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 analysis of the peptidoglycan editing factor PdeF from Bacillus cereus ATCC 14579.

The coding gene for peptidoglycan editing factor (pdeF) is located in the division and cell wall (dcw) cluster, and encodes a protein that has an editing function for misplaced amino acids in peptidoglycan in E. coli. In this study, we determined the crystal structure of PdeF from Bacillus cereus (BcPdeF) at a 1.60 Å resolution. BcPdeF exists as a monomer in solution and consists of two domains: a core domain containing a Pfam motif DUF152 and a smaller subdomain. The X-ray fluorescence spectrum of BcPdeF crystal elucidated that the protein has a Zn2+ ion in its active site and the metal ion was coordinated by two histidine and one cysteine residue. We also performed docking calculations of the N-acetylmuramate (MurNAc)-L-Ser-D-iGlu ligand in the BcPdeF structure and revealed the substrate binding mode of the enzyme. Furthermore, structural comparisons between BcPdeF and human fatty acid metabolism-immunity nexus (FAMIN), which also contains the DUF152 motif in its core domain, provided a structural basis how the two structurally similar proteins have completely different physiological functions.

Structural basis for stereospecificity to d-amino acid of glycine oxidase from Bacillus cereus ATCC 14579.

Glycine oxidase (GO) is an enzyme that catalyzes the oxidation of the primary and secondary amines of various chemicals, including glycine, and the enzyme has been applied in a variety of fields, such as biosensor and genetically modified glyphosate resistance plants. Here, we report that the gene product of BC0747 from Bacillus cereus (BcGO) shows oxidase activity for glycine and small d-amino acids, such as d-proline and d-alanine. We also determined the crystal structure of BcGO complexed with the FAD cofactor at a 2.36 Å resolution and revealed how the cofactor binds to the deep pocket of the enzyme. We performed the molecular docking calculation of the glycine substrate to the BcGO structure and identified how the carboxyl- and amine-groups of the d-amino acid are stabilized at the substrate binding site. Structural analysis of BcGO also provided information on the structural basis for the stereospecificity of the enzyme to d-amino acids. In addition, we placed the glyphosate molecule, a plant herbicide, at the substrate binding site, and explained how the mutation of Gly51 to arginine enhances enzyme activity.

Cohnella saccharovorans sp. nov., isolated from ginseng soil.

A novel bacterial strain, CJ22T, was isolated from soil of a ginseng field located in Anseong, Korea. Cells of strain CJ22T were aerobic, Gram-stain-positive, endospore-forming, motile, oxidase- and catalase-positive and rod-shaped. The isolate grew optimally at pH 7 and 30 °C. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain CJ22T belonged to the genus Cohnella, displaying highest sequence similarity of 97.3% with Cohnella panacarvi Gsoil 349T. DNA-DNA relatedness between strain CJ22T and its closest relative was 35.5 % (reciprocal value, 23.8%). The phenotypic features of strain CJ22T also distinguished it from related species of the genus Cohnella. The diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. The major isoprenoid quinone was menaquinone MK-7 and the major polar lipids were phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, lysyl-phosphatidylglycerol, two unidentified phospholipids and two unidentified aminophospholipids. The predominant cellular fatty acids of strain CJ22T were anteiso-C15 : 0, iso-C16:0 and C16:0. The DNA G+C content was 63.1 mol%. Based on data from this polyphasic taxonomic study, strain CJ22T is considered to represent a novel species of the genus Cohnella, for which the name Cohnella saccharovorans sp. nov. is proposed. The type strain is CJ22T (=KACC 17501T=JCM 19227T).

Lysobacter panacisoli sp. nov., isolated from ginseng soil.

A novel bacterial strain, designated CJ29(T), was isolated from ginseng soil of Anseong in South Korea. Cells of strain CJ29(T) were Gram-stain-negative, facultatively anaerobic, rod-shaped and non-motile. Strain CJ29(T) grew optimally at 28-30 °C and pH 7.0. Based on 16S rRNA gene sequence analysis, strain CJ29(T) was shown to belong to the genus Lysobacter within the class Gammaproteobacteria and was related most closely to Lysobacter soli DCY21(T) (98.5% similarity) and Lysobacter niastensis GH41-7(T) (98.2%). DNA-DNA relatedness between strain CJ29(T) and its closest relatives was below 55.6%. The predominant cellular fatty acids of strain CJ29(T) were iso-C15 : 0, iso-C16 : 0 and iso-C17 : 1ω9c. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The major isoprenoid quinone was ubiquinone 8 (Q-8). The G+C content of the genomic DNA was 65.6 mol%. Phenotypic, genotypic and phylogenetic characteristics strongly supported the differentiation of strain CJ29(T) from related species of the genus Lysobacter. On the basis of data from this polyphasic taxonomic study, strain CJ29(T) is considered to represent a novel species of the genus Lysobacter, for which the name Lysobacter panacisoli sp. nov. is proposed. The type strain is CJ29(T) ( = KACC 17502(T) = JCM 19212(T)).

