Phylogenetic analysis of uroporphyrinogen III synthase (UROS) gene

The uroporphyrinogen III synthase (UROS) enzyme (also known as hydroxymethylbilane hydrolyase) catalyzes the cyclization of hydroxymethylbilane to uroporphyrinogen III during heme biosynthesis. A deficiency of this enzyme is associated with the very rare Gunther''s disease or congenital erythropoietic porphyria, an autosomal recessive inborn error of metabolism. The current study investigated the possible role of UROS (Homo sapiens [EC: 4.2.1.75; 265 aa; 1371 bp mRNA; Entrez Pubmed ref {"type":"entrez-protein","attrs":{"text":"NP_000366.1","term_id":"4557873","term_text":"NP_000366.1"}}NP_000366.1, {"type":"entrez-nucleotide","attrs":{"text":"NM_000375.2","term_id":"171543867","term_text":"NM_000375.2"}}NM_000375.2]) in evolution by studying the phylogenetic relationship and divergence of this gene using computational methods. The UROS protein sequences from various taxa were retrieved from GenBank database and were compared using Clustal-W (multiple sequence alignment) with defaults and a first-pass phylogenetic tree was built using neighbor-joining method as in DELTA BLAST 2.2.27+ version. A total of 163 BLAST hits were found for the uroporphyrinogen III synthase query sequence and these hits showed putative conserved domain, HemD superfamily (as on 14^th Nov 2012). We then narrowed down the search by manually deleting the proteins which were not UROS sequences and sequences belonging to phyla other than Chordata were deleted. A repeat phylogenetic analysis of 39 taxa was performed using PhyML and TreeDyn software to confirm that UROS is a highly conserved protein with approximately 85% conserved sequences in almost all chordate taxons emphasizing its importance in heme synthesis.     

Cloning, sequencing, and expression of the uroporphyrinogen III methyltransferase cobA gene of Propionibacterium freudenreichii (shermanii).

We cloned, sequenced, and overexpressed cobA, the gene encoding uroporphyrinogen III methyltransferase in Propionibacterium freudenreichii, and examined the catalytic properties of the enzyme. The methyltransferase is similar in mass (27 kDa) and homologous to the one isolated from Pseudomonas denitrificans. In contrast to the much larger isoenzyme encoded by the cysG gene of Escherichia coli (52 kDa), the P. freudenreichii enzyme does not contain the additional 22-kDa peptide moiety at its N-terminal end bearing the oxidase-ferrochelatase activity responsible for the conversion of dihydrosirohydrochlorin (precorrin-2) to siroheme. Since it does not contain this moiety, it is not a likely candidate for synthesis of a cobalt-containing early intermediate that has been proposed for the vitamin B12 biosynthetic pathway in P. freudenreichii. Uroporphyrinogen III methyltransferase of P. freudenreichii not only catalyzes the addition of two methyl groups to uroporphyrinogen III to afford the early vitamin B12 intermediate, precorrin-2, but also has an overmethylation property that catalyzes the synthesis of several tri- and tetra-methylated compounds that are not part of the vitamin B12 pathway. The enzyme catalyzes the addition of three methyl groups to uroporphyrinogen I to form trimethylpyrrocorphin, the intermediate necessary for biosynthesis of the natural products, factors S1 and S3, previously isolated from this organism. A second gene found upstream from the cobA gene encodes a protein homologous to CbiO of Salmonella typhimurium, a membrane-bound, ATP-dependent transport protein thought to be part of the cobalt transport system involved in vitamin B12 synthesis. These two genes do not appear to constitute part of an extensive cobalamin operon.     

