Genomic organization and expression analysis of a farnesyl diphosphate synthase gene (FPPS2) in apples (Malus domestica Borkh.)

A farnesyl diphosphate synthase gene (FPPS2), which contains 11 introns and 12 exons, was isolated from the apple cultivar “White Winter Pearmain”. When it was compared to our previously reported FPPS1, its each intron size was different, its each exon size was the same as that of FPPS1 gene, 30 nucleotide differences were found in its coding sequence. Based on these nucleotide differences, specific primers were designed to perform expression analysis; the results showed that it expressed in both fruit and leaf, its expression level was obviously lower than that of FPPS1 gene in fruit which was stored at 4 °C for 5 weeks. This is the first report concerning two FPPS genes and their expression comparison in apples.     

Evolution of the salivary apyrases of blood-feeding arthropods

Phylogenetic analyses of three families of arthropod apyrases were used to reconstruct the evolutionary relationships of salivary-expressed apyrases, which have an anti-coagulant function in blood-feeding arthropods. Members of the 5′nucleotidase family were recruited for salivary expression in blood-feeding species at least five separate times in the history of arthropods, while members of the Cimex-type apyrase family have been recruited at least twice. In spite of these independent events of recruitment for salivary function, neither of these families showed evidence of convergent amino acid sequence evolution in salivary-expressed members. On the contrary, in the 5′-nucleotide family, salivary-expressed proteins conserved ancestral amino acid residues to a significantly greater extent than related proteins without salivary function, implying parallel evolution by conservation of ancestral characters. This unusual pattern of sequence evolution suggests the hypothesis that purifying selection favoring conservation of ancestral residues is particularly strong in salivary-expressed members of the 5′-nucleotidase family of arthropods because of constraints arising from expression within the vertebrate host.     

Characterization of a novel nm23 gene and its potential roles in gametogenesis in the prawn Macrobrachium rosenbergii (de Man, 1879) (Crustacea: Decapoda)

Nm23 is a family of genes encoding the nucleoside diphosphate (NDP) kinase, which functions in a wide variety of biological processes, including growth, development, differentiation and tumor metastasis. In this study, a novel nm23 gene, designated as Mrnm23, was identified from the freshwater giant prawn Macrobrachium rosenbergii. The full-length cDNA was 776 bp in length, encoding for a protein of 176 amino acids with one typical NDP kinase domain that harbored all the crucial residues for nucleotide binding and enzymatic activity. Like human novel nm23-H1B, the putative protein contained a unique 21-amino-acid NH₂-terminal extension as compared to human nm23 (nm23-H1) homologs. Further, 3 extra amino acid residues prolonged the COOH-terminus. The Mrnm23 was ubiquitously expressed in all tissues examined, including androgenic gland, gill, heart, liver, muscle, ovary, and testis. In situ hybridization to gonad sections indicated that the Mrnm23 mRNA was localized in the cytoplasm of cup-base of differentiating spermatids, in the spike of the umbrella-shaped spermatozoa and in the cytoplasm of the early previtellogenic oocytes, suggesting that the Mrnm23 has potential roles in spermiogenesis and early differentiation of oocyte.     

Methylerythritol phosphate pathway to isoprenoids: Kinetic modeling and in silico enzyme inhibitions in Plasmodium falciparum

The methylerythritol phosphate (MEP) pathway of Plasmodium falciparum (P. falciparum) has become an attractive target for anti-malarial drug discovery. This study describes a kinetic model of this pathway, its use in validating 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) as drug target from the systemic perspective, and additional target identification, using metabolic control analysis and in silico inhibition studies. In addition to DXR, 1-deoxy-d-xylulose 5-phosphate synthase (DXS) can be targeted because it is the first enzyme of the pathway and has the highest flux control coefficient followed by that of DXR. In silico inhibition of both enzymes caused large decrement in the pathway flux. An added advantage of targeting DXS is its influence on vitamin B1 and B6 biosynthesis. Two more potential targets, 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase, were also identified. Their inhibition caused large accumulation of their substrates causing instability of the system.     

Structure of the (E)-4-hydroxy-3-methyl-but-2-enyl-diphosphate reductase from Plasmodium falciparum

Terpenoid precursor biosynthesis occurs in human and many pathogenic organisms via the mevalonate and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways, respectively. We determined the X-ray structure of the Fe/S containing (E)-4-hydroxy-3-methyl-but-2-enyl-diphosphate reductase (LytB) of the pathogenic protozoa Plasmodium falciparum which catalyzes the terminal step of the MEP pathway. The cloverleaf fold and the active site of P. falciparum LytB corresponds to those of the Aquifex aeolicus and Escherichia coli enzymes. Its distinct electron donor [2Fe–2S] ferredoxin was modeled to its binding site by docking calculations. The presented structural data provide a platform for a rational search of anti-malarian drugs.     

