Hiroshi Matsubara

Cloning, sequencing, characterization, and expression of a β-glucosidase cDNA from the indigo plant ☆

Leaves of the indigo plant (Polygonum tinctorium) contain a β-glucosidase with a high activity for the substrate indican. The β-glucosidase cDNA was cloned from a leaf cDNA library. The cDNA consists of 1813 nucleotides including 1533 nucleotides of an open reading frame (ORF). The ORF in the cDNA encoded a total of 511 amino acids containing 483 amino acids of a mature protein and 28 of a transit peptide. Interestingly, an internal homologous region spanning the transit peptide and N-terminal sequence was found. The deduced amino acid sequence of the mature enzyme showed a high homology with that of β-glucosidases from several other plants and bacteria, and a well conserved region of several amino acids that forms the pocket of the active site. The cDNA was expressed in E. coli and the cell extract was confirmed to have β-glucosidase activity.

Isolation and Characterization of Glutathione Reductase from Physarum polycephalum and Stage-Specific Expression of the Enzyme in Life-Cycle Stages with Different Oxidation-Reduction Levels

Abstract Physarum polycephalum has a life cycle with several distinct phases that have different oxidation-reduction requirements. To investigate the relationship between the life cycle and the oxidation-reduction state, we isolated glutathione reductase (GR; EC 1.6.4.2) from Physarum microplasmodia. The enzyme was found to be a homodimer with a subunit Mr of 49,000, and Km values for oxidized glutathione and NADPH of 40 and 28.6 μM, respectively. We then constructed a cDNA library from microplasmodium mRNA and cloned GR cDNA from the library. The isolated cDNA consisted of 1,475 bp encoding a polypeptide of 452 amino acids. The amino acid sequence similarity was about 50% with GRs of other organisms, and several conserved sequence motifs thought to be necessary for activity are evident in the Physarum enzyme. Escherichia coli transformed with an expression vector containing the cDNA synthesized the active GR. Genomic Southern blot analysis indicated that the GR gene is present as a single copy in the Physarum genome. Immunoblot analysis and RT-PCR analysis detected GR mRNA expression in the microplasmodium, plasmodium, and sclerotium, but not in the spore or flagellate. GR activity was low in the spore and flagellate. These results suggest that the glutathione oxidation-reduction system relates to the Physarum life cycle.

Three iron-sulfur proteins encoded by three ORFs in chloroplasts and cyanobacteria.

A brief review is presented on the gene products of frxA, frxB and frxC found in chloroplasts. The product of frxA shows high sequence homologies to bacterial 2[4Fe-4S] ferredoxins, but it functions as iron-sulfur centers A and B in Photosystem I, transferring electrons to [2Fe-2S] ferredoxin. This protein is located on surface of the thylakoid membranes in a state being covered by two other proteins. Proteins homologous to frxB product are found in mitochondrial respiratory Complex I and the product of frxB may function in chlororespiration, but at present no clear function of this protein is known. The frxC gene product is found to function in light-independent chlorophyll synthesis as one of the subunits of protochlorophyllide reductase and is reviewed in comparison to nitrogenase. Several problems and future research direction in these areas are also presented.