Identification and characterization of the Arabidopsis gene encoding the tetrapyrrole biosynthesis enzyme uroporphyrinogen III synthase

UROS (uroporphyrinogen III synthase; EC 4.2.1.75) is the enzyme responsible for the formation of uroporphyrinogen III, the precursor of all cellular tetrapyrroles including haem, chlorophyll and bilins. Although UROS genes have been cloned from many organisms, the level of sequence conservation between them is low, making sequence similarity searches difficult. As an alternative approach to identify the UROS gene from plants, we used functional complementation, since this does not require conservation of primary sequence. A mutant of Saccharomyces cerevisiae was constructed in which the HEM4 gene encoding UROS was deleted. This mutant was transformed with an Arabidopsis thaliana cDNA library in a yeast expression vector and two colonies were obtained that could grow in the absence of haem. The rescuing plasmids encoded an ORF (open reading frame) of 321 amino acids which, when subcloned into an Escherichia coli expression vector, was able to complement an E. coli hemD mutant defective in UROS. Final proof that the ORF encoded UROS came from the fact that the recombinant protein expressed with an N-terminal histidine-tag was found to have UROS activity. Comparison of the sequence of AtUROS (A. thaliana UROS) with the human enzyme found that the seven invariant residues previously identified were conserved, including three shown to be important for enzyme activity. Furthermore, a structure-based homology search of the protein database with AtUROS identified the human crystal structure. AtUROS has an N-terminal extension compared with orthologues from other organisms, suggesting that this might act as a targeting sequence. The precursor protein of 34 kDa translated in vitro was imported into isolated chloroplasts and processed to the mature size of 29 kDa. Confocal microscopy of plant cells transiently expressing a fusion protein of AtUROS with GFP (green fluorescent protein) confirmed that AtUROS was targeted exclusively to chloroplasts in vivo.     

一株对羟基苯甲酸酯海洋降解菌的关键酶基因克隆与表达

【背景】对羟基苯甲酸及其酯类常作为合成多种芳香族化合物的前体物质广泛应用于多个领域,但其难以自然降解给环境造成了污染问题,同时这些污染物随着洋流迁移到海洋中破坏海洋生态环境。【目的】从海洋环境中筛选对羟基苯甲酸酯高效降解菌,通过全基因组测序及注释分析,预测对羟基苯甲酸酯代谢通路,确定其代谢过程中的关键酶并进行功能研究。【方法】通过富集培养从海洋环境中分离对羟基苯甲酸酯降解菌,利用基因克隆技术将降解对羟基苯甲酸酯关键酶基因在大肠杆菌中高效表达,探究重组蛋白活性及酶学特征。【结果】从海底泥沙中筛选到一个菌株,经16S rRNA基因测序鉴定为硝化柠檬球菌(Citricoccus nitrophenolicus);该菌株能够利用多种对羟基苯甲酸酯类物质进行生长,在甲酯为碳源条件下生长状态最好;将羧酸酯酶基因和单加氧酶基因在大肠杆菌中进行高效表达,重组表达的羧酸酯酶最适反应条件为：pH 8.0,30℃反应30 min;重组表达的单加氧酶活性表达依赖于辅酶,Mg²⁺、Mn²⁺、Zn²⁺和Fe3+可增强该酶活性;经荧光定量PCR进一步...     

Cloning and expression of Chlorobium vibrioforme uroporphyrinogen decarboxylase gene in a Salmonella heme auxotroph

To study the post-uroporphyrin steps in heme and chlorophyll biosynthesis in Chlorobium, we attempted to clone the uroporphyrinogen decarboxylase ( hemE) gene. A Chlorobium genomic library was used to transform a restriction-minus Salmonella typhimurium strain. The recombinant DNA molecules were transduced into an auxotrophic Salmonella double mutant ( hemA(-) hemE(-)) by phage P22. Faster-growing colonies indicated complementation of the hemE mutation. Each clone was tested by backcross transduction of the mutant. Growth rates of the confirmed clones in LB medium were comparable to wild-type Salmonella. HPLC analysis of the substrate (uroporphyrinogen) and the product (coproporphyrinogen) of the decarboxylase activity was performed in one such clone. This clone showed an active hemE gene within a 4-kb insert.     

