MpFAE3, a beta-ketoacyl-CoA synthase gene in the liverwort Marchantia polymorpha L., is preferentially involved in elongation of palmitic acid to stearic acid

Fatty acid chain elongation is a crucial step in the biosynthesis of long chain fatty acids. An essential reaction in the elongation process is condensation of malonyl-CoA with acyl-CoA, which is catalyzed by beta-ketoacyl-CoA synthase (KCS) in plants. We have isolated and characterized the MpFAE3 gene, one of the KCS gene family in the liverwort Marchantia polymorpha. Transgenic M. polymorpha plants overexpressing MpFAE3 accumulate fatty acids 18:0, 20:0, and 22:0. In these plants, the amount of 16:0 is reduced to 50% of wild type. In a heterologous assay, transgenic methylotrophic yeast expressing the MpFAE3 gene accumulates fatty acid 18:0 and generates several longer fatty acids which are not detectable in the control, accompanied by a decrease of 16:0. These observations indicate that the MpFAE3 protein is preferentially involved in the elongation of 16:0 to 18:0 and also in the subsequent steps of 18:0 to 20:0 and 20:0 to 22:0 in M. polymorpha.     

Cyclic peptide analysis of the biologically active loop region in the laminin alpha3 chain LG4 module demonstrates the importance of peptide conformation on biological activity

The laminin alpha3 chain LG4 module (alpha3LG4 module) has cell adhesion, heparin binding, migration, and neurite outgrowth activities. The LG4 module consists of a 14-stranded beta-sheet (A-N) sandwich structure. Previously, we identified the A3G756 sequence (KNSFMALYLSKGRLVFALG in the human laminin alpha3 chain 1411-1429) as a biologically active site in the alpha3LG4 module. The A3G756 sequence is located on the E and F strands based on a crystal structure-based sequence alignment. The Lys1421 and Arg1423 residues, critical amino acids for the biological activity of A3G756, are located on the E-F connecting loop region as a KGR sequence. In this study, we focused on the KGR sequence and investigated the structural requirements of the E-F connecting loop region in the alpha3LG4 module. We synthesized three linear peptides containing the KGR sequence at the middle and the N and C termini and also prepared three cyclic analogues corresponding to the linear peptides. cyclo-hEF3A (CLYLSKGRLVFAC), which is a cyclic peptide containing the KGR sequence at the middle, showed the strongest inhibitory effect on both the heparin binding and the cell attachment to the recombinant alpha3LG4 module protein. The cyclo-hEF3A peptide was more active for syndecan-4 binding and neurite outgrowth than the linear form. Furthermore, we found that the structure of cyclo-hEF3A is similar to that of the connecting E-F loop region in human laminin alpha3LG4 module by structural analysis using molecular dynamics simulations. These results suggest that the loop structure of the E-F connecting region of the alpha3LG4 module is important for its biological activities. The cyclo-hEF3A peptide may be useful for the development of therapeutic reagents especially for wound healing and nerve regeneration.     

Expression, purification and characterization of isoforms of peripheral stalk subunits of human V-ATPase

The vacuolar-type H+-ATPase (V-ATPase) is a multi-subunit proton pump that is involved in both intra- and extracellular acidification processes throughout human body. Subunits constituting the peripheral stalk of the V-ATPase are known to have several isoforms responsible for tissue/cell specific different physiological roles. To study the different interaction of these isoforms, we expressed and purified the isoforms of human V-ATPase peripheral stalk subunits using Escherichia coli cell-free protein synthesis system: E1, E2, G1, G2, G3, C1, C2, H and N-terminal soluble part of a1 and a2 isoforms. The purification conditions were different depending on the isoforms, maybe reflecting the isoform specific biochemical characteristics. The purified proteins are expected to facilitate further experiments to study about the cell specific interaction and regulation and thus provide insight into physiological meaning of the existence of several isoforms of each subunit in V-ATPase.     

Indigestible dextrin stimulates glucoamylase production in submerged culture of <Emphasis Type="Italic">Aspergillus kawachii</Emphasis>

Submerged batch cultures of Aspergillus kawachii grown on indigestible dextrin were investigated for potential improvements in glucoamylase (GA) production. In flask culture, specific GA productivities per dry weight biomass using dextrin and indigestible dextrin were 11.0 and 56.1 mU/mg-DW, respectively. Indigestible dextrin was a poor substrate for enzymatic hydrolysis. Rates of glucose formation from dextrin and indigestible dextrin by enzymatic hydrolysis were 0.477 and 0.100 mg-glucose/ml/h, respectively. For this reason, residual glucose concentrations in batch cultures grown on indigestible dextrin remained below 1.32 mg/ml where glucose-limiting conditions were easily maintained. Batch culture using indigestible dextrin had the same residual glucose profile as dextrin fed-batch culture, and nearly the same GA activity was obtained after 42.5 h of growth. However, between 42.5 and 66 h, the GA production rate of the indigestible dextrin batch culture (11.5 mU/ml/h) was higher than that of the dextrin fed-batch culture (6.5 mU/ml/h). During this period, a high amount of residual maltooligosaccharide was detected in the culture supernatant grown on indigestible dextrin. The high GA productivity observed in the indigestible dextrin batch culture may have resulted from the absence of glucose and the simultaneous presence of maltooligosaccharides throughout growth.