Yoshiko Minami

Crystal structure of Escherichia coli SufA involved in biosynthesis of iron-sulfur clusters : Implications for a functional dimer

IscA and SufA are paralogous proteins that play crucial roles in the biosynthesis of Fe–S clusters, perhaps through a mechanism involving transient Fe–S cluster formation. We have determined the crystal structure of E. coli SufA at 2.7 Å resolution. SufA exists as a homodimer, in contrast to the tetrameric organization of IscA. Furthermore, a C‐terminal segment containing two essential cysteine residues (Cys‐Gly‐Cys), which is disordered in the IscA structure, is clearly visible in one molecule (the α1 subunit) of the SufA homodimer. Although this segment is disordered in the other molecule (the α2 subunit), computer modeling of this segment based on the well‐defined conformation of α1 subunit suggests that the four cysteine residues (Cys114 and Cys116 in each subunit) in the Cys‐Gly‐Cys motif are positioned in close proximity at the dimer interface. The arrangement of these cysteines together with the nearby Glu118 in SufA dimer may allow coordination of an Fe–S cluster and/or an Fe atom.

Crystal structure of Escherichia coli SufC, an ABC‐type ATPase component of the SUF iron–sulfur cluster assembly machinery

SufC is an ATPase component of the SUF machinery, which is involved in the biosynthesis of Fe–S clusters. To gain insight into the function of this protein, we have determined the crystal structure of Escherichia coli SufC at 2.5 Å resolution. Despite the similarity of the overall structure with ABC‐ATPases (nucleotide‐binding domains of ABC transporters), some key differences were observed. Glu171, an invariant residue involved in ATP hydrolysis, is rotated away from the nucleotide‐binding pocket to form a SufC‐specific salt bridge with Lys152. Due to this salt bridge, D‐loop that follows Glu171 is flipped out to the molecular surface, which may sterically inhibit the formation of an active dimer. Thus, the salt bridge may play a critical role in regulating ATPase activity and preventing wasteful ATP hydrolysis. Furthermore, SufC has a unique Q‐loop structure on its surface, which may form a binding site for its partner proteins, SufB and/or SufD.

Molecular Dynamism of Fe–S Cluster Biosynthesis Implicated by the Structure of the SufC2–SufD2 Complex

Maturation of iron-sulfur (Fe-S) proteins is achieved by the SUF machinery in a wide number of eubacteria and archaea, as well as eukaryotic chloroplasts. This machinery is encoded in Escherichia coli by the sufABCDSE operon, where three Suf components, SufB, SufC, and SufD, form a complex and appear to provide an intermediary site for the Fe-S cluster assembly. Here, we report the quaternary structure of the SufC(2)-SufD(2) complex in which SufC is bound to the C-terminal domain of SufD. Comparison with the monomeric structure of SufC revealed conformational change of the active-site residues: SufC becomes competent for ATP binding and hydrolysis upon association with SufD. The two SufC subunits were spatially separated in the SufC(2)-SufD(2) complex, whereas cross-linking experiments in solution have indicated that two SufC molecules associate with each other in the presence of Mg(2+) and ATP. Such dimer formation of SufC may lead to a gross structural change of the SufC(2)-SufD(2) complex. Furthermore, genetic analysis of SufD revealed an essential histidine residue buried inside the dimer interface, suggesting that conformational change may expose this crucial residue. These findings, together with biochemical characterization of the SufB-SufC-SufD complex, have led us to propose a model for the Fe-S cluster biosynthesis in the complex.

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.

Properties, intracellular localization, and stage-specific expression of membrane-bound β-glucosidase. BglM1, from Physarum polycephalum

