Structural basis for the enantiospecificities of R- and S-specific phenoxypropionate/alpha-ketoglutarate dioxygenases

(R)- and (S)-dichlorprop/alpha-ketoglutarate dioxygenases (RdpA and SdpA) catalyze the oxidative cleavage of 2-(2,4-dichlorophenoxy)propanoic acid (dichlorprop) and 2-(4-chloro-2-methyl-phenoxy)propanoic acid (mecoprop) to form pyruvate plus the corresponding phenol concurrent with the conversion of alpha-ketoglutarate (alphaKG) to succinate plus CO2. RdpA and SdpA are strictly enantiospecific, converting only the (R) or the (S) enantiomer, respectively. Homology models were generated for both enzymes on the basis of the structure of the related enzyme TauD (PDB code 1OS7). Docking was used to predict the orientation of the appropriate mecoprop enantiomer in each protein, and the predictions were tested by characterizing the activities of site-directed variants of the enzymes. Mutant proteins that changed at residues predicted to interact with (R)- or (S)-mecoprop exhibited significantly reduced activity, often accompanied by increased Km values, consistent with roles for these residues in substrate binding. Four of the designed SdpA variants were (slightly) active with (R)-mecoprop. The results of the kinetic investigations are consistent with the identification of key interactions in the structural models and demonstrate that enantiospecificity is coordinated by the interactions of a number of residues in RdpA and SdpA. Most significantly, residues Phe171 in RdpA and Glu69 in SdpA apparently act by hindering the binding of the wrong enantiomer more than the correct one, as judged by the observed decreases in Km when these side chains are replaced by Ala.     

Characterization and expression profiling of 4-hydroxyphenylpyruvate dioxygenase gene (<Emphasis Type="Italic">Smhppd</Emphasis>) from <Emphasis Type="Italic">Salvia</Emphasis> <Emphasis Type="Italic">miltiorrhiza</Emphasis> hairy root cultures

A novel 4-hydroxyphenylpyruvate dioxygenase gene (designated as Smhppd) was cloned from hairy roots of Salvia miltiorrhiza Bung. The full-length cDNA of Smhppd was 1,736 bp long with an ORF (open reading frame) that putatively encoded a polypeptide of 481 amino acids, with a predicted molecular mass of 52.54 kDa. The deduced amino acid sequence of the Smhppd gene shared high homology with other known HPPDs. Analysis of Smhppd genomic DNA revealed that it contained two exons and one intron. The analysis of Smhppd promoter region was also presented. Southern-blot analysis revealed that the Smhppd was a low-copy gene in S. miltiorrhiza. Real-time quantitative PCR analysis indicated that Smhppd was constitutively expressed in roots, stems and leaves of S. miltiorrhiza, with the high expression in roots. In addition, Smhppd expreession level under different stress condition was also analyzed during the hairy root culture period, including signaling components for plant defence responses, such as methyl jasmonate and salicylic acid, as well as an abiotic elicitor, Ag⁺ and a biotic elicitor, yeast extract. This study will enable us to further understand the role Smhppd plays in the synthesis of active pharmaceutical compounds in S. miltiorrhiza at molecular level.     

Mass-spectrometric characterization of two posttranslational modifications of cysteine dioxygenase

Recent crystal structures of cysteine dioxygenase (CDO) suggest the presence of two posttranslational modifications adjacent to the catalytic iron center: a thioether cross-link between Cys93 and Tyr157 and extra electron density at Cys164 which was variously explained as cystine or cysteine sulfinic acid. Purification of recombinant rat CDO yields “mature” and “immature” forms with distinct electrophoretic mobilities. We have positively identified and characterized the two modifications in the products of three sequential proteolytic digestions using liquid chromatography coupled with tandem mass spectrometry. The cross-link is unique to the mature form and was identified in an ion of m/z 3,225.403, consistent with a Tyr-Cys cross-link of peptides Gly80-Phe94 with His155-Phe167. The cross-link is liable to cleavage by in-source decay and the resulting separate peptides were sequenced by collision-induced dissociation tandem mass spectrometry. Mass-spectrometric analysis of these same and overlapping peptides in the presence or absence of reductants and alkylating agents identified the second modification to be a cystine formed between Cys164 and exogenous cysteine as proposed earlier. Both modifications have been shown to form in the presence of high levels of cysteine and iron. This and the presence of small amounts of an apparently off-pathway cystine at position Cys93 suggest that although these conditions promote CDO maturation, they may actually arise via nonenzymatic, nonphysiological processes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cloning the bacterial bphC gene into Nicotiana tabacum to improve the efficiency of PCB phytoremediation

