Reversible and nonoxidative gamma-resorcylic acid decarboxylase: characterization and gene cloning of a novel enzyme catalyzing carboxylation of resorcinol, 1,3-dihydroxybenzene, from Rhizobium radiobacter

We found a gamma-resorcylic acid (gamma-RA, 2,6-dihydroxybenzoic acid) decarboxylase, as a novel enzyme applicable to carboxylation of resorcinol (RE, 1,3-dihydroxybenzene) to form gamma-RA, in a bacterial strain Rhizobium radiobacter WU-0108 isolated through the screening of gamma-RA degrading microorganisms. The activities for carboxylation of RE and decarboxylation of gamma-RA were detected in the cell-free extracts of R. radiobacter WU-0108 grown aerobically with gamma-RA. The enzyme, gamma-RA decarboxylase, was purified to homogeneity on SDS-PAGE through the steps of one ion-exchange chromatography and two kinds of hydrophobic chromatography. The molecular weight of the enzyme was estimated to be 130 kDa by gel-filtration, and that of the subunit was determined to be 34 kDa by SDS-PAGE, suggesting that the enzyme is a homotetrameric structure. The enzyme catalyzed the decarboxylation of gamma-RA, but not alpha-RA or beta-RA. Without addition of any cofactors, the enzyme catalyzed the regio-selective carboxylation of RE to form gamma-RA, without formation of alpha-RA and beta-RA, and of catechol to 2,3-dihydroxybenzoic acid. In the presence of oxygen, this gamma-RA decarboxylase showed no decrease in both of the activities as for decarboxylation of gamma-RA and carboxylation of RE, different from other decarboxylases reported so far. The gene, rdc, encoding the gamma-RA decarboxylase was cloned into Escherichia coli, sequenced, and subjected to over-expression. The deduced amino acid sequence of the rdc gene consists of 327 amino acid residues corresponding to 34 kDa protein, and shows 42% and 30% identity to those of a 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus niger and a 5- carboxyvanillate decarboxylase from Sphingomonas paucimobilis SYK-6. A site-directed mutagenesis study revealed the two histidine residues at positions of 164 and 218 in Rdc to be essential for the catalytic activities of decarboxylation of gamma-RA and carboxylation of RE.     

Light-induced structural changes in a putative blue-light receptor with a novel FAD binding fold sensor of blue-light using FAD (BLUF); Slr1694 of synechocystis sp. PCC6803

The sensor of blue-light using FAD (BLUF) domain is the flavin-binding fold categorized to a new class of blue-light sensing domain found in AppA from Rhodobacter sphaeroides and PAC from Euglena gracilis, but little is known concerning the mechanism of blue-light perception. An open reading frame slr1694 in a cyanobacterium Synechocystis sp. PCC6803 encodes a protein possessing the BLUF domain. Here, a full-length Slr1694 protein retaining FAD was expressed and purified and found to be present as an oligomeric form (trimer or tetramer). Using the purified Slr1694, spectroscopic properties of Slr1694 were characterized. Slr1694 was found to show the same red-shift of flavin absorption and quenching of flavin fluorescence by illumination as those of AppA. These changes reversed in the dark although the rate of dark state regeneration was much faster in Slr1694 than AppA, indicating that Slr1694 is a blue-light receptor based on BLUF with the similar photocycle to that of AppA. The dark decay in D(2)O was nearly four times slower than in H(2)O. Light-induced Fourier transform infrared (FTIR) difference spectroscopy was applied to examine the light-induced structure change of a chromophore and apo-protein with deuteration and universal (13)C and (15)N isotope labeling. The FTIR results indicate that light excitation induced distinct changes in the amide I modes of peptide backbone but relatively limited changes in flavin chromophore. Light excitation predominantly weakened the C(4)=O and C(2)=O bonding and strengthened the N1C10a and/or C4aN5 bonding, indicating formational changes of the isoalloxazine ring II and III of FAD but little formational change in the isoalloxazine ring I. The photocycle of the BLUF is unique in the sense that light excitation leads to the structural rearrangements of the protein moieties coupled with a minimum formational change of the chromophore.     

