11 °C increase in its TmD value. The PET depolymerization test across a temperature range low to high (30-70 °C) confirmed that Z1-PETase exhibits high accessibility of mesophilic PET hydrolase and rapid depolymerizing rate at higher temperature in accordance with the thermal behaviors of polymer and enzyme. Additionally, structural interpretation indicated that the stabilization of specific active site loops in Z1-PETase contributes to enhanced thermostability without adversely impacting enzymatic activity. In a pH-stat bioreactor, Z1-PETase depolymerized > 90% of both transparent and colored post-consumer PET powders within 24 and 8 h at 40 °C and 55 °C, respectively, demonstrating that the utility of this IsPETase variant in the bio-recycling of PET.

Novel FAM20A mutations in hypoplastic amelogenesis imperfecta.

Amelogenesis imperfecta (AI) is a genetically and clinically heterogeneous group of inherited dental enamel defects without any other nonoral symptoms. Recently, a disease-causing nonsense mutation (c.406C>T) in a novel gene, FAM20A, was identified in a large consanguineous family affected by AI with gingival hyperplasia. We performed mutational analyses on nine AI families with similar phenotypes and identified three homozygous mutations (c.34_35delCT, c.813-2A>G, c.1175_1179delGGCTC) in three families and a compound heterozygous mutation (c.[590-2A>G] + [c.826C>T]) in one family. An in vitro splicing assay with a minigene confirmed the mutations located in the splicing acceptor site caused the deletion of exons 3 and 6, respectively. Taking into consideration the locations of the nonsense and frameshift mutations, the mutant transcripts are most likely degraded by nonsense-mediated mRNA degradation and it results in a loss of the FAM20A protein. This study confirms the importance of the FAM20A protein in enamel biomineralization as well as tooth eruption.

Structural and functional characterization of an auxiliary domain-containing PET hydrolase from Burkholderiales bacterium.

Biodegradation of polyethylene terephthalate (PET) is one of fundamental ways to solve plastic pollution. As various microbial hydrolases have an extra domain unlike PETase from Ideonella sakaiensis (IsPETase), research on the role of these extra domain in PET hydrolysis is crucial for the identification and selection of a novel PET hydrolase. Here, we report that a PET hydrolase from Burkholderiales bacterium RIFCSPLOWO2_02_FULL_57_36 (BbPETase) with an additional N-terminal domain (BbPETaseAND) shows a similar hydrolysis activity toward microcrystalline PET and a higher thermal stability than IsPETase. Based on detailed structural comparisons between BbPETase and IsPETase, we generated the BbPETaseS335N/T338I/M363I/N365G variant with an enhanced PET-degrading activity and thermal stability. We further revealed that BbPETaseAND contributes to the thermal stability of the enzyme through close contact with the core domain, but the domain might hinder the adhesion of enzyme to PET substrate. We suggest that BbPETase is an enzyme in the evolution of efficient PET degradation and molecular insight into a novel PET hydrolase provides a novel strategy for the development of biodegradation of PET.

Implications for the PET decomposition mechanism through similarity and dissimilarity between PETases from Rhizobacter gummiphilus and Ideonella sakaiensis.

The development of a superb polyethylene terephthalate (PET) hydrolyzing enzyme requires an accurate understanding of the PET decomposition mechanism. However, studies on PET degrading enzymes, including the PET hydrolase from Ideonella sakaiensis (IsPETase), have not provided sufficient knowledge of the molecular mechanisms for the hardly accessible substrate. Here, we report a novel PET hydrolase from Rhizobacter gummiphilus (RgPETase), which has a hydrolyzing activity similar to IsPETase toward microcrystalline PET but distinct behavior toward low crystallinity PET film. Structural analysis of RgPETase reveals that the enzyme shares the key structural features of IsPETase for high PET hydrolysis activity but has distinguished structures at the surface-exposed regions. RgPETase shows a unique conformation of the wobbling tryptophan containing loop (WW-loop) and change of the electrostatic surface charge on the loop dramatically affects the PET-degrading activity. We further show that effect of the electrostatic surface charge to the activity varies depending on locations. This work provides valuable information underlying the uncovered PET decomposition mechanism.

Extra disulfide and ionic salt bridge improves the thermostability of lignin peroxidase H8 under acidic condition.

