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1.
Microb Cell Fact ; 19(1): 118, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32487216

RESUMO

BACKGROUND: Hydrogenobyrinic acid is a key intermediate of the de-novo aerobic biosynthesis pathway of vitamin B12. The introduction of a heterologous de novo vitamin B12 biosynthesis pathway in Escherichia coli offers an alternative approach for its production. Although E. coli avoids major limitations that currently faced by industrial producers of vitamin B12, such as long growth cycles, the insufficient supply of hydrogenobyrinic acid restricts industrial vitamin B12 production. RESULTS: By designing combinatorial ribosomal binding site libraries of the hemABCD genes in vivo, we found that their optimal relative translational initiation rates are 10:1:1:5. The transcriptional coordination of the uroporphyrinogen III biosynthetic module was realized by promoter engineering of the hemABCD operon. Knockdown of competitive heme and siroheme biosynthesis pathways by RBS engineering enhanced the hydrogenobyrinic acid titer to 20.54 and 15.85 mg L-1, respectively. Combined fine-tuning of the heme and siroheme biosynthetic pathways enhanced the hydrogenobyrinic acid titer to 22.57 mg L-1, representing a remarkable increase of 1356.13% compared with the original strain FH215-HBA. CONCLUSIONS: Through multi-level metabolic engineering strategies, we achieved the metabolic balance of the uroporphyrinogen III biosynthesis pathway, eliminated toxicity due to by-product accumulation, and finally achieved a high HBA titer of 22.57 mg L-1 in E. coli. This lays the foundation for high-yield production of vitamin B12 in E. coli and will hopefully accelerate its industrial production.


Assuntos
Proteínas de Escherichia coli , Escherichia coli/metabolismo , Engenharia Metabólica , Uroporfirinas/biossíntese , Vitamina B 12/biossíntese , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Óperon
2.
FEBS J ; 276(21): 6399-411, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19796169

RESUMO

The synthesis of the modified tetrapyrrole known as d(1) haem requires several dedicated proteins which are coded for by a set of genes that are often found adjacent to the structural gene, nirS, for cytochrome cd(1) nitrite reductase. NirE, the product of the first gene in the nir biogenesis operon, was anticipated to catalyse the conversion of uroporphyrinogen III into precorrin-2; this was confirmed, but it was shown that this enzyme is less sensitive to product inhibition than similar enzymes that function in other biosynthetic pathways. Sequence analysis suggesting that one of these proteins, NirN, is a c-type cytochrome, and has similarity to the part of cytochrome cd(1) that binds d(1), was validated by recombinant production and characterization of NirN. A NirN-d(1) haem complex was demonstrated to release the cofactor to a semi-apo form of cytochrome cd(1) from which d(1) was extracted, suggesting a role for NirN in the assembly of cytochrome cd(1) (NirS). However, inactivation of nirN surprisingly led to only a marginal attenuation of growth of Paracoccus pantotrophus under anaerobic denitrifying conditions. As predicted, NirC is a c-type cytochrome; it was shown in vitro to be an electron donor to the NirN-d(1) complex.


Assuntos
Bactérias/metabolismo , Heme/biossíntese , Nitrito Redutases/fisiologia , Proteínas de Transporte de Ânions/fisiologia , Citocromos/fisiologia , Proteínas de Escherichia coli/fisiologia , Heme/análogos & derivados , Paracoccus pantotrophus/genética , Paracoccus pantotrophus/crescimento & desenvolvimento , Uroporfirinogênios/metabolismo , Uroporfirinas/biossíntese
3.
Exp Parasitol ; 118(4): 629-36, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18164705

RESUMO

To further develop the Leishmania model for porphyria based on their deficiencies in heme biosynthesis, three Old World species were doubly transfected as before for Leishmania amazonensis with cDNAs, encoding the 2nd and 3rd enzymes in the pathway. Expression of the transgenes was verified immunologically at the protein level and functionally by uroporphyrin neogenesis that occurs only after exposure of the double-transfectants to delta-aminolevulinate. All species examined were equally deficient in heme biosynthesis, as indicated by the accumulation of uroporphyrin as the sole porphyrin and the production of coproporphyrin upon further transfection of one representative species with the downstream gene. The results obtained thus demonstrate that at least the first five enzymes for heme biosynthesis are absent in all species examined, rendering their transfectants inducible with aminolevulinate to accumulate porphyrins and thus useful as cellular models for human porphyrias.


