Actinocatenispora comari sp. nov., an endophytic actinomycete isolated from aerial parts of Comarum salesowianum.

A novel actinomycete, designated NUM-2625T, was isolated as an endophytic bacterium in aerial parts of Comarum salesowianum, an endemic species in the Altai, Himalaya mountain chain area, collected from Khasagt Khairkhan Mountain in Mongolia. The 16S rRNA gene sequence of strain NUM-2625T showed the highest similarity to Actinocatenispora thailandica TT2-10T (99.4 %), Actinocatenispora sera KV-744T (99.3 %), and Actinocatenispora rupis CS5-AC17T (97.7 %). Chemotaxonomic properties of strain NUM-2625T were essentially consistent with those of the genus Actinocatenispora, such as the presence of meso-diaminopimelic acid as the diagnostic diamino acid of the peptidoglycan, MK-9(H4) and MK-9(H6) as the major menaquinones, and iso-C16 : 0, iso-C15 : 0, iso-C14 : 0 3-OH, and anteiso-C17 : 0 as the major fatty acids. Meanwhile, digital DNA-DNA hybridization and average nucleotide identity values revealed a low relatedness between strain NUM-2625T and the other type strains of the genus Actinocatenispora. In addition, strain NUM-2625T exhibited several phenotypic properties that could be used to distinguish it from its closest relatives. Based on the results of polyphasic analyses, strain NUM-2625T represents a novel species in the genus Actinocatenispora, for which the name Actinocatenispora comari sp. nov. is proposed. The type strain is NUM-2625T (=NBRC 114660T=TBRC 13496T).

Structural basis of substrate specificity and regiochemistry in the MycF/TylF family of sugar O-methyltransferases.

Sugar moieties in natural products are frequently modified by O-methylation. In the biosynthesis of the macrolide antibiotic mycinamicin, methylation of a 6'-deoxyallose substituent occurs in a stepwise manner first at the 2'- and then the 3'-hydroxyl groups to produce the mycinose moiety in the final product. The timing and placement of the O-methylations impact final stage C-H functionalization reactions mediated by the P450 monooxygenase MycG. The structural basis of pathway ordering and substrate specificity is unknown. A series of crystal structures of MycF, the 3'-O-methyltransferase, including the free enzyme and complexes with S-adenosyl homocysteine (SAH), substrate, product, and unnatural substrates, show that SAM binding induces substantial ordering that creates the binding site for the natural substrate, and a bound metal ion positions the substrate for catalysis. A single amino acid substitution relaxed the 2'-methoxy specificity but retained regiospecificity. The engineered variant produced a new mycinamicin analog, demonstrating the utility of structural information to facilitate bioengineering approaches for the chemoenzymatic synthesis of complex small molecules containing modified sugars. Using the MycF substrate complex and the modeled substrate complex of a 4'-specific homologue, active site residues were identified that correlate with the 3' or 4' specificity of MycF family members and define the protein and substrate features that direct the regiochemistry of methyltransfer. This classification scheme will be useful in the annotation of new secondary metabolite pathways that utilize this family of enzymes.

A new structural form in the SAM/metal-dependent o­methyltransferase family: MycE from the mycinamicin biosynthetic pathway.

O-linked methylation of sugar substituents is a common modification in the biosynthesis of many natural products and is catalyzed by multiple families of S-adenosyl-L-methionine (SAM or AdoMet)-dependent methyltransferases (MTs). Mycinamicins, potent antibiotics from Micromonospora griseorubida, can be methylated at two positions on a 6-deoxyallose substituent. The first methylation is catalyzed by MycE, a SAM- and metal-dependent MT. Crystal structures were determined for MycE bound to the product S-adenosyl-L-homocysteine (AdoHcy) and magnesium, both with and without the natural substrate mycinamicin VI. This represents the first structure of a natural product sugar MT in complex with its natural substrate. MycE is a tetramer of a two-domain polypeptide, comprising a C-terminal catalytic MT domain and an N-terminal auxiliary domain, which is important for quaternary assembly and for substrate binding. The symmetric MycE tetramer has a novel MT organization in which each of the four active sites is formed at the junction of three monomers within the tetramer. The active-site structure supports a mechanism in which a conserved histidine acts as a general base, and the metal ion helps to position the methyl acceptor and to stabilize a hydroxylate intermediate. A conserved tyrosine is suggested to support activity through interactions with the transferred methyl group from the SAM methyl donor. The structure of the free enzyme reveals a dramatic order-disorder transition in the active site relative to the S-adenosyl-L-homocysteine complexes, suggesting a mechanism for product/substrate exchange through concerted movement of five loops and the polypeptide C-terminus.

Characteristics of cesium accumulation in the filamentous soil bacterium Streptomyces sp. K202.

A filamentous soil bacterium, strain K202, was isolated from soil where an edible mushroom (Boletopsis leucomelas) was growing and identified as belonging to the genus Streptomyces on the basis of its morphological characteristics and the presence of LL-2, 6-diaminopimelic acid. We studied the existence states of Cs and its migration from extracellular to intracellular fluid in the mycelia of Streptomyces sp. K202. The results indicated that Cs accumulated in the cells through at least 2 steps: in the first step, Cs(+) was immediately and non-specifically adsorbed on the negatively charged cell surface, and in the second step, this adsorbed Cs(+) was taken up into the cytoplasm, and a part of the Cs entering the cytoplasm was taken up by an energy-dependent transport system(s). Further, we confirmed that a part of the Cs(+) was taken up into the mycelia competitively with K(+), because K(+) uptake into the intact mycelia of the strain was significantly inhibited by the presence of Cs(+) in the culture media. This suggested that part of the Cs is transported by the potassium transport system. Moreover, (133)Cs-NMR spectra and SEM-EDX spectra of the mycelia that accumulated Cs showed the presence of at least 2 intracellular Cs states: Cs(+) trapped by intercellular materials such as polyphosphate and Cs(+) present in a cytoplasmic pool.

Functional analysis of MycE and MycF, two O-methyltransferases involved in the biosynthesis of mycinamicin macrolide antibiotics.

Mg motors: We characterized the in vitro function of MycE and MycF, two O-methyltransferases involved in the biosynthesis of mycinamicin antibiotics. Each enzyme was confirmed to be an S-adenosyl-L-methionine (SAM)-dependent deoxysugar methyltransferase. Their optimal activities require the presence of Mg(2+). With the reconstituted in vitro assays, the order of mycinamicin VI-->III-->IV in the post-PKS (polyketide synthase) tailoring pathway of mycinamicin was unambiguously determined.

Accumulation and localization of cesium in edible mushroom (Pleurotus ostreatus) mycelia.

The characteristics of Cs accumulation and localization in edible mushrooms were examined using the mycelia of Pleurotus ostreatus-Y1. Scanning electron microscope images revealed the existence of white spots, and energy dispersive X-ray microanalyzer analysis indicated the presence of larger amounts of Cs and P in these spots in mycelia cultured on medium containing 25 mM CsCl. The (137)Cs activities in the mycelia were approximately 4-6 times higher than those in water used for (137)Cs elution. Higher Cs concentrations in the sediment fraction including vacuolar pellets were obtained compared to the upper fractions. It was observed that yellowish spots caused by the fluorescence of 4',6-diamidino-2-phenylindole (DAPI)-stained polyphosphate were localized in the mycelia. The higher fluorescence intensity of the yellowish-grained spots was measured in comparison with other regions in the mycelium. These results suggested that Cs in the mycelia was trapped by polyphosphate in vacuoles or other organelles.