A novel Bacillus ligniniphilus catechol 2,3-dioxygenase shows unique substrate preference and metal requirement

Identification of novel enzymes from lignin degrading microorganisms will help to develop biotechnologies for biomass valorization and aromatic hydrocarbons degradation. Bacillus ligniniphilus L1 grows with alkaline lignin as the single carbon source and is a great candidate for ligninolytic enzyme identification. The first dioxygenase from strain L1 was heterologously expressed, purified, and characterized with an optimal temperature and pH of 32.5 °C and 7.4, respectively. It showed the highest activity with 3-ethylcatechol and significant activities with other substrates in the decreasing order of 3-ethylcatechol > 3-methylcatechol > 3-isopropyl catechol > 2, 3-dihydroxybiphenyl > 4-methylcatechol > catechol. It did not show activities against other tested substrates with similar structures. Most reported catechol 2,3-dioxygenases (C23Os) are Fe2+-dependent whereas Bacillus ligniniphilus catechol 2,3-dioxygenase (BLC23O) is more Mn2+- dependent. At 1 mM, Mn2+ led to 230-fold activity increase and Fe2+ led to 22-fold increase. Sequence comparison and phylogenetic analyses suggested that BL23O is different from other Mn-dependent enzymes and uniquely grouped with an uncharacterized vicinal oxygen chelate (VOC) family protein from Paenibacillus apiaries. Gel filtration analysis showed that BLC23O is a monomer under native condition. This is the first report of a C23O from Bacillus ligniniphilus L1 with unique substrate preference, metal-dependency, and monomeric structure.

Structural basis for substrate specificity of heteromeric transporters of neutral amino acids

Despite having similar structures, each member of the heteromeric amino acid transporter (HAT) family shows exquisite preference for the exchange of certain amino acids. Substrate specificity determines the physiological function of each HAT and their role in human diseases. However, HAT transport preference for some amino acids over others is not yet fully understood. Using cryo–electron microscopy of apo human LAT2/CD98hc and a multidisciplinary approach, we elucidate key molecular determinants governing neutral amino acid specificity in HATs. A few residues in the substrate-binding pocket determine substrate preference. Here, we describe mutations that interconvert the substrate profiles of LAT2/CD98hc, LAT1/CD98hc, and Asc1/CD98hc. In addition, a region far from the substrate-binding pocket critically influences the conformation of the substrate-binding site and substrate preference. This region accumulates mutations that alter substrate specificity and cause hearing loss and cataracts. Here, we uncover molecular mechanisms governing substrate specificity within the HAT family of neutral amino acid transporters and provide the structural bases for mutations in LAT2/CD98hc that alter substrate specificity and that are associated with several pathologies.

Two new lecanoroid lichen species from the forested wetlands of South Korea, with a key for Korean Protoparmeliopsis species

Lecanora parasymmicta Lee & Hur and Protoparmeliopsis crystalliniformis Lee & Hur are described as new lichen species to science from the forested wetlands in southern South Korea. Molecular analyses employing internal transcribed spacer (ITS) and mitochondrial small subunit (mtSSU) sequences strongly support the two lecanoroid species to be distinct in their genera. Lecanora parasymmicta is included in the Lecanora symmicta group. It is morphologically distinguished from Lecanora symmicta (Ach.) Ach., its most similar species, by areolate-rimose thallus, blackish hypothallus, larger apothecia, absence of thalline excipulum from the beginning, narrower paraphyses, larger ascospores, smaller pycnoconidia, and the presence of placodiolic acid. The second new species Protoparmeliopsis crystalliniformis is included in a clade with Protoparmeliopsis bipruinosa (Fink) S.Y. Kondr. and P. nashii (B.D. Ryan) S.Y. Kondr., differs from Protoparmeliopsis ertzii Bungartz & Elix, its most morphologically similar species, by whitish thallus, flat to concave and paler disc, longer ascospores, thallus K+ yellow reaction, presence of atranorin and rhizocarpic acid, and the substrate preference to sandstone or basalt. A key is provided to assist in the identification of Protoparmeliopsis species in Korea.

High-Throughput Mutagenesis and Cross-Complementation Experiments Reveal Substrate Preference and Critical Residues of the Capsule Transporters in Streptococcus pneumoniae

All licensed pneumococcal vaccines target the capsular polysaccharide (CPS). This layer is highly variable and is important for virulence in many bacterial pathogens.