The aim of this work is to increase the efficiency of the biodegradation of polychlorinated biphenyls (PCBs) by the introduction of bacterial genes into the plant genome. For this purpose, we selected the bphC gene encoding 2,3-dihydroxybiphenyl-1,2-dioxygenase from Pseudomonas testosteroni B-356 to be cloned into tobacco plants. The dihydroxybiphenyldioxygenase enzyme is the third enzyme in the biphenyl degradation pathway, and its unique function is the cleavage of biphenyl. Three different constructs were designed and prepared in E. coli: the bphC gene being fused with the beta-glucuronidase (GUS) gene, with the luciferase (LUC) gene, and with histidine tail in three separate plant cloning vectors. The GUS and LUC genes were chosen because they can be used as markers for the easy detection of transgenic plants, while histidine tail better enables the isolation of protein expressed in plant tissue. The prepared vectors were then introduced into cells of Agrobacterium tumefaciens. The transient expression of the prepared genes was first studied in cells of Nicotiana tabacum. Once this ability had been established, model tobacco plants were transformed by agrobacterial infection with the bphC/GUS, bphC/LUC, and bphC/His genes. The transformed regenerants were selected on media using a selective antibiotic, and the presence of transgenes and mRNA was determined by PCR and RT-PCR. The expression of the fused proteins BphC/GUS and BphC/LUC was confirmed histochemically by analysis of the expression of their detection markers. Western blot analysis was performed to detect the presence of the BphC/His protein immunochemically using a mouse anti-His antibody. Growth and viability of transgenic plants in the presence of PCBs was compared with control plants.     

Arabidopsis thaliana expresses a second functional flavonol synthase

Arabidopsis thaliana L. produces flavonoid pigments, i.e. flavonols, anthocyanidins and proanthocyanidins, from dihydroflavonol substrates. A small family of putative flavonol synthase (FLS) genes had been recognized in Arabidopsis, and functional activity was attributed only to FLS1. Nevertheless, other FLS activities must be present, because A. thalianafls1 mutants still accumulate significant amounts of flavonols. The recombinant FLSs and leucoanthocyanidin dioxygenase (LDOX) proteins were therefore examined for their enzyme activities, which led to the identification of FLS3 as a second active FLS. This enzyme is therefore likely responsible for the formation of flavonols in the ldox/fls1-2 double mutant. These double mutant and biochemical data demonstrate for the first time that LDOX is capable of catalyzing the in planta formation of flavonols.     

洋葱伯克霍尔德氏菌L68双加氧酶区基因克隆及序列分析

克隆洋葱伯克霍尔德氏菌L68双加氧酶区基因并对其进行序列分析。采用邻苯二酚喷洒方法从洋葱伯克霍尔德氏菌L68的基因文库中筛选到了1株含有双加氧酶区基因的重组子,其重组质粒命名为pB2k。重组质粒pB2k含有8030bp的L68基因片断,经BLAST比对,该片断含有11个与已报道的ORF相近的ORF。在该片断的5’端,ORF1和ORF2分别编码两个转移酶基因,tomA1t、omA2t、omA3和tomA4编码酚羟化酶组份,tomA5编码氧化还原酶,phnT编码铁氧还蛋白,phnE编码邻苯二酚2,3-双加氧酶,ORF3编码未知功能蛋白,phnG编码部分2-羟粘糠酸半醛脱氢酶。     

Simplified cysteine dioxygenase activity assay allows simultaneous quantitation of both substrate and product

A high-performance liquid chromatography (HPLC) method for enzyme activity assays using a hydrophilic interaction liquid chromatography (HILIC) column in combination with an evaporative light scattering detector was developed. The method was used to measure the activity of the non-heme mono-iron enzyme cysteine dioxygenase. The substrate cysteine and the product cysteine sulfinic acid are very weak chromophores, making direct ultraviolet (UV) detection without derivatization rather insensitive; moreover, derivatization of cysteine is often not efficient. Using the system described, underivatized substrate and product in samples from cysteine dioxygenase activity assays could be separated and analyzed. Furthermore, it was possible to quantify cysteic acid, the noncatalytic oxidation product of cysteine sulfinic acid. Acetone was used both to stop the enzymatic reaction by protein precipitation and as an organic mobile phase, making sample preparation very easy and the assay highly reproducible.     

