(E)-2-Bromo-4-[(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)imino­meth­yl]-6-meth­oxy­phenyl 4-methyl­benzene­sulfonate

In the title compound, C26H24BrN3O5S, the central benzene ring makes dihedral angles of 6.27 (6), 33.63 (6) and 69.31 (5)°, respectively, with the pyrazolone ring, the bromo­benzene ring and the terminal phenyl ring. An intra­molecular C—H⋯O hydrogen bond occurs. The crystal packing features weak non-classical C—Br⋯O inter­actions [Br⋯O = 3.222 (2) Å] that form inversion-related dimers

New sesquiterpene and other constituents from Senecio flammeus

607-60

Structural and molecular basis for the substrate positioning mechanism of a new PL7 subfamily alginate lyase from the Arctic

Alginate lyases play important roles in alginate degradation in the ocean. Although a large number of alginate lyases have been characterized, little is yet known about those in extremely cold polar environments, which may have unique mechanisms for environmental adaptation and for alginate degradation. Here, we report the characterization of a novel PL7 alginate lyase AlyC3 from Psychromonas sp. C-3 isolated from the Arctic brown alga Laminaria, including its phylogenetic classification, catalytic properties and structure. We propose the establishment of a new PM-specific subfamily of PL7 (subfamily 6) represented by AlyC3 based on phylogenetic analysis and enzymatic properties. Structural and biochemical analyses showed that AlyC3 is a dimer, representing the first dimeric endo-alginate lyase structure. AlyC3 is activated by NaCl and adopts a novel salt-activated mechanism, that is, salinity adjusts the enzymatic activity by affecting its aggregation states. We further solved the structure of an inactive mutant H127A/Y244A in complex with a dimannuronate molecule, and proposed the catalytic process of AlyC3 based on structural and biochemical analyses. We show that Arg82 and Tyr190 at the two ends of the catalytic canyon help the positioning of the repeated units of the substrate, and that His127, Tyr244, Arg78, and Gln125 mediate the catalytic reaction. Our study uncovers, for the first time, the amino acid residues for alginate positioning in an alginate lyase, and demonstrate that such residues involved in alginate positioning are conserved in other alginate lyases. This study provides a better understanding of the mechanisms of alginate degradation by alginate lyases

Characterization of a cryptic plasmid pSM429 and its application for heterologous expression in psychrophilic Pseudoalteromonas

<p>Abstract</p> <p>Background</p> <p><it>Pseudoalteromonas </it>is an important genus widespread in marine environment, and a lot of psychrophilic <it>Pseudoalteromonas </it>strains thrive in deep sea and polar sea. By now, there are only a few genetic systems for <it>Pseudoalteromonas </it>reported and no commercial <it>Pseudoalteromonas </it>genetic system is available, which impedes the study of <it>Pseudoalteromonas</it>, especially for psychrophilic strains. The aim of this study is to develop a heterologous expression system for psychrophilic <it>Pseudoalteromonas</it>.</p> <p>Results</p> <p>A cryptic plasmid pSM429 isolated from psychrophilic <it>Pseudoalteromonas </it>sp. BSi20429 from the Arctic sea ice, was sequenced and characterized. The plasmid pSM429 is 3874 bp in length, with a G+C content of 28%. Four putative open reading frames (ORFs) were identified on pSM429. Based on homology, the ORF4 was predicted to encode a replication initiation (Rep) protein. A shuttle vector (<it>Escherichia coli, Pseudoalteromonas</it>), pWD, was constructed by ligating pSM429 and pUC19 and inserting a chloramphenicol acetyl transferase (CAT) cassette conferring chloramphenicol resistance. To determine the minimal replicon of pSM429 and to check the functionality of identified ORFs, various pWD derivatives were constructed. All derivatives except the two smallest ones were shown to allow replication in <it>Pseudoalteromonas </it>sp. SM20429, a plasmid-cured strain of <it>Pseudoalteromonas </it>sp. BSi20429, suggesting that the <it>orf4 </it>and its flanking intergenic regions are essential for plasmid replication. Although not essential, the sequence including some repeats between <it>orf1 </it>and <it>orf2 </it>plays important roles in segregational stability of the plasmid. With the aid of pWD-derived plasmid pWD2, the erythromycin resistance gene and the <it>cd </it>gene encoding the catalytic domain of a cold-adapted cellulase were successfully expressed in <it>Pseudoalteromonas </it>sp. SM20429.</p> <p>Conclusions</p> <p>Plasmid pSM429 was isolated and characterized, and the regions essential for plasmid replication and stability were determined, helping the development of pSM429-based shuttle vectors. The shuttle vectors pWD and its derivatives could be used as cloning vectors for <it>Pseudoalteromonas</it>, offering new perspectives in the genetic manipulation of <it>Pseudoalteromonas </it>strains. With the aid of pWD-derived vector and its host, the erythromycin resistance gene and the <it>cd </it>gene of a cold-adapted protein were successfully expressed, indicating that the potential use of this system for recombinant protein production, especially for cold-adapted proteins.</p

Mechanistic Insights into Dimethylsulfoniopropionate Lyase DddY, a New Member of the Cupin Superfamily