Assuntos
Ácido Aminolevulínico/farmacologia , Leishmania/metabolismo , Porfirinas/biossíntese , Animais , Western Blotting , Cromatografia em Camada Fina , DNA Complementar/genética , Heme/biossíntese , Heme/genética , Hidroximetilbilano Sintase/metabolismo , Leishmania/enzimologia , Leishmania/genética , Sintase do Porfobilinogênio/metabolismo , Porfirinas/análise , Porfirinas/genética , Espectrometria de Fluorescência , Transfecção , Transgenes/fisiologia , Uroporfirinas/biossíntese , Uroporfirinas/genética
4.
Arch Biochem Biophys ; 439(1): 1-11, 2005 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-15946643

RESUMO

Primary cultures of chick embryo hepatocytes have been used to study the mechanisms by which various drugs and other chemicals cause accumulation of porphyrin intermediates of the heme pathway. When these cultures are incubated with the heme precursor, 5-aminolevulinic acid (ALA), there is a major accumulation of protoporphyrin. However, in the presence of ALA, addition of insulin caused a striking increase in accumulation of uroporphyrin I and coproporphyrin III, whereas addition of glucagon mainly caused an increase in uroporphyrin I. Treatment with both insulin and glucagon resulted in additive increases in uroporphyrin, but not coproporphyrin. Antioxidants abolished the uroporphyrin I accumulation and increased coproporphyrin III. Insulin caused an increase in uptake of ALA and an increase in porphobilinogen accumulation, suggesting that the accumulation of uroporphyrin I is due to increased flux through the heme pathway. Apparently, this increased flux could particularly affect the utilization of the intermediate hydroxymethylbilane, which would result in accumulation of uroporphyrin I.


Assuntos
Ácido Aminolevulínico/metabolismo , Coproporfirinas/biossíntese , Fármacos Gastrointestinais/farmacologia , Glucagon/farmacologia , Hepatócitos/metabolismo , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Uroporfirinas/biossíntese , Ácido Aminolevulínico/farmacologia , Animais , Células Cultivadas , Embrião de Galinha , Hepatócitos/efeitos dos fármacos , Fármacos Fotossensibilizantes/farmacologia
5.
Toxicol Appl Pharmacol ; 189(1): 28-38, 2003 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-12758057

RESUMO

Hepatic uroporphyria can be readily induced by a variety of treatments in mice of the C57BL strains, whereas DBA/2 mice are almost completely resistant. However, feeding of the protoporphyrinogen oxidase-inhibiting herbicide fomesafen (0.25% in the diet for 18 weeks) induced hepatic uroporphyria in male DBA/2N mice (liver porphyrin content up to 150 nmol/g, control animals 1 nmol/g), whereas fomesafen-treated male C57BL/6N mice displayed only a slight elevation of liver porphyrins (approximately 5 nmol/g). The profile of accumulated hepatic porphyrins in fomesafen-treated DBA/2N mice resembled the well-characterised uroporphyria induced by polyhalogenated aromatic hydrocarbons, while histological examination confirmed the presence of uroporphyria-specific cytoplasmic inclusions in the hepatocytes. Uroporphyrinogen decarboxylase activity decreased to about 30% of control values in fomesafen-treated DBA/2N mice; microsomal methoxyresorufin O-dealkylase activity was slightly reduced. The amount of CYP1A1 and CYP1A2 mRNA, as determined by real-time PCR, was not significantly changed; mRNA encoding the housekeeping 5-aminolevulinic acid synthase was elevated 10-fold. Total liver iron was slightly increased. A similar uroporphyria was induced by the herbicide formulation Blazer, containing a structurally related herbicide acifluorfen, when fed to DBA/2N mice at a dose corresponding to 0.25% of acifluorfen in the diet. Since DBA/2 mice are almost completely resistant to all well-characterised porphyrogenic chemicals, the results suggest the possible existence of a yet unknown mechanism of uroporphyria induction, to which the DBA/2 mouse strain is more sensitive than the C57BL strain.