Functional Analysis of Type 1 Isopentenyl Diphosphate Isomerase from Halobacterium sp. NRC-1

Here we report the characterization of the type-1 isopentenyl diphosphate isomerase derived from Halobacterium sp. NRC-1. The expressed purified enzyme showed maximum isomerase activity in the presence of 1 M NaCl at 37 °C at pH 6.0. This type-1 enzyme appears to be the first for which the Co²⁺ ion is required for activity.     

The methylerythritol phosphate pathway for isoprenoid biosynthesis in coccidia: presence and sensitivity to fosmidomycin

The apicoplast is a recently discovered, plastid-like organelle present in most apicomplexa. The methylerythritol phosphate (MEP) pathway involved in isoprenoid biosynthesis is one of the metabolic pathways associated with the apicoplast, and is a new promising therapeutic target in Plasmodium falciparum. Here, we check the presence of isoprenoid genes in four coccidian parasites according to genome database searches. Cryptosporidium parvum and C. hominis, which have no plastid genome, lack the MEP pathway. In contrast, gene expression studies suggest that this metabolic pathway is present in several development stages of Eimeria tenella and in tachyzoites of Toxoplasma gondii. We studied the potential of fosmidomycin, an antimalarial drug blocking the MEP pathway, to inhibit E. tenella and T. gondii growth in vitro. The drug was poorly effective even at high concentrations. Thus, both fosmidomycin sensitivity and isoprenoid metabolism differs substantially between apicomplexan species.     

An Escherichia coli mutant producing a truncated inactive form of GlgC synthesizes glycogen: further evidences for the occurrence of various important sources of ADPglucose in enterobacteria

AC70R1-504 Escherichia coli mutants possess a glgC* gene with a nucleotide change resulting in a premature stop codon that renders a truncated, inactive form of GlgC. Cells over-expressing the wild type glgC, but not those over-expressing the AC70R1-504 glgC*, accumulated high ADPglucose and glycogen levels. AC70R1-504 mutants accumulated glycogen, whereas DeltaglgCAP deletion mutants lacking the whole glycogen biosynthetic machinery displayed a glycogen-less phenotype. AC70R1-504 cells with enhanced glycogen synthase activity accumulated high glycogen levels. By contrast, AC70R1-504 cells with high ADPG hydrolase activity accumulated low glycogen. These data further confirm that enterobacteria possess various sources of ADPglucose linked to glycogen biosynthesis.     

Catalase-1 (CAT-1) and nucleoside diphosphate kinase-1 (NDK-1) play an important role in protecting conidial viability under light stress in <Emphasis Type="Italic">Neurospora crassa</Emphasis>

Recently we reported that Catalase-1 (CAT-1) played an important role in protecting conidial viability in Neurospora crassa, and interacted with a light signal transducer, nucleoside diphosphate kinase-1 (NDK-1). To disclose the functional interaction between CAT-1 and NDK-1 at the genetic level, we created CAT-1 and NDK-1 double mutants, cat-1;ndk-1-1 and cat-1;ndk-1-2, by crossing single mutants of cat-1 ^RIP and ndk-1 ^P72H previously isolated in our laboratory. The double mutant strains grew normally, but showed increased CAT-2 activity. In cat-1 ^RIP , NDK activity was increased when dCDP was used as a substrate. ndk-1 ^P72H , cat-1;ndk-1-1, and cat-1;ndk-1-2 were more sensitive to riboflavin than the wild type and cat-1 ^RIP under strong light (100 μE m⁻² s⁻¹). The pull-down experiment suggests that His-tagged NDK-1 is bound to [³²P]NADH. However, his-tagged NDK-1^P72H was not bound to [³²P]NADH. The double mutants showed much lower conidial viability and lost all conidial germination ability much more rapidly than cat-1 ^RIP , when they were cultured under continuous light for more than 2 weeks. These results indicate that the interaction of CAT-1 with NDK-1 plays an important role in supporting the survival of conidia under oxidative and light-induced stress including singlet oxygen, and confirm our former conclusion that reactive oxygen species play an important role in light signal transduction via NDK-1 at the genetic level. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.