Bombyx mori Pyridoxal Kinase cDNA Cloning and Enzymatic Characterization

Pyridoxal kinase （PLK） （EC 2.7.1.35） catalyzes the ATP-dependent phosphorylation of pyridoxal, generating pyridoxal-5＇-phosphate （PLP）, an important cofactor for many enzymatic reactions. Bombyx mori, similar to mammals, relies on a nutritional source of vitamin B6 to synthesize PLP. This article describes how a cDNA encoding PLK was cloned from Bombyx mori using the PCR method （GenBank accession number： DQ452397）. The cDNA has an 894 bp open reading frame and encodes a protein of 298 amino acid residues with a molecular mass of 33.1 k.Da. The amino acid sequence shares 48.6% identity with that of human PLK, and it also contains signature conserved motifs of the PLK family. However, the protein is 10 or more amino acids shorter than the PLK from mammals and plants, and several amino acid residues conserved in the PLK from mammals and plants are changed in the protein. The cDNA cloned was expressed successfully in Escherichia coli using the T7 promoter/T7 RNA polymerase expression system, and the crude extracts containing the expressed product were found to have strong PLK enzymatic activity with a value of 30 nmol/min/mg, confirming that the cDNA encodes the functional PLK of Bombyx mori. This is the first identification of a gene encoding PLK in insects.

pyrG is required for maintaining stable cellular uracil level and normal sporulation pattern under excess uracil stress in Aspergillus nidulans

Tight control of the intracellular uracil level is believed to be important to reduce the occurrence of uracil incorporation into DNA. The pyrG gene ofAspergillus niduIans encodes orotidine 5＇-phosphate decarboxylase, which catalyzes the conversion of orotidine monophosphate （OMP） to uridine monophosphate （UMP）. In this study, we found that pyrG is critical for maintaining uracil at a low concentration in A. nidulans cells in the presence of exogenous uracil. Excess uracil and its derivatives had a stronger inhibitory effect on the growth of the pyrG89 mutant with defective OMP decarboxylase activity than on the growth of wild type, and induced sexual development in the pyrG89 mutant but not in wild type. Analysis of transcriptomic responses to excess uracil by digital gene expression profiling （DGE） revealed that genes related to sexual development were transcrip- tionally activated in the pyrG89 mutant but not in wild type. Quantitative analysis by HPLC showed that the cellular uracil level was 6.5 times higher in the pyrG89 mutant than in wild type in the presence of exogenous uracil. This study not only provides new information on uracil recycling and adaptation to excess uracil but also reveals the potential effects of OMP decarboxylase on fungal growth and development.

Chiral nanoparticle-induced amplification in optical activity of molecules with chiral centers

Sensitive differentiation of an enantiomer from its mirror image(ie,enantiodifferentiation),a perennial challenge for pharmaceutical production and disease diagnosis,is technically limited by the weak optical activity(OA)of enantiomers,mainly due to their dimensional mismatch with light wavelengths in the ultraviolet(UV)-visible region.Here we use silver chiral nanoparticles(Ag CNPs)with nominally sub-5 nm helical pitch(P)to amplify the OA of(20R,30R,40S)-riboflavin-50-phosphate sodium salts(RP),which have been found to indirectly affect metabolic processes,through the formation of an RP thin film(TF)covering a close-packed array of Ag CNPs.The OA of the RP in the deep-UV region can be amplified up to 80-fold,ascribed to the aggregation of RP in the TFs and the interactions between RP and the atomically chiral lattices at the CNPs'surfaces.The former contribution,not associated with the chiral Ag topographies,plays a dominant role by thickening the RP TFs,so that the observed amplification has no enantioselective dependence on the chirality of the Ag CNPs.This study extends progress in the sensitive detection of bio-enantiomers,which is highly desired for advanced bio-detection in disease diagnosis and production of single-enantiomer pharmaceuticals.