Assuntos
Benzamidas/toxicidade , Herbicidas/toxicidade , Éteres Fenílicos/toxicidade , Porfirias Hepáticas/induzido quimicamente , Uroporfirinas/biossíntese , Animais , Citocromo P-450 CYP1A2/biossíntese , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Reação em Cadeia da Polimerase , Porfirias Hepáticas/enzimologia , Porfirias Hepáticas/metabolismo , Porfirias Hepáticas/patologia , RNA Mensageiro/biossíntese , Especificidade da Espécie , Uroporfirinas/análise
6.
Nat Prod Rep ; 19(4): 390-412, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12195810

RESUMO

Vitamin B12, or cobalamin, is one of the most structurally complex small molecules made in Nature. Major progress has been made over the past decade in understanding how this synthesis is accomplished. This review covers some of the most important findings that have been made and provides the reader with a complete description of the transformation of uroporphyrinogen III into adenosylcobalamin (AdoCbl). 183 references are cited.


Assuntos
Bactérias/metabolismo , Uroporfirinogênios/biossíntese , Uroporfirinogênios/química , Vitamina B 12 , Catálise , Cobalto/metabolismo , Cobamidas/metabolismo , Ciclização , Genes Bacterianos , Metiltransferases/química , Metiltransferases/metabolismo , Estrutura Molecular , Nucleotidiltransferases/metabolismo , Conformação Proteica , Uroporfirinas/biossíntese , Uroporfirinas/química , Vitamina B 12/análogos & derivados , Vitamina B 12/biossíntese , Vitamina B 12/química
7.
Vitam Horm ; 61: 267-97, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-11153269

RESUMO

The manner in which vitamin B12 is synthesized is detailed with emphasis on the different mechanisms for ring contraction encountered in aerobic and anaerobic organisms. The aerobic process utilizes two enzymes and is dependent on molecular oxygen, in stark contrast to the anaerobic mechanism which is controlled by cobalt and requires only one enzyme.


Assuntos
Vitamina B 12/análogos & derivados , Vitamina B 12/biossíntese , Ácido Aminolevulínico/metabolismo , Anaerobiose , Oxigênio/metabolismo , Porfirinas/biossíntese , Porfirinas/metabolismo , Pseudomonas/genética , Salmonella typhimurium/genética , Uroporfirinas/biossíntese , Uroporfirinas/metabolismo , Vitamina B 12/metabolismo
8.
Arch Biochem Biophys ; 384(2): 383-90, 2000 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-11368328

RESUMO

Porphyria cutanea tarda is a liver disease characterized by excess production of uroporphyrin. We previously reported that acetone, an inducer of CYP2E1, enhances hepatic uroporphyrin accumulation in mice treated with iron dextran (Fe) and 5-aminolevulinic acid (ALA). Cyp2e1(-/-) mice treated with Fe and ALA were used to investigate whether CYP2E1 is required for the acetone effect. Hepatic uroporphyrin accumulation was stimulated by acetone in Cyp2e1(-/-) mice to the same extent as in wild-type mice. In the absence of acetone, uroporphyrin accumulated in Cyp2e1(-/-) mice treated with Fe and ALA, but less than in wildtype mice. However, in Cypla2(-/-) mice, uroporphyrin accumulation caused by Fe and ALA, with or without acetone, was completely prevented. Acetone was not an inducer of hepatic CYP1A2 in the wild-type mice. Although acetone is an inducer of CYP2E1, CYP1A2 appears to have the essential role in acetone-enhancement of uroporphyria.


Assuntos
Acetona/farmacologia , Citocromo P-450 CYP1A2/fisiologia , Citocromo P-450 CYP2E1/fisiologia , Uroporfirinas/biossíntese , Ácido Aminolevulínico/farmacologia , Animais , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP2E1/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Ferro/metabolismo , Ferro/farmacologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Oxirredutases/metabolismo , Porfiria Cutânea Tardia/enzimologia , Uroporfirinogênios/metabolismo
9.
Bioorg Med Chem ; 7(10): 2215-9, 1999 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-10579529