The development of a lignin peroxidase (LiP) that is thermostable even under acidic pH conditions is a main issue for efficient enzymatic lignin degradation due to reduced repolymerization of free phenolic products at acidic pH (< 3). Native LiP under mild conditions (half-life (t1/2) of 8.2 days at pH 6) exhibits a marked decline in thermostability under acidic conditions (t1/2 of only 14 min at pH 2.5). Thus, improving the thermostability of LiP in acidic environments is required for effective lignin depolymerization in practical applications. Here, we show the improved thermostability of a synthetic LiPH8 variant (S49C/A67C/H239E, PDB: 6ISS) capable of strengthening the helix-loop interactions under acidic conditions. This variant retained excellent thermostability at pH 2.5 with a 10-fold increase in t1/2 (2.52 h at 25 °C) compared with that of the native enzyme. X-ray crystallography analysis showed that the recombinant LiPH8 variant is the only unique lignin peroxidase containing five disulfide bridges, and the helix-loop interactions of the synthetic disulfide bridge and ionic salt bridge in its structure are responsible for stabilizing the Ca2+-binding region and heme environment, resulting in an increase in overall structural resistance against acidic conditions. Our work will allow the design of biocatalysts for ligninolytic enzyme engineering and for efficient biocatalytic degradation of plant biomass in lignocellulose biorefineries.

Immediate reconstruction of mandibular defect after treatment of medication-related osteonecrosis of the jaw (MRONJ) with rhBMP-2/ACS and miniplate: Review of 3 cases.

INTRODUCTION: The purpose of this study was to pursue, and to report the results of, mandibular reconstruction and rehabilitation of medication-related osteonecrosis of the jaw (MRONJ) patients having large critical-sized defects of the mandible using a combination of recombinant human bone morphogenetic protein-2 (rhBMP-2) and absorbable collagen sponge (ACS) with surgical miniplate without any grafting materials. CASE PRESENTATION: Three (3) patients aged 67 and 86 (2 patients) presented due to discomfort on the mandible. They all had a medical history of bisphosphonate and steroids treatment orally or intravenously, and all had been diagnosed as MRONJ stage 3. Sequestrectomy and saucerization were performed, and then a surgical miniplate (Hansolmedical, Korea) was adapted and fixed on the sound portion of the mandible. rhBMP-2 was loaded onto an ACS at a dose of 1.5 mg/cc. Several rhBMP-2 (Cowellmedi, Korea)/ACS (Ateloplug, TRMkorea, Korea) were placed into the bony defect with a surgical miniplate. All 3 patients recovered without complications. They all exhibited radiographic evidence of bone formation by 3 months postoperatively in every case. CONCLUSIONS: All 3 patients were treated successfully with rhBMP-2/ACS and miniplate without any complications. This protocol reported herein represents a new approach to the surgical treatment of maxillofacial bone defects and deficiencies, especially in MRONJ patients.

Structural bioinformatics-based protein engineering of thermo-stable PETase from Ideonella sakaiensis.

Poly(ethylene terephthalate) (PET), a widely used plastic around the world, causes various environmental and health problems. Several groups have been extensively conducting research to solve these problems through enzymatic degradation of PET at high temperatures around 70 °C. Recently, Ideonella sakaiensis, a bacterium that degrades PET at mild temperatures, has been newly identified, and further protein engineering studies on the PET degrading enzyme from the organism (IsPETase) have also been conducted to overcome the low thermal stability of the enzyme. In this study, we performed structural bioinformatics-based protein engineering of IsPETase to optimize the substrate binding site of the enzyme and developed two variants, IsPETaseS242T and IsPETaseN246D, with higher enzymatic activity at both 25 and 37 °C compared with IsPETaseWT. We also developed the IsPETaseS121E/D186H/S242T/N246D variant by integrating the S242 T and N246D mutations into the previously reported IsPETaseS121E/D186H/R208A variant. At the 37 °C incubation, the quadruple variant maintained the PET degradation activity for 20 days, unlike IsPETaseWT that lost its activity within a day. Consequently, this study exhibited 58-fold increase in the activity compared with IsPETaseWT.

Microbial Polyhydroxyalkanoates and Nonnatural Polyesters.