Cloning and embryonic expression of zebrafish neuropilin genes

Neuropilin (Nrp), a cell surface receptor for class 3 semaphorins and for certain heparin forms of vascular endothelial growth factors, functions in many biological processes including axon guidance, neural cell migration and angiogenesis in the development of the nervous system and the cardiovascular system. To understand the role of neuropilins in zebrafish embryogenesis, we have cloned three zebrafish neuropilin homologues, nrp1b, nrp2a and nrp2b. Based on synteny, zebrafish nrp1b and the previously cloned nrp1a are orthologous to human nrp1, and zebrafish nrp2a and 2b orthologous to human nrp2. We have characterized the expression patterns of these four zebrafish neuropilin genes in wild type embryos from the beginning of somitogenesis to 48 h post-fertilization. Zebrafish nrp1a is expressed in the neural tube including telencephalon, epithalamus, cells along the axonal trajectory of the posterior commissure and the medial longitudinal fascicle, hindbrain neurons, vagus motor neurons and spinal motoneurons. Zebrafish nrp1b is expressed in the nose, the cranial neural crest cell (NCC) derived tissue underlying the hypothalamus, endothelial precursors and the trunk and tail vasculature. Zebrafish nrp2a is expressed in telencephalon, anterior pituitary, oculomotor and trochlear motor neurons, cells along the supra-optic and posterior commissures, hindbrain rhombomere 1, hindbrain neurons, cranial NCCs and sclerotome. Zebrafish nrp2b is expressed in telencephalon, thalamus, hypothalamus, epiphysis, cells along the anterior and posterior commissures, post-optic and supra-optic commissures and the olfactory axonal trajectory, hindbrain neurons, cranial NCCs, somites and spinal cord neurons.     

Purification and properties of uroporphyrinogen III synthase (co-synthetase) from Euglena gracilis.

Uroporphyrinogen III synthase (co-synthetase) purified from Euglena gracilis is a monomer of Mr 38 500 by gel-filtration studies and 31 000 by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The pI is apparently in the range 4.8-5.1. No evidence for any cofactors was found, and folate derivatives were shown to be absent; no metal ions appear to be present in the enzyme. The Km for hydroxymethylbilane is in the range 12-40 microM, and the product, uroporphyrinogen III, is an inhibitor. Modification studies suggest that arginine residues are essential for the activity of co-synthetase; lysine residues may also be essential, but histidine, cysteine and tyrosine residues are not.     

马铃薯多酚氧化酶基因克隆及反义表达载体构建

从甘农薯1号马铃薯试管苗叶片中提取总DNA,根据Genebank报道的马铃薯多酚氧化酶基因(PPT32)序列,设计合成了带有BamHⅠ、SacⅠ特定酶切位点的2对特异引物,通过PCR获得l840bP和476bP的扩增产物。测序结果表明,l840bp的扩增产物与Oenebank中发表的序列一致,476bp的扩增产物正是欲扩增的马铃薯多酚氧化酶基因的同源片段。将2个扩增产物反向连接到表达载体PC3中,通过双酶切鉴定后,按直接导入法实现反义基因表达载体质粒向农杆菌EHA105的导人;并经PCR鉴定证明,此质粒已整合到农杆菌Ti上。     

Cloning and expression of genes encoding meta-cleavage enzymes from 4,6-dimethyldibenzothiophene-degrading Sphingomonas strain TZS-7

Sphingomonas strain TZS-7 was reported as the first strain to have the ability to degrade 4,6-dimethyldibenzothiophene (4,6-dmDBT) by the ring-destructive pathway. Two genes for meta-cleavage dioxygenases were cloned from strain TZS-7. Expression of each gene showed that one enzyme was specific for 2,3-dihydroxybiphenyl while another was more specific for catechol. The genes for the two enzymes were named dmdC and catA. The analysis of deduced amino acid sequences indicates that CatA falls into the class of meta-cleavage dioxygenases acting on dihydroxylated monocyclic compounds and DmdC falls into the class of meta-cleavage dioxygenases acting on dihydroxylated polycyclic compounds.     