RESUMO

The construction of a new recombinant strain of Escherichia coli in which two vitamin B12 biosynthetic genes, cobA and cobI, from Pseudomonas denitrificans are simultaneously overexpressed has resulted in the in vivo synthesis and accumulation of Factor III, an isobacteriochlorin not normally synthesized in E. coli. A lysate of the new strain can take the place of two lysates normally required to provide uroporphyrinogen III methyltransferase (cobA) and precorrin-2 methyltransferase (cobI) in an anaerobic five-enzyme synthesis of the early B12 intermediate, precorrin-3 (the reduced form of Factor III) from delta-aminolevulinic acid.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Genética/métodos , Uroporfirinas/biossíntese , Sequência de Bases , Metiltransferases/genética , Metiltransferases/metabolismo , Dados de Sequência Molecular , Pseudomonas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
10.
Microbiology (Reading) ; 145 ( Pt 3): 529-538, 1999 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10217486

RESUMO

Clusters of genes encoding enzymes for tetrapyrrole biosynthesis were cloned from Bacillus sphaericus, Bacillus stearothermophilus, Brevibacillus brevis and Paenibacillus macerans. The sequences of all hemX genes found, and of a 6.3 kbp hem gene cluster from P. macerans, were determined. The structure of the hem gene clusters was compared to that of other Gram-positive bacteria. The Bacillus and Brevibacillus species have a conserved organization of the genes hemAXCDBL, required for biosynthesis of uroporphyrinogen III (UroIII) from glutamyl-tRNA. In P. macerans, the hem genes for UroIII synthesis are also closely linked but their organization is different: there is no hemX gene and the gene cluster also contains genes, cysG8 and cysG(A)-hemD, encoding the enzymes required for synthesis of sirohaem from UroIII. Bacillus subtilis contains genes for three proteins, NasF, YInD and YInF, with sequence similarity to Escherichia coli CysG, which is a multi-functional protein catalysing sirohaem synthesis from UroIII. It is shown that YInF is required for sirohaem synthesis and probably catalyses the precorrin-2 to sirohaem conversion. YInD probably catalyses precorrin-2 synthesis from UroIII and NasF seems to be specific for nitrite reduction.


Assuntos
Bacillaceae/genética , Proteínas de Bactérias/genética , Genes Bacterianos , Bactérias Gram-Positivas/genética , Pirróis/metabolismo , Sequência de Aminoácidos , Bacillus/genética , Clonagem Molecular , Escherichia coli/genética , Teste de Complementação Genética , Heme/análogos & derivados , Heme/biossíntese , Metiltransferases/genética , Dados de Sequência Molecular , Família Multigênica , Aminoacil-RNA de Transferência/metabolismo , Homologia de Sequência de Aminoácidos , Tetrapirróis , Uroporfirinogênios/biossíntese , Uroporfirinas/biossíntese
11.
Pharmacogenetics ; 8(6): 485-94, 1998 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9918132

RESUMO

Genetic susceptibility to toxic chemicals is of major importance but most studies concentrate on candidate genes and searches for unknown susceptibility genes are uncommon. Human sporadic porphyria cutanea tarda is usually precipitated by alcohol, oestrogens, hepatitis viruses, HIV or haemodialysis. The mechanism is not known but there is a role for iron metabolism and an underlying genetic predisposition is suspected. A similar porphyria in humans has also been caused by hexachlorobenzene. These human porphyrias can be modelled in iron-loaded mice exposed to hexachlorobenzene, in which C57BL/10ScSn is a prototype susceptible strain whereas DBA/2 mice are extremely resistant. A search for susceptibility genes was undertaken using complex trait analysis with DNA microsatellite markers of 'high' and 'low' responders from an F2 intercross. Correlation of markers with susceptibility, defined as accumulation of uroporphyrin in the liver, was assessed by chi-squared test for the proportion of C57BL/10ScSn and DBA/2 alleles present. Susceptibility loci on chromosomes 12, 14 and 17 were identified. Further analysis of markers on chromosomes 14 and 17 by MAPMAKER/EXP and MAPMAKER/QTL gave LOD scores of 7.3 and 3.6, respectively. Typing of chromosome 12 for the Ahr gene, using a restriction fragment length polymorphism distinguishing between the b-1 and d alleles, gave significant but not perfect linkage. However, no strong association between alleles or expression of Cyp1a1/2 genes, regulated by Ahr, and susceptibility for porphyria was detected. The results demonstrate that the porphyria induced by hexachlorobenzene in C57BL/10ScSn mice is a complex trait determined by at least three genes, which may be of relevance to susceptibility in the development of sporadic porphyria cutanea tarda and unknown aspects of liver damage.