Production of various phenolic aldehyde compounds using the 4CL-FCHL biosynthesis platform.

Vanillin (3-methoxy-4-hydroxybenzaldehyde) is one of the most important flavoring substances used in the cosmetic and food industries. Feruloyl-CoA hydratase/lyase (FCHL) is an enzyme that catalyzes the production of vanillin from feruloyl-CoA. In this study, we report kinetic parameters and biochemical properties of FCHL from Sphingomonas paucimobilis SYK-6 (SpFCHL). Also, the crystal structures of an apo-form of SpFCHL and two complexed forms with acetyl-CoA and vanillin/CoA was present. Comparing the apo structure to its complexed forms of SpFCHL, a gate loop with an "open and closed" role was observed at the entrance of the substrate-binding site. With vanillin and CoA complexed to SpFCHL, we captured a conformational change in the feruloyl moiety-binding pocket that repositions the catalytic SpFCHLE146 and other key residues. This binding pocket does not tightly fit the vanillin structure, suggesting substrate promiscuity of this enzyme. This observation is in good agreement with assay results for phenylpropanoid-CoAs and indicates important physicochemical properties of the substrate for the hydratase/lyase reaction mechanism. In addition, we showed that various phenolic aldehydes could be produced using the 4CL-FCHL biosynthesis platform.

Crystal Structures of 6-Phosphogluconate Dehydrogenase from Corynebacterium glutamicum.

Corynebacterium glutamicum (C. glutamicum) has been considered a very important and meaningful industrial microorganism for the production of amino acids worldwide. To produce amino acids, cells require nicotinamide adenine dinucleotide phosphate (NADPH), which is a biological reducing agent. The pentose phosphate pathway (PPP) can supply NADPH in cells via the 6-phosphogluconate dehydrogenase (6PGD) enzyme, which is an oxidoreductase that converts 6-phosphogluconate (6PG) to ribulose 5-phosphate (Ru5P), to produce NADPH. In this study, we identified the crystal structure of 6PGD_apo and 6PGD_NADP from C. glutamicum ATCC 13032 (Cg6PGD) and reported our biological research based on this structure. We identified the substrate binding site and co-factor binding site of Cg6PGD, which are crucial for understanding this enzyme. Based on the findings of our research, Cg6PGD is expected to be used as a NADPH resource in the food industry and as a drug target in the pharmaceutical industry.

Structural studies of a novel auxiliary-domain-containing phenylalanine hydroxylase from Bacillus cereus ATCC 14579.

Phenylalanine hydroxylase (PAH), which belongs to the aromatic amino-acid hydroxylase family, is involved in protein synthesis and pyomelanine production through the hydroxylation of phenylalanine to tyrosine. In this study, the crystal structure of PAH from Bacillus cereus ATCC 14579 (BcPAH) with an additional 280 amino acids in the C-terminal region was determined. The structure of BcPAH consists of three distinct domains: a core domain with two additional inserted α-helices and two novel auxiliary domains: BcPAH-AD1 and BcPAH-AD2. Structural homologues of BcPAH-AD1 and BcPAH-AD2 are known to be involved in mRNA regulation and protein-protein interactions, and thus it was speculated that BcPAH might utilize the auxiliary domains for interaction with its partner proteins. Furthermore, phylogenetic tree analysis revealed that the three-domain PAHs, including BcPAH, are completely distinctive from both conventional prokaryotic PAHs and eukaryotic PAHs. Finally, biochemical studies of BcPAH showed that BcPAH-AD1 might be important for the structural integrity of the enzyme and that BcPAH-AD2 is related to enzyme stability and/or activity. Investigations into the intracellular functions of the two auxiliary domains and the relationship between these functions and the activity of PAH are required.

Chimeric Antigen Receptor T Cells With Modified Interleukin-13 Preferentially Recognize IL13Rα2 and Suppress Malignant Glioma: A Preclinical Study.