Cloning and expression of the soybean DapA gene encoding dihydrodipicolinate synthase

The rate-limiting step in the pathway for lysine synthesis in plants is catalyzed by the enzyme dihydrodipicolinate synthase (DS). We have cloned the portion of the soybean (Glycine max cv. Century) DapA cDNA that encodes the mature DS protein. Expression of the cloned soybean cDNA as a lacZ fusion protein was selected in a dapA ^- Escherichia coli auxotroph. The DS activity of the fusion protein was characterized in E. coli extracts. The DS activity of the fusion protein was inhibited by lysine concentrations that also inhibited native soybean DS, while E. coli DS activity was much less sensitive to inhibition by lysine.     

Regulation of expression of the genes encoding steroidogenic enzymes

In recent years it has become apparent that tropic hormones involved in steroidogenesis act to regulate the expression of the enzymes involved in the various steroidogenic pathways. This is particularly evident in the ovary where the episodic secretion of steroids throughout the ovarian cycle is regulated largely by changes in the levels of the particular enzymes involved in each step of the steroid biosynthetic pathways. Recently, the genes for the various cytochrome P450 species involved in ovarian steroidogenesis, namely cholesterol side-chain cleavage P450 (P450SCC), 17 alpha-hydroxylase P450 (P450(17 alpha], and aromatase cytochrome P450 (P450AROM) have been isolated and characterized, making it possible to study the regulation of expression at the molecular level. To this end, a series of chimeric constructs have been prepared in which fragments of the 5'-untranslated region of bovine P450(17 alpha) and P450SCC have been inserted upstream of the chloramphenicol acetyl transferase (CAT) and beta-globin reporter genes. These constructs have been used to transfect primary cultures of bovine luteal and thecal cells. The results indicate that cAMP responsiveness lies within defined regions of genes which do not contain a classical CRE, similar to previous results utilizing adrenal cells in culture. Furthermore, although constructs containing both the P450(17 alpha) and P450SCC 5'-upstream regions are expressed in both luteal and thecal cell cultures, only those containing the P450SCC sequences are expressed in luteal cells. Studies on the expression of P450AROM indicate that the promoter which is responsible for its expression in human placenta is not operative in the corpus luteum. Thus estrogen biosynthesis may be regulated by the differential use of tissue specific promoters, thus accounting for the complexity and multifactorial nature of the expression of this activity.     

Cloning and expression of genes encoding pheromone-inducible antigens of Enterococcus (Streptococcus) faecalis.

Fragments, generated by restriction enzyme digestion, of the 58-kilobase Enterococcus (Streptococcus) faecalis tetracycline resistance plasmid pCF10 were cloned and introduced into Escherichia coli and E. faecalis to characterize the pheromone-inducible conjugation system encoded by this plasmid. Western blot (immunoblot) analyses revealed that a 130-kilodalton (kDa) antigen, identical to the Tra130 antigen shown previously to be involved in pCF10-mediated pheromone-inducible surface exclusion, was produced by both bacterial hosts carrying the recombinant plasmid pINY1825 (cloned EcoRI C fragment). Both bacterial hosts carrying pINY1825 also produced various amounts of immunologically related 118- to 125-kDa antigens (designated pre-Tra130) that resembled antigens produced by E. faecalis cells carrying pCF10. An additional 150-kDa antigen, Tra150, probably involved in pheromone-induced cellular aggregation, was produced by Escherichia coli and E. faecalis hosts carrying pINY1801 (cloned EcoRI C and E fragments). The coding sequences for the Tra150 and Tra130 antigens were further localized in the TRA region of pCF10 by transposon insertion mutagenesis. Western blot analyses of the recombinant strains, and of strains carrying derivatives of pCF10 or various recombinant plasmids containing Tn5 or Tn917 insertions, suggested that the portion of pCF10 comprising the tra3 through -6 segments (previously defined by Tn917 insertional mutagenesis) contained several genes that are involved in regulating the synthesis of Tra130 and Tra150.     