Assuntos
Mapeamento Cromossômico , Ligação Genética , Hexaclorobenzeno/toxicidade , Ferro/toxicidade , Porfiria Cutânea Tardia/genética , Animais , Sequência de Bases , Primers do DNA , Sinergismo Farmacológico , Marcadores Genéticos , Predisposição Genética para Doença , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Porfiria Cutânea Tardia/induzido quimicamente , Porfiria Cutânea Tardia/metabolismo , Especificidade da Espécie , Uroporfirinas/biossíntese , Uroporfirinas/metabolismo
12.
Chem Biol ; 4(9): 659-66, 1997 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9331403

RESUMO

BACKGROUND: During the biosynthesis of vitamin B12, the aerobic bacterium Pseudomonas denitrificans uses two enzymes, CobG and CobJ, to convert precorrin-3 to the ring-contracted intermediate, precorrin-4. CobG is a monooxygenase that adds a hydroxyl group, derived from molecular oxygen, to C-20, whereas CobJ is bifunctional, inserting a methyl group at C-17 of the macrocycle and catalyzing ring contraction. Molecular oxygen is not available to vitamin B12-producing anaerobic bacteria and members of the ancient Archaea, so the question arises of how these microbes accomplish the key ring-contraction process. RESULTS: Cloning and overexpression of Salmonella typhimurium genes has led to the discovery that a single enzyme, CbiH, is responsible for ring contraction during anaerobic biosynthesis of vitamin B12. The process occurs when CbiH is incubated with precorrin-3, but only in the presence of cobalt. CbiH functions as a C-17 methyltransferase and mediates ring contraction and lactonization to yield the intermediate, cobalt-precorrin-4, isolated as cobalt-factor IV. 13C labeling studies have proved that cobalt-precorrin-4 is incorporated into cobyrinic acid, thereby confirming that cobalt-precorrin-4 is an intermediate in vitamin B12 biosynthesis. CONCLUSIONS: Two distinct mechanisms exist in nature for the ring contraction of porphyrinoids to corrinoids-an ancient anaerobic pathway that requires cobalt complexation prior to nonoxidative rearrangement, and a more recent aerobic route in which molecular oxygen serves as the cofactor. The present results offer a rationale for the main differences between aerobic and anaerobic biosynthesis of vitamin B12. Thus, in anaerobes there is exchange of oxygen at the C-27 acetate site, extrusion of acetaldehyde and early insertion of cobalt, whereas the aerobes show no exchange of oxygen at C-27, extrude acetic acid and insert cobalt very late in the biosynthetic pathway, after ring contraction has occurred. These parallel routes to vitamin B12 have now been clearly distinguished by their differing mechanisms for ring contraction.


Assuntos
Oxigênio/metabolismo , Porfirinas/biossíntese , Vitamina B 12/biossíntese , Anaerobiose , Cobalto/metabolismo , Corrinoides , Metilação , Metiltransferases/metabolismo , Conformação Molecular , Porfirinas/metabolismo , Pseudomonas/enzimologia , Pseudomonas/metabolismo , Frações Subcelulares/metabolismo , Uroporfirinas/biossíntese , Uroporfirinas/metabolismo , Vitamina B 12/metabolismo
13.
Xenobiotica ; 27(7): 681-92, 1997 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-9253145