Microorganisms produce diverse polymers for various purposes such as storing genetic information, energy, and reducing power, and serving as structural materials and scaffolds. Among these polymers, polyhydroxyalkanoates (PHAs) are microbial polyesters synthesized and accumulated intracellularly as a storage material of carbon, energy, and reducing power under unfavorable growth conditions in the presence of excess carbon source. PHAs have attracted considerable attention for their wide range of applications in industrial and medical fields. Since the first discovery of PHA accumulating bacteria about 100 years ago, remarkable advances have been made in the understanding of PHA biosynthesis and metabolic engineering of microorganisms toward developing efficient PHA producers. Recently, nonnatural polyesters have also been synthesized by metabolically engineered microorganisms, which opened a new avenue toward sustainable production of more diverse plastics. Herein, the current state of PHAs and nonnatural polyesters is reviewed, covering mechanisms of microbial polyester biosynthesis, metabolic pathways, and enzymes involved in biosynthesis of short-chain-length PHAs, medium-chain-length PHAs, and nonnatural polyesters, especially 2-hydroxyacid-containing polyesters, metabolic engineering strategies to produce novel polymers and enhance production capabilities and fermentation, and downstream processing strategies for cost-effective production of these microbial polyesters. In addition, the applications of PHAs and prospects are discussed.

Structural insight into molecular mechanism of poly(ethylene terephthalate) degradation.

Plastics, including poly(ethylene terephthalate) (PET), possess many desirable characteristics and thus are widely used in daily life. However, non-biodegradability, once thought to be an advantage offered by plastics, is causing major environmental problem. Recently, a PET-degrading bacterium, Ideonella sakaiensis, was identified and suggested for possible use in degradation and/or recycling of PET. However, the molecular mechanism of PET degradation is not known. Here we report the crystal structure of I. sakaiensis PETase (IsPETase) at 1.5 Å resolution. IsPETase has a Ser-His-Asp catalytic triad at its active site and contains an optimal substrate binding site to accommodate four monohydroxyethyl terephthalate (MHET) moieties of PET. Based on structural and site-directed mutagenesis experiments, the detailed process of PET degradation into MHET, terephthalic acid, and ethylene glycol is suggested. Moreover, other PETase candidates potentially having high PET-degrading activities are suggested based on phylogenetic tree analysis of 69 PETase-like proteins.

Dental treatment of a patient with long QT syndrome under moderate sedation with target-controlled infusion of propofol.

Long QT syndrome (LQTs) is a rare congenital disorder of the heart's electrical activity. Patients with LQTs are at increased risk of developing fatal ventricular arrhythmias. Elevated levels of sympathetic stimulation can exacerbate this risk. Successful behavior management is indispensable in the treatment of patients with LQTs. However, many drugs involved in pharmacologic behavior management are known to adversely affect the QT interval. Therefore, careful selection of a sedative drug is essential in avoiding such incidences. A 10-year-old boy with a known diagnosis of LQTs required restorative treatment due to dental caries at the permanent molar. He required sedation since treatment was painful and dental phobia can trigger sympathetic stimulation, creating a dangerous situation for patients with LQTs. Therefore, the treatment was performed over two sessions under moderate sedation involving propofol combined with nitrous oxide. Restorative treatment was successful without any complications under sedation with a target-controlled infusion (TCI) of propofol. There was no significant QT prolongation during pulpal treatment. Propofol TCI may be a good candidate for sedation in patients with LQTs.

Crystal structure of the periplasmic component of a tripartite macrolide-specific efflux pump.

In Gram-negative bacteria, type I protein secretion systems and tripartite drug efflux pumps have a periplasmic membrane fusion protein (MFP) as an essential component. MFPs bridge the outer membrane factor and an inner membrane transporter, although the oligomeric state of MFPs remains unclear. The most characterized MFP AcrA connects the outer membrane factor TolC and the resistance-nodulation-division-type efflux transporter AcrB, which is a major multidrug efflux pump in Escherichia coli. MacA is the periplasmic MFP in the MacAB-TolC pump, where MacB was characterized as a macrolide-specific ATP-binding-cassette-type efflux transporter. Here, we report the crystal structure of E. coli MacA and the experimentally phased map of Actinobacillus actinomycetemcomitans MacA, which reveal a domain orientation of MacA different from that of AcrA. Notably, a hexameric assembly of MacA was found in both crystals, exhibiting a funnel-like structure with a central channel and a conical mouth. The hexameric MacA assembly was further confirmed by electron microscopy and functional studies in vitro and in vivo. The hexameric structure of MacA provides insight into the oligomeric state in the functional complex of the drug efflux pump and type I secretion system.