An effective chromatographic separation of chicken red blood cell coproporphyrinogen oxidase and uroporphyrinogen decarboxylase, two enzymes in heme biosynthesis

Of the heme biosynthetic pathway enzymes, coproporphyrinogen oxidase is one of the least understood. Substrate recognition studies [Prepr. Biochem. Biotech.1997, 27, 47, J. Org. Chem.1999, 64, 464] have been done using chicken blood hemolysates (CBH) as the source of this enzyme. However, the enzyme uroporphyrinogen decarboxylase is also present in these preparations and separation of these two enzymes from CBH had not yet been achieved. Thus, a substrate ligand column was developed by covalently linking coproporphyrin-III to a sepharose resin following a similar procedure previously used for the purification of uroporphyrinogen decarboxylase [Int. J. Biochem.1992, 24, 105]. The ligand-resin chromatography step rapidly separates coproporphyrinogen oxidase from uroporphyrinogen decarboxylase as well as the majority of the hemoglobin.     

Cloning and expression of a cDNA encoding homospermidine synthase from Senecio vulgaris (Asteraceae) in Escherichia coli

The enzyme homospermidine synthase catalyzes the NAD⁺-dependent conversion of 2 mol putrescine into homospermidine. Instead of putrescine, spermidine can substitute for the first putrescine moiety in plants, in which case diaminopropane instead of ammonia is released. The enzyme facilitates the formation of the ‘uncommon’ polyamine homospermidine which is an important precursor in the biosynthesis of pyrrolizidine alkaloids. The first plant homospermidine synthase was purified to apparent chemical homogenity from the root tissue culture Senecio vernalis (Asteraceae) ( Böttcher et al. 1994 , Can. J. Chem. 72, 80–85; Ober 1997 , Dissertation). Four endopeptidase LysC fragments were sequenced from the purified protein. With the aid of degenerate primers against these peptides, a cDNA encoding homospermidine synthase was now cloned and characterized from Senecio vulgaris. The nucleotide sequence of the cloned cDNA revealed an open reading frame of 1155-base pairs containing 385 amino acids with a predicted M_r of 44500. GenBank research revealed that the deduced amino acid sequence shows 59% identity to human deoxyhypusine synthase. The homospermidine synthase encoding cDNA was subcloned into the expression vector pet15b and overexpressed in E. coli. The recombinant enzyme formed upon expression catalyzed homospermidine synthesis.     

Cloning and phylogenetic analysis of the genes encoding acetohydroxyacid synthase from the archaeon Methanococcus aeolicus

The gene for acetohydroxyacid synthase (AHAS) was cloned from the archaeon Methanococcus aeolicus. Contrary to biochemical studies [Xing, R. and Whitman, W.B. (1994) J. Bacteriol. 176, 1207–1213] the enzyme was encoded by two open reading frames (ORFs). Based on sequence homology, these ORFs were designated ilvB and ilvN for the large and small subunits of AHAS, respectively. A putative methanogen promoter preceded ilvB-ilvN, and a potential internal promoter was found upstream of ilvN. ilvB encoded a 65-kDa protein, which agreed well with the measured value for the purified enzyme. ilvN encoded a 19-kDa protein, which fell within the range of M_r of small subunits from other sources. Phylogenetic analysis of the deduced amino acid sequence of ilvB showed a close relationship between the AHAS of Bacteria and Archaea, to the exclusion of other enzymes in this family, including pyruvate oxidase, glyoxylate carboligase, pyruvate decarboxylase, and the acetolactate synthase found in fermentative Bacteria. Thus, this family of enzymes probably arose prior to the divergence of the Bacteria and Archaea. Moreover, the higher plant AHAS and the red algal AHAS were related to the AHAS II of Escherichia coli and the cyanobacterial AHAS, respectively. For this reason, these genes appear to have been acquired by the Eucarya during the endosymbiosis that gave rise to the mitochondrion and chloroplast, respectively. One of the ORFs in the Methanococcus jannaschii genome possesses high similarity to the M. aeolicus ilvB, indicating that it is an authentic AHAS.