Physarum polycephalum expresses a membrane-bound beta-glucosidase (BglM1) with a molecular mass of 130 kDa. The primary structure of BglM1 consists of a glycosyl hydrolase family 3 domain at an amino-terminal domain and a carboxyl-terminal region without homology to the sequence of known glycosidases. The latter region contains two calx-beta motifs known as Ca(2+)-binding sites; an RGD sequence, which is known to be a cell attachment sequence; and a transmembrane region. The molecular mass calculated from the amino acid sequence is 130 kDa, but that in the crude extract was estimated by SDS-PAGE to be 230 kDa, and decreased to 130 kDa during purification. However, when BglM1 was purified in the presence of calcium ion, the molecular mass remained 230 kDa. The biochemical characteristics of the 130- and 230-kDa BglM1 forms were analyzed: differences were found in the kinetic data for some substrates specific for both these enzymes; however, no difference was found in their intrinsic characteristics such as optimum pH and temperature. In addition, the molecular mass of native BglM1 with a calcium ion was estimated to be 1,000 kDa or larger by gel filtration. These results suggest that the calcium ion influences the conformation of BglM1. The evidence that BglM1 localizes on the plasma membrane of plasmodia was confirmed using immunofluorescence microscopy. Although Physarum BglM1 was expressed in microplasmodia and plasmodia, little expression was detected in other stages. BglM1 may have some function only in multinuclear cells.

Structure of Physarum polycephalum cytochrome b5 reductase at 1.56 A resolution.

Physarum polycephalum cytochrome b(5) reductase catalyzes the reduction of cytochrome b(5) by NADH. The structure of P. polycephalum cytochrome b(5) reductase was determined at a resolution of 1.56 A. The molecular structure was compared with that of human cytochrome b(5) reductase, which had previously been determined at 1.75 A resolution [Bando et al. (2004), Acta Cryst. D60, 1929-1934]. The high-resolution structure revealed conformational differences between the two enzymes in the adenosine moiety of the FAD, the lid region and the linker region. The structural properties of both proteins were inspected in terms of hydrogen bonding, ion pairs, accessible surface area and cavity volume. The differences in these structural properties between the two proteins were consistent with estimates of their thermostabilities obtained from differential scanning calorimetry data.

RNA-Seq analysis for indigo biosynthesis pathway genes in Indigofera tinctoria and Polygonum tinctorium.

Natural indigo is the most important blue dye for textile dyeing and valuable secondary metabolite biosynthesized in Indigofera tinctoria and Polygonum tinctorium plants. Present investigation is made to generation of gene resource for pathway enrichment and to understand possible gene expression involved in indigo biosynthesis. The data about raw reads and the transcriptome assembly project has been deposited at GenBank under the accessions SRA180766 and SRX692542 for I. tinctoria and P. tinctorium, respectively.

Transcriptome analysis for identification of indigo biosynthesis pathway genes in Polygonum tinctorium

Abstract Indigo is the most important blue dye for textile dyeing and is biosynthesized in Polygonum tinctorium. Some biochemical studies related to biosynthesis are available. However, genomic and transcriptome studies have not received sufficient attention. Here, we report de novo assembly of transcriptome datasets and its comprehensive analysis. A total of 60,395 unigenes were annotated using BLAST search against the different databases. At least 23,721 unigenes mapped onto different pathways using KEGG database. We found that 3,323 genes are involved in biosynthesis of secondary metabolites, 117 phenylalanine, tyrosine and tryptophan biosynthesis and 147 tryptophan metabolisms. Apart from this, indigo biosynthesis pathway genes viz., dioxygenase, monooxygenase, and glucosyltransferase have also been identified. Fourteen genes encoding cytochrome P450 monooxygenase, 26 glucoside dioxygenase, 9 UDP-glucose D-glucosyltransferase and 52 were β-D-glucosidase. These findings provide a foundation for further analysis of this pathway with potential to enhance the synthesis of indican in P. tinctorium

Structure and properties of the recombinant NADH-cytochrome b5 reductase of Physarum polycephalum

A cDNA for NADH–cytochrome b 5 reductase of Physarum polycephalum was cloned from a cDNA library, and the nucleotide sequence of the cDNA was determined (accession no. AB259870). The DNA of 943 base pairs contains 5′- and 3′-noncoding sequences, including a polyadenylation sequence, and a coding sequence of 843 base pairs. The amino acid sequence (281 residues) deduced from the nucleotide sequence was 25 residues shorter than those of vertebrate enzymes. Nevertheless, the recombinant Physarum enzyme showed enzyme activity comparable to that of the human enzyme. The recombinant Physarum enzyme showed a pH optimum of around 6.0, and apparent K m values of 2 μM and 14 μM for NADH and cytochrome b 5 respectively. The purified recombinant enzyme showed a typical FAD-derived absorption peak of cytochrome b 5 reductase at around 460 nm, with a shoulder at 480 nm. These results suggest that the Physarum enzyme plays an important role in the organism.