Diversity of 16S rRNA and dioxygenase genes detected in coal‐tar‐contaminated site undergoing active bioremediation

Aims: In order to develop effective bioremediation strategies for polyaromatic hydrocarbons (PAHs) degradation, the composition and metabolic potential of microbial communities need to be better understood, especially in highly PAH contaminated sites in which little information on the cultivation‐independent communities is available. Methods and Results: Coal‐tar‐contaminated soil was collected, which consisted of 122·5 mg g^?1 total extractable PAH compounds. Biodegradation studies with this soil indicated the presence of microbial community that is capable of degrading the model PAH compounds viz naphthalene, phenanthrene and pyrene at 50 ppm each. PCR clone libraries were established from the DNA of the coal‐tar‐contaminated soil, targeting the 16S rRNA to characterize (i) the microbial communities, (ii) partial gene fragment encoding the Rieske iron sulfur center (α‐subunit) common to all PAH dioxygenase enzymes and (iii) β‐subunit of dioxygenase. Phylotypes related to Proteobacteria (Alpha‐, Epsilon‐ and Gammaproteobacteria), Acidobacteria, Actinobacteria, Firmicutes, Gemmatimonadetes and Deinococci were detected in 16S rRNA derived clone libraries. Many of the gene fragment sequences of α‐subunit and β‐subunit of dioxygenase obtained from the respective clone libraries fell into clades that are distinct from the reference dioxygenase gene sequences. Presence of consensus sequence of the Rieske type [2Fe‐2S] cluster binding site suggested that these gene fragments encode for α‐subunit of dioxygenase gene. Conclusions: Sequencing of the cloned libraries representing α‐subunit gene fragments (Rf1) and β‐subunit of dioxygenase showed the presence of hitherto unidentified dioxygenase in coal‐tar‐contaminated soil. Significance and Impact of the Study: The combination of the Rieske primers and bacterial community profiling represents a powerful tool for both assessing bioremediation potential and the exploration of novel dioxygenase genes in a contaminated environment.     

Effects of mitochondria and o-methoxybenzoylalanine on 3-hydroxyanthranilic acid dioxygenase activity and quinolinic acid synthesis

The use of o-methoxybenzoylalanine, a selective kynureninase inhibitor, has been proposed with the aim of reducing brain synthesis of quinolinic acid, an excitotoxic tryptophan metabolite. In liver homogenates, however, this compound caused unexpected accumulation of 3-hydroxyanthranilic acid, the product of kynureninase activity and the precursor of quinolinic acid. To explain this observation, we investigated the interaction(s) of o-methoxybenzoylalanine with 3-hydroxyanthranilic acid dioxygenase, the enzyme responsible for quinolinic acid formation. When the purified enzyme or partially purified cytosol preparations were used, o-methoxybenzoylalanine did not affect 3-hydroxyanthranilic acid dioxygenase activity. However, a significant reduction of this enzymatic activity did occur when o-methoxybenzoylalanine was tested in the presence of mitochondria. It is interesting that addition of purified mitochondria to 3-hydroxyanthranilic acid dioxygenase preparations reduced the enzymatic activity and the synthesis of quinolinic acid. In vivo, administration of o-methoxybenzoylalanine significantly reduced quinolinic acid synthesis and content in both blood and brain of mice. Our results suggest that mitochondrial protein(s) interact(s) with soluble 3-hydroxyanthranilic acid dioxygenase and cause(s) modifications in the enzyme resulting in a decrease in its activity. These modifications also allow the enzyme to interact with o-methoxybenzoylalanine, thus leading to a further reduction in quinolinic acid synthesis.     

Cloning and sequence analyses of a 2,3-dihydroxybiphenyl 1,2-dioxygenase gene (<Emphasis Type="Italic">bphC</Emphasis>) from<Emphasis Type="Italic"> Comamonas</Emphasis> sp. SMN4 for phylogenetic and structural analysis

A genomic library of biphenyl-degrading Comamonas sp. SMN4 for isolating fragments containing the 2,3-dihydroxybiphenyl 1,2-dioxygenase (23DBDO) gene was constructed. The smallest subclone (pNPX9) encoding 23DBDO activity was sequenced and analyzed. The C-terminal domain of 23DBDO from Comamonas sp. SMN4 had five catalytically essential residues and was more highly conserved than the N-terminal domain. Phylogenetic and structural relationships of 23DBDO from Comamonas sp. SMN4 were analyzed. Electronic Publication