The marine osmolyte dimethylsulfoniopropionate (DMSP) is one of Earth's most abundant organosulfur molecules. Bacterial DMSP lyases cleave DMSP, producing acrylate and dimethyl sulfide (DMS), a climate-active gas with roles in global sulfur cycling and atmospheric chemistry. DddY is the only known periplasmic DMSP lyase and is present in β-, γ-, δ- and ε-proteobacteria. Unlike other known DMSP lyases, DddY has not been classified into a protein superfamily, and its structure and catalytic mechanism are unknown. Here, we determined the crystal structure of DddY from the γ-proteobacterium Acinetobacter bereziniae originally isolated from human clinical specimens. This structure revealed that DddY contains a cap domain and a catalytic domain with a Zn2 + bound at its active site. We also observed that the DddY catalytic domain adopts a typical β-barrel fold and contains two conserved cupin motifs. Therefore, we concluded that DddY should belong to the cupin superfamily. Using structural and mutational analyses, we identified key residues involved in Zn2 + coordination, DMSP binding and the catalysis of DMSP cleavage, enabling elucidation of the catalytic mechanism, in which the residue Tyr271 of DddY acts as a general base to attack DMSP. Moreover, sequence analysis suggested that this proposed mechanism is common to DddY proteins from β-, γ-, δ- and ε-proteobacteria. The DddY structure and proposed catalytic mechanism provide a better understanding of how DMSP is catabolized to generate the important climate-active gas DMS

A pathway for chitin oxidation in marine bacteria

Oxidative degradation of chitin, initiated by lytic polysaccharide monooxygenases (LPMOs), contributes to microbial bioconversion of crystalline chitin, the second most abundant biopolymer in nature. However, our knowledge of oxidative chitin utilization pathways, beyond LPMOs, is very limited. Here, we describe a complete pathway for oxidative chitin degradation and its regulation in a marine bacterium, Pseudoalteromonas prydzensis. The pathway starts with LPMO-mediated extracellular breakdown of chitin into C1-oxidized chitooligosaccharides, which carry a terminal 2-(acetylamino)−2-deoxy-D-gluconic acid (GlcNAc1A). Transmembrane transport of oxidized chitooligosaccharides is followed by their hydrolysis in the periplasm, releasing GlcNAc1A, which is catabolized in the cytoplasm. This pathway differs from the known hydrolytic chitin utilization pathway in enzymes, transporters and regulators. In particular, GlcNAc1A is converted to 2-keto-3-deoxygluconate 6-phosphate, acetate and NH3 via a series of reactions resembling the degradation of D-amino acids rather than other monosaccharides. Furthermore, genomic and metagenomic analyses suggest that the chitin oxidative utilization pathway may be prevalent in marine Gammaproteobacteria

The RET C611Y mutation causes MEN 2A and associated cutaneous lichen amyloidosis

Background: Cutaneous lichen amyloidosis (CLA) has been reported in some multiple endocrine neoplasia type 2A (MEN 2A) families affected by specific germline RET mutations C634F/G/R/W/Y or V804M, as a characteristic of the clinical manifestation in ‘MEN 2A with CLA’, one of four variants of MEN 2A, which was strictly located in the scapular region of the upper back. Patient Findings: This study reports a large south-eastern Chinese pedigree with 17 individuals carrying the MEN 2A-harboring germline C611Y (c.1832G>A) RET mutation by Sanger sequencing. One individual presented MEN 2A-related clinical features, including typical CLA in the interscapular region; another individual exhibited neurological pruritus and scratching in the upper back but lacked CLA skin lesions. Both subjects presented with CLA or pruritic symptoms several years before the onset of medullary thyroid carcinoma (MTC) and/or pheochromocytoma. The remaining 15 RET mutation carriers did not exhibit CLA; of these, one presented with MTC and pheochromocytoma, nine with MTC only, two with elevated serum calcitonin and three younger subjects with normal serum calcitonin levels. This family’s clinical data revealed a later diagnosis of MTC (mean age, 45.9 (range: 23–73) years), a lower penetrance of pheochromocytoma (2/17, 11.8%) and CLA (1/17, 5.9%). However, no hyperparathyroidism and Hirschsprung disease were reported in this family. Summary and Conclusions: This is the first description of a family with MEN 2A-related CLA due to a germline RET C611Y mutation, which might exhibit a novel and diversified genotype–phenotype spectrum in MEN 2A

Nucleotide variation in ATHK1 region of Arabidopsis thaliana and its association study with drought tolerance

The ATHK1 gene in Arabidopsis encodes a putative histidine kinase that is transcriptionally upregulated in response to changes in external osmolarity. In this work, we investigated the nucleotide variability of the ATHK1 gene in a sample of 32 core Arabidopsis accessions originating from different ecoclimatic regions and their drought tolerance. The results showed that different accessions had quite difference in adaptation to drought stress. Thirty-two Arabidopsis accessions were clustered into four groups according to their drought tolerance capacity. Relative water content of the leaves (RWC) combined anyone of membrane permeability of leaves (MP) and water retain capacity of detached leaves (WRC) were selected as two representative physiological indexes for evaluation of comprehensive drought tolerance. Sequencing 5 515 bp encompassing ATHK1 coding region in 32 core accessions revealed 39 polymorphisms, which formed 24 haplotypes. The polymorphism (including single nucleotide polymorphism (SNP) and insertion/deletion (Indel)) frequency was 1 SNP per 131.2 bp. In coding region of ATHK1, the ratio of average number of nucleotide difference π n /π s ratio was 0.727, suggesting that the ATHK1 protein is not constrained against amino acid changes within the species and this gene belonged to the middle evolution rate gene. Using ANOVA analysis, it showed that the 1199 site amino acid (Ser stop) variation of the eleventh haplotype (257 and 266av) was associated with not only RWC but also WRC, indicating that the change of Ser stop is associated with comprehensive drought tolerance of 257 and 266av. This amino acid change may cause 257 and 266 av accessions originating from moist ecoclimatic region to be sensitive to dry climate, and likely be the evidence of adaptive evolution