RESUMO

1. The effect of Irgasan DP 300 (5-chloro-2-(2,4-dichlorophenoxy)phenol) on cytochrome P450 (P450) induction and haem biosynthesis was studied in rat hepatocytes cultured on Matrigel. 2. Irgasan DP 300 significantly induced 7-benzyloxyresorufin O-debenzylase activity, followed by 7-pentoxyresorufin O-depentylase and 7-ethoxyresorufin O-deethylase activities. 4-Nitrophenol hydroxylase, testosterone 6 beta-hydroxylase and methoxyresorufin O-demethylase activities were also slightly increased. The maximum induction of these enzyme activities was obtained at the same concentration of 125 microM in the culture medium. 3. Immunochemical blots using anti-rat cytochrome P450 antibodies revealed that Irgasan DP 300 preferably induced CYP2B1/2 along with a slight increase in 3A. These results indicate that Irgasan DP 300 is a phenobarbital-type inducer. 4. In the absence of exogenous 5-aminolevulinic acid (ALA), slight increases in protoporphyrin IX (2.6-fold) and coproporphyrin III (1.3-fold) were observed in the Irgasan DP 300-treated cultures. In contrast, when 75 microM ALA was present, Irgasan DP 300 (250 microM) caused an extensive accumulation of uroporphyrin I (13-fold). 5. Irgasan DP 300 inhibited rat hepatic uroporphyrinogen III synthase in vitro. 6. These results indicate that Irgasan DP 300 produced accumulation of hydroxymethylbilane in rat hepatocytes by inhibiting uroporphyrinogen III synthase, and consequently an accumulation of uroporphyrin I.


Assuntos
Anti-Infecciosos Locais/farmacologia , Carbanilidas/farmacologia , Sistema Enzimático do Citocromo P-450/biossíntese , Heme/antagonistas & inibidores , Fígado/metabolismo , Ácido Aminolevulínico/farmacologia , Animais , Carbanilidas/química , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Colágeno/fisiologia , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Combinação de Medicamentos , Indução Enzimática/efeitos dos fármacos , Matriz Extracelular/fisiologia , Heme/biossíntese , Laminina/fisiologia , Fígado/citologia , Fígado/efeitos dos fármacos , Masculino , Proteoglicanas/fisiologia , Ratos , Ratos Wistar , Uroporfirinogênio III Sintetase/antagonistas & inibidores , Uroporfirinogênios/biossíntese , Uroporfirinas/biossíntese
14.
Chem Biol ; 4(6): 445-51, 1997 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9224567

RESUMO

BACKGROUND: In order to study the biosynthesis of vitamin B12, it is necessary to produce various intermediates along the biosynthetic pathway by enzymic methods. Recently, information on the organisation of the biosynthetic pathway has permitted the selection of the set of enzymes needed to biosynthesise any specific identified intermediate. The aim of the present work was to use recombinant enzymes in reconstituted multi-enzyme systems to biosynthesise particular intermediates. RESULTS: The products of the cobG and cobJ genes from Pseudomonas denitrificans were expressed heterologously in Escherichia coli to afford good levels of activity of the corresponding enzymes, CobG and CobJ. Aerobic incubation of precorrin-3A with the CobG enzyme alone yielded precorrin-3B. When CobJ and S-adenosyl-L-methionine were included in the incubation, the product was precorrin-4. Both precorrin 3B and precorrin-4 are known precursors of vitamin B12 and their availability has allowed new mechanistic studies of enzymic transformations. CONCLUSIONS: Our results show that the expression of the CobG and CobJ enzymes has been successful, thus facilitating the biosynthesis of two precursors of vitamin B12. This lays the foundation for the structure determination of CobG and CobJ as well as future enzymic experiments focusing on later steps of vitamin B12 biosynthesis.


Assuntos
Proteínas de Bactérias , Metiltransferases/metabolismo , Complexos Multienzimáticos/metabolismo , Oxigenases/metabolismo , Pseudomonas/enzimologia , Uroporfirinas/biossíntese , Vitamina B 12/biossíntese , Clonagem Molecular , Escherichia coli/genética , Expressão Gênica , Genes Bacterianos , Metiltransferases/genética , Estrutura Molecular , Complexos Multienzimáticos/genética , Mutagênese Sítio-Dirigida/genética , Oxigenases/genética , Plasmídeos , Proteínas Recombinantes/metabolismo , Uroporfirinas/genética , Uroporfirinas/metabolismo
15.
Toxicol Appl Pharmacol ; 147(2): 171-9, 1997 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9439713