Background: Interleukin-13 receptor α 2 (IL13Rα2) is a promising tumor-directed antigen of malignant glioma (MG). Here, we examine the efficacy and safety of T cells containing a YYB-103 chimeric antigen receptor (CAR) that can preferentially bind to IL13Rα2 on MG cells. Methods: IL13 was modified on the extracellular domain by substitution of amino acids with E13K, R66D, S69D, and R109K and stably transfected into human T cells using a retroviral vector. The in vitro efficacy of YYB-103 CAR T cells was tested in cell lines with differing IL13Rα1 and IL13Rα2 expression. The in vivo efficacy of intracerebroventricular (i.c.v.) and intravenous (i.v.) routes of YYB-103 CAR T-cell administration were tested in orthotopic MG mouse models. Immunohistochemical staining of MG was performed using WHO grade 3/4 surgical specimens from 53 patients. IL13Rα2 expression was quantified by H-score calculated from staining intensity and percentage of positive cells. Results: Binding affinity assay of YYB-103 verified apparently nil binding to IL13Rα1, which was more selective than previously reported IL13 modification (E13Y). YYB-103 CAR T cells showed selective toxicity toward co-cultured U87MG (IL13Rα1+/IL13Rα2+) cells but not A431 (IL13Rα1+/IL13Rα2-) cells. Consistently, YYB-103 CAR T cells suppressed tumor growth in nude mice receiving orthotopic injection of U87 MG cells. Both i.c.v. and i.v. injections of YYB-103 CAR T cells reduced tumor volume and prolonged overall survival of tumor-bearing mice. The median H-score for IL13Rα2 in patient-derived MG tissue was 5 (mean, 57.5; SD, 87.2; range, 0 to 300). Conclusion: This preclinical study demonstrates the efficacy of IL13Rα2-targeted YYB-103 CAR T cells against MG cells. The use of modified IL13 to construct a CAR facilitated the selective targeting of IL13Rα2-expressing MG cells while sparing IL13Rα1-expressing cells. Notably, YYB-103 CAR T cells exhibited effective blood-brain barrier crossing, suggesting compatibility with i.v. administration rather than intracranial injection. Additionally, the high H-score for IL13Rα2 in glioblastoma, especially in conjunction with the poor prognostic markers of wild-type isocitrate dehydrogenase-1 (IDH-1) and unmethylated O6-methyl guanine methyl-transferase (MGMT), could be used to determine the eligibility of patients with recurrent glioblastoma for a future clinical trial of YYB-103 CAR T cells.

Enhanced succinic acid production by Mannheimia employing optimal malate dehydrogenase.

Succinic acid (SA), a dicarboxylic acid of industrial importance, can be efficiently produced by metabolically engineered Mannheimia succiniciproducens. Malate dehydrogenase (MDH) is one of the key enzymes for SA production, but has not been well characterized. Here we report biochemical and structural analyses of various MDHs and development of hyper-SA producing M. succiniciproducens by introducing the best MDH. Corynebacterium glutamicum MDH (CgMDH) shows the highest specific activity and least substrate inhibition, whereas M. succiniciproducens MDH (MsMDH) shows low specific activity at physiological pH and strong uncompetitive inhibition toward oxaloacetate (ki of 67.4 and 588.9 µM for MsMDH and CgMDH, respectively). Structural comparison of the two MDHs reveals a key residue influencing the specific activity and susceptibility to substrate inhibition. A high-inoculum fed-batch fermentation of the final strain expressing cgmdh produces 134.25 g L-1 of SA with the maximum productivity of 21.3 g L-1 h-1, demonstrating the importance of enzyme optimization in strain development.

Atropine injection followed by coronary artery spasm with ventricular tachycardia during spinal anesthesia -A case report-.

Bradycardia may occur during spinal anesthesia with atropine commonly used as a treatment. A 44-year-old female with no known history of any underlying diseases, developed a coronary spasm following ventricular tachycardia when 0.5 mg of atropine was injected intravenously to treat bradycardia during spinal anesthesia. The imbalance caused by atropine in the sympathovagal activity may predispose the coronary artery to develop spasms with ventricular tachycardia. Therefore prudent use of atropine should be accompanied by close monitoring.

Severe desaturation while attempting one-lung ventilation for congenital cystic adenomatoid malformation with respiratory distress syndrome in neonate -A case report-.

There are many methods for achieving one-lung ventilation (OLV) during thoracic surgery in neonates and the accuracy of OLV may affect postoperative outcome. The authors have performed OLV using a 5 Fr Arndt endobronchial blocker (AEB, Cook Inc., Bloomington, IN, USA) on a neonate diagnosed with congenital cystic adenomatoid malformation and respiratory distress syndrome (RDS) associated with marked mediastinal shift. In spite of sufficient preoxygenation, sudden and severe fall in oxygen saturation had occurred. Since neonates with RDS may develop sudden and severe desaturation, rapid intubation with anticipation of potential difficulty is necessary as well as sufficient preoxygenation.