Recognition of sphingomyelin by lysenin and lysenin-related proteins

Lysenin is a sphingomyelin (SM)-specific toxin isolated from the coelomic fluid of the earthworm Eisenia foetida. Lysenin comprises a family of proteins together with lysenin-related protein 1 (LRP-1, lysenin 2) and LRP-2 (lysenin 3). In the present study, we characterized LRP-1 and LRP-2 together with lysenin using maltose-binding-protein-tagged recombinant proteins. LRP-2 specifically bound SM and induced hemolysis like lysenin. In contrast the binding and hemolytic activities of LRP-1 were 10 times less than those of lysenin and LRP-2. Lysenin and LRP-2 share 30 common sites of aromatic amino acids. Among them, only one position, phenylalanine 210, is substituted for isoleucine in LRP-1. The activity of LRP-1 was dramatically increased by introducing a single amino acid substitution of isoleucine 210 to phenylalanine, suggesting the importance of this aromatic amino acid in biological activities of lysenin and LRPs. The importance of aromatic amino acids was further indicated by a systematic tryptophan to alanine mutation of lysenin. Lysenin contains six tryptophan residues of which five are conserved in LRP-1 and -2. We showed that the conserved tryptophans but not the nonconserved one were required both in the recognition of SM and in the hemolytic activity of lysenin. Our results suggest the importance of tryptophan in the toxin function likely due to a direct recognition of SM or in maintaining the protein structure.     

Transgenic mouse expressing human mutant alpha-galactosidase A in an endogenous enzyme deficient background: a biochemical animal model for studying active-site specific chaperone therapy for Fabry disease

Fabry disease is an inborn error of glycosphingolipid metabolism caused by the deficiency of lysosomal alpha-galactosidase A (alpha-Gal A). We have established transgenic mice that exclusively express human mutant alpha-Gal A (R301Q) in an alpha-Gal A knock-out background (TgM/KO mice). This serves as a biochemical model to study and evaluate active-site specific chaperone (ASSC) therapy for Fabry disease, which is specific for those missense mutations that cause misfolding of alpha-Gal A. The alpha-Gal A activities in the heart, kidney, spleen, and liver of homozygous TgM/KO mice were 52.6, 9.9, 29.6 and 44.4 unit/mg protein, respectively, corresponding to 16.4-, 0.8-, 0.6- and 1.4-fold of the endogenous enzyme activities in the same tissues of non-transgenic mice with a similar genetic background. Oral administration of 1-deoxygalactonojirimycin (DGJ), a competitive inhibitor of alpha-Gal A and an effective ASSC for Fabry disease, at 0.05 mM in the drinking water of the mice for 2 weeks resulted in 13.8-, 3.3-, 3.9-, and 2.6-fold increases in enzyme activities in the heart, kidney, spleen and liver, respectively. No accumulation of globotriaosylceramide, a natural substrate of alpha-Gal A, could be detected in the heart of TgM/KO mice after DGJ treatment, indicating that degradation of the glycolipid in the heart was not inhibited by DGJ at that dosage. The alpha-Gal A activity in homozygous or heterozygous fibroblasts established from TgM/KO mice (TMK cells) was approximately 39 and 20 unit/mg protein, respectively. These TgM/KO mice and TMK cells are useful tools for studying the mechanism of ASSC therapy, and for screening ASSCs for Fabry disease.     

A small deletion hotspot in the type II keratin gene mK6irs1/Krt2-6g on mouse chromosome 15, a candidate for causing the wavy hair of the caracul (Ca) mutation

A new mutation has arisen in a colony of mice transgenic for human alpha-galactosidase. The mutation is independent of the transgenic insertion, autosomal dominant, and morphologically very similar to the classical wavy coat mutation, caracul (Ca), on chromosome 15. Therefore, we designated this locus the caracul Rinshoken (Ca(Rin)). Applying a positional cloning approach, we identified the mK6irs1/Krt2-6g gene as a strong candidate for Ca(Rin) because among five Ca alleles examined mutations always occurred in the highly conserved positions of the alpha-helical rod domain (1A and 2B subdomain) of this putative gene product. The most striking finding is that four independently discovered alleles, the three preexistent alleles Ca(J), Ca(9J), Ca(10J), and our allele Ca(Rin), all share one identical amino acid deletion (N 140 del) and the fifth, Ca(medJ), has an amino acid substitution (A 431 D). These findings indicate that a mutation hotspot exists in the Ca locus. Additionally, we describe a Ca mutant allele induced by ENU mutagenesis, which also possesses an amino acid substitution (L 424 W) in the mK6irs1/Krt2-6g gene. The identification of the Ca candidate gene enables us to further define the nature of the genetic pathway required for hair formation and provides an important new candidate that may be implicated in human hair and skin diseases.     

Targeted gene disruption by homologous recombination in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1