RESUMO

Uroporphyrin (URO) accumulation occurs in chick embryo hepatocytes treated with a number of polyhalogenated aromatic hydrocarbons (PHAHs) that are known inducers of cytochrome P4501As (CYP1A). Previous dose response studies had shown that URO accumulation does not begin until CYP1A, as indicated by ethoxyresorufin O-deethylase (EROD) activity, is maximally induced. The reason why the concentrations of PHAHs required for URO accumulation were higher than those required to induce EROD had not been explained. PHAHs, such as 3,3',4,4'-tetrachlorobiphenyl (PCB77, IUPAC nomenclature, TCB) stimulate uroporphyrinogen (UROGEN) oxidation by microsomes from 3-methylcholanthrene (MC)-treated chick embryos. Here we used a new protocol to investigate whether the requirement for more TCB to stimulate in vitro microsomal UROGEN oxidation extended to TCB-induced URO accumulation in intact cultured hepatocytes. Cultures were treated with increasing concentrations of TCB or other PHAHs to induce CYP1As, then with cycloheximide (CX) to prevent further P450 synthesis. The CX treatment was shown to block any further increases in CYP1A as determined by immunoblots. 5-Aminolevulinic acid and a high concentration of TCB ("postinduction TCB") were then added to stimulate intracellular UROGEN oxidation. Using the protocol with postinduction TCB, the inducing concentrations of TCB which caused URO to begin to accumulate were now much lower than in the absence of postinduction TCB. Increases in CYP1A proteins, measured immunochemically, were detected at about the same inducing TCB concentrations that began to increase URO accumulation. The new protocol, with postinduction TCB, using URO accumulation as the end point, greatly increased the sensitivity of the culture system for detection of PHAHs with EC50s (nM) for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), TCB, 3,3',4,4',5,5'-hexachlorobiphenyl, MC, and hexachlorobenzene being about 0.003, 0.11, 0.75, 3.5, and 30, respectively. As little as 2-4 fmol TCDD per culture dish caused detectible increases in URO accumulation. We conclude that URO accumulation in chick hepatocyte cultures is limited not only by the induction of CYP1A, but also by the stimulation of intracellular UROGEN oxidation.


Assuntos
Citocromo P-450 CYP1A1/biossíntese , Fígado/efeitos dos fármacos , Bifenilos Policlorados/farmacologia , Dibenzodioxinas Policloradas/farmacologia , Uroporfirinas/biossíntese , Animais , Células Cultivadas , Embrião de Galinha , Galinhas , Indução Enzimática , Fígado/metabolismo , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo
16.
Bioorg Med Chem ; 4(12): 2179-85, 1996 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9022980

RESUMO

The problems inherent in the enzymatic and chemical synthesis of S-adenosyl-L-methionine (SAM) led us to develop an efficient, simple method for the synthesis of large amounts of labeled SAM. Previously, we reported that the problem of product inhibition of E. coli SAM synthetase encoded by the metK gene was successfully overcome in the presence of sodium p-toluenesulfonate (pTsONa). This research has now been expanded to demonstrate that product inhibition of this enzyme can also be overcome by adding a high concentration of beta-mercaptoethanol (beta ME), acetonitrile, or urea. In addition a recombinant strain of E. coli has been constructed that expresses the yeast SAM synthetase encoded by the sam2 gene. The yeast enzyme does not have the problem of product inhibition seen with the E. coli enzyme. Complete conversion of 10 mM methionine to SAM was achieved in incubations with either the recombinant yeast enzyme and 1 molar potassium ion or the E. coli enzyme in the presence of additives such as beta ME, acetonitrile, urea, or pTsONa. The recombinant yeast SAM synthetase was used to generate SAM in situ for use in the multi-enzymatic synthesis of precorrin 2.


Assuntos
Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismo , S-Adenosilmetionina/biossíntese , Escherichia coli/genética , Escherichia coli/metabolismo , Microbiologia Industrial/métodos , Metionina Adenosiltransferase/efeitos dos fármacos , Desnaturação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sais , Solventes , Uroporfirinas/biossíntese , Leveduras/enzimologia , Leveduras/genética
17.
Arch Biochem Biophys ; 329(2): 163-74, 1996 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-8638948

