Characterization of 3-isopropylmalate dehydrogenase from extremely halophilic archaeon Haloarcula japonica

3-Isopropylmalate dehydrogenase (IPMDH) catalyzes oxidative decarboxylation of (2R, 3S)-3-isopropylmalate to 2-oxoisocaproate in leucine biosynthesis. In this study, recombinant IPMDH (HjIPMDH) from an extremely halophilic archaeon, Haloarcula japonica TR-1, was characterized. Activity of HjIPMDH increased as KCl concentration increased, and the maximum activity was observed at 3.0 M KCl. Analytical ultracentrifugation revealed that HjIPMDH formed a homotetramer at high KCl concentrations, and it dissociated to a monomer at low KCl concentrations. Additionally, HjIPMDH was thermally stabilized by higher KCl concentrations. This is the first report on haloarchaeal IPMDH.

Protein complex formation in methionine chain-elongation and leucine biosynthesis

During the past two decades, glucosinolate (GLS) metabolic pathways have been under extensive studies because of the importance of the specialized metabolites in plant defense against herbivores and pathogens. The studies have led to a nearly complete characterization of biosynthetic genes in the reference plant Arabidopsis thaliana. Before methionine incorporation into the core structure of aliphatic GLS, it undergoes chain-elongation through an iterative three-step process recruited from leucine biosynthesis. Although enzymes catalyzing each step of the reaction have been characterized, the regulatory mode is largely unknown. In this study, using three independent approaches, yeast two-hybrid (Y2H), coimmunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC), we uncovered the presence of protein complexes consisting of isopropylmalate isomerase (IPMI) and isopropylmalate dehydrogenase (IPMDH). In addition, simultaneous decreases in both IPMI and IPMDH activities in a leuc:ipmdh1 double mutants resulted in aggregated changes of GLS profiles compared to either leuc or ipmdh1 single mutants. Although the biological importance of the formation of IPMI and IPMDH protein complexes has not been documented in any organisms, these complexes may represent a new regulatory mechanism of substrate channeling in GLS and/or leucine biosynthesis. Since genes encoding the two enzymes are widely distributed in eukaryotic and prokaryotic genomes, such complexes may have universal significance in the regulation of leucine biosynthesis.

Prevalence of Enterotoxigenic Bacteroides Fragilis in Stool Specimens Collected from Children Less Than 5 Years Of Age in Iraq

Although Bacteroides fragilis is a bacterium present within gut microbiota, the toxin producer strain, known as enterotoxigenic B. fragilis (ETBF), is associated with diarrhea in children less than 5 years of age. This study includes 69 diarrheal and 29 non-diarrheal (control) samples collected from children less than 5 years old. DNA was extracted directly from stool specimens and directed to conventional PCR targeting beta-isopropylmalate dehydrogenase (leuB) gene, used for detection of B. fragilis, and Bacteroides fragilis toxin (bft) gene, used for the detection of ETBF. The results showed that the prevalence of leuB gene was 78 (79.6%) including 56 (81.2%) in diarrheal and 22 (75.9%) in non-diarrheal subjects, while that of bft gene was only 3 (3.1%) including 2 (2.9%) in diarrheal and 1 (3.4%) in non-diarrheal subjects. Based on sequencing of bft-positive specimens, both bft-1 and bft-2 isoforms were represented in diarrheal specimens, whereas only bft-1 was found in the control specimens. In conclusion, this study examined for the first time the leuB and bft gene in a specimen of Iraqi children with diarrhea and showed no significant differences between diarrheal and control groups in both genes.