In contrast to the high accumulation in sequence data for hyperthermophilic archaea, methodology for genetically manipulating these strains is still at an early stage. This study aimed to develop a gene disruption system for the hyperthermophilic euryarchaeon Thermococcus kodakaraensis KOD1. Uracil-auxotrophic mutants with mutations in the orotidine-5'-monophosphate decarboxylase gene (pyrF) were isolated by positive selection using 5-fluoroorotic acid (5-FOA) and used as hosts for further transformation experiments. We then attempted targeted disruption of the trpE locus in the host strain by homologous recombination, as disruption of trpE was expected to result in tryptophan auxotrophy, an easily detectable phenotype. A disruption vector harboring the pyrF marker within trpE was constructed for double-crossover recombination. The host cells were transformed with the exogenous DNA using the CaCl(2) method, and several transformants could be selected based on genetic complementation. Genotypic and phenotypic analyses of a transformant revealed the unique occurrence of targeted disruption, as well as a phenotypic change of auxotrophy from uracil to tryptophan caused by integration of the wild-type pyrF into the host chromosome at trpE. As with the circular plasmid, gene disruption with linear DNA was also possible when the homologous regions were relatively long. Shortening these regions led to predominant recombination between the pyrF marker in the exogenous DNA and the mutated allele on the host chromosome. In contrast, we could not obtain trpE disruptants by insertional inactivation using a vector designed for single-crossover recombination. The gene targeting system developed in this study provides a long-needed tool in the research on hyperthermophilic archaea and will open the way to a systematic, genetic approach for the elucidation of unknown gene function in these organisms.     

Cloning of the genes for the pituitary glycoprotein hormone alpha and follicle-stimulating hormone beta subunits in the Japanese crested ibis,Nipponia nippon

We have isolated a part of the gene for the pituitary glycoprotein hormone common alpha subunit (PGHalpha) and the whole gene for the follicle-stimulating hormone beta subunit (FSHbeta) in the Japanese crested ibis (Nipponia nippon), a critically endangered bird species in East Asia. The nucleotide sequence of a part of the PGHalpha gene (5026 bp) contained three exons holding the whole coding and 3' untranslated regions, but lacked a 5' untranslated region. Its exon-intron structure was similar to that in mammals, but different from that in teleosts in the location of the second intron. For the FSHbeta gene, the nucleotide sequence of 7633 bp was assembled from two phage clones. The exon-intron structure of three exons and two introns was similar to that observed in mammals and teleosts. In the putative promoter region of the ibis FSHbeta gene, a progesterone responsive element (PRE)-like sequence and two AP-1 responsive element-like sequences reported in the ovine FSHbeta gene were not conserved in complete form. The increased number of ATTTA motifs in the putative 3' untranslated region in comparison with those in Japanese quail and chicken FSHbeta cDNA suggested that more rapid degradation of FSHbeta mRNA occurs in this species. Deduced amino acid sequences of the ibis PGHalpha and FSHbeta showed high similarities with those of the corresponding subunits of other avian species. This is the first report on the genomic sequences of the PGHalpha and FSHbeta in an avian species.     

A rationale for the shift in colour towards blue in transgenic carnation flowers expressing the flavonoid 3',5'-hydroxylase gene

Recently marketed genetically modified violet carnations cv. Moondust and Moonshadow (Dianthus caryophyllus) produce a delphinidin type anthocyanin that native carnations cannot produce and this was achieved by heterologous flavonoid 3',5'-hydroxylase gene expression. Since wild type carnations lack a flavonoid 3',5'-hydroxylase gene, they cannot produce delphinidin, and instead accumulate pelargonidin or cyanidin type anthocyanins, such as pelargonidin or cyanidin 3,5-diglucoside-6"-O-4, 6"'-O-1-cyclic-malyl diester. On the other hand, the anthocyanins in the transgenic flowers were revealed to be delphinidin 3,5-diglucoside-6"-O-4, 6"'-O-1-cyclic-malyl diester (main pigment), delphinidin 3,5-diglucoside-6"-malyl ester, and delphinidin 3,5-diglucoside-6",6"'- dimalyl ester. These are delphinidin derivatives analogous to the natural carnation anthocyanins. This observation indicates that carnation anthocyanin biosynthetic enzymes are versatile enough to modify delphinidin. Additionally, the petals contained flavonol and flavone glycosides. Three of them were identified by spectroscopic methods to be kaempferol 3-(6"'-rhamnosyl-2"'-glucosyl-glucoside), kaempferol 3-(6"'-rhamnosyl-2"'-(6-malyl-glucosyl)-glucoside), and apigenin 6-C-glucosyl-7-O-glucoside-6"'-malyl ester. Among these flavonoids, the apigenin derivative exhibited the strongest co-pigment effect. When two equivalents of the apigenin derivative were added to 1 mM of the main pigment (delphinidin 3,5-diglucoside-6"-O-4,6"'-O-1-cyclic-malyl diester) dissolved in pH 5.0 buffer solution, the lambda(max) shifted to a wavelength 28 nm longer. The vacuolar pH of the Moonshadow flower was estimated to be around 5.5 by measuring the pH of petal. We conclude that the following reasons account for the bluish hue of the transgenic carnation flowers: (1). accumulation of the delphinidin type anthocyanins as a result of flavonoid 3',5'-hydroxylase gene expression, (2). the presence of the flavone derivative strong co-pigment, and (3). an estimated relatively high vacuolar pH of 5.5.