RESUMO

Hepatic uroporphyria is a well-known effect of halo- genated aromatic hydrocarbons in mammalian and avian systems, including primary cell cultures, but attempts to produce uroporphyria in vertebrate (mammalian) hepatoma lines have been unsuccessful. In this study, the ability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), and selected chlorobiphenyl congeners to cause uroporphyria was examined in a fish hepatoma cell line (PLHC-1) that expresses aryl hydrocarbon (Ah) receptors and an inducible cytochrome P4501A (CYP1A). Dose-dependent accumulation of porphyrins was observed in cells treated for 48 h with TCDD or 3,3',4,4'-tetrachlorobiphenyl (3,3',4,4'-TCB; IUPAC 77) when the heme precursor delta-aminolevulinic acid (ALA) was present during the last 5 h of treatment. HPLC analysis identified the porphyrins as uroporphyrin (approximately 80%) and heptacarboxylporphyrin (approximately 20%). Uroporphyria did not occur in cells treated with TCDD or 3,3',4,4'-TCB in the absence of added ALA. ALA-dependent porphyrin accumulation was also seen following treatment of PLHC-1 cells with TCDF or with the non-ortho-substituted chlorobiphenyls 3,4,4',5-tetrachlorobiphenyl (IUPAC 81) and 3,3',4,4',5-pentachlorobiphenyl (IUPAC 126). Neither of the mono-ortho-substituted chlorobiphenyls 2,3,3',4,4'-pentachlorobiphenyl (IUPAC 105) or 2,3',4,4'5-pentachlorobiphenyl (IUPAC 118) increased the porphyrin content of PLHC-1 cells. The ability of the PCB congeners to cause porphyria correlated with their ability to induce the CYP1A catalytic activity ethoxyresorufin 0-deethylase (EROD) and immunodetectable CYP1A protein in these cells, suggesting direct or indirect regulation of porphyrin accumulation via the Ah receptor and/or the induced CYP1A. Induction of EROD activity by TCDD, TCDF, and the planar polychlorinated biphenyls was biphasic, with increases at lower concentrations of inducer followed by decreased induction at higher concentrations, as seen previously. EC50 values for porphyrin accumulation were similar to, or slightly higher than, the concentrations at which peak EROD activities were obtained, suggesting a relationship between the decline in EROD activity and enhanced porphyrin accumulation. alpha-Naphthoflavone inhibited TCDD-induced EROD activity and porphyrin accumulation, providing further evidence for the involvement of a fish CYP1A in the mechanism of this prophyria. Addition of 3,3',4,4'-TCB to TCDD-treated cells also inhibited EROD activity, but enhanced porphyrin accumulation, suggesting that an interaction between the halogenated inducer and the induced CYP1A is necessary for the porphyrogenic response. PLHC-1 cells grown in medium supplemented with ALA may be a useful model System for studying mechanisms of chemical uroporphyria induced by Ah receptor agonists.


Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , Peixes/metabolismo , Hidrocarbonetos Clorados/farmacologia , Fígado/metabolismo , Oxirredutases/biossíntese , Receptores de Hidrocarboneto Arílico/agonistas , Uroporfirinas/biossíntese , Animais , Carcinoma Hepatocelular/metabolismo , Citocromo P-450 CYP1A1 , Indução Enzimática , Fígado/citologia , Neoplasias Hepáticas/metabolismo , Bifenilos Policlorados/farmacologia , Dibenzodioxinas Policloradas/farmacologia , Relação Estrutura-Atividade , Células Tumorais Cultivadas
18.
Biochem J ; 313 ( Pt 1): 335-42, 1996 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-8546704

RESUMO

The Bacillus subtilis genes hemB, hemC and hemD, encoding respectively the enzymes porphobilinogen synthase, hydroxymethylbilane synthase and uroporphyrinogen III synthase, have been expressed in Escherichia coli using a single plasmid construct. An enzyme preparation from this source converts 5-aminolaevulinic acid (ALA) preparatively and in high yield into uroporphyrinogen III. The Pseudomonas denitrificans genes cobA and cobI, encoding respectively the enzymes S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT) and S-adenosyl-L-methionine:precorrin-2 methyltransferase (SP2MT), were also expressed in E. coli. When SUMT was combined with the coupled-enzyme system that produces uroporphyrinogen III, precorrin-2 was synthesized from ALA, and when SP2MT was also added the product from the coupling of five enzymes was precorrin-3A. Both of these products are precursors of vitamin B12, and they can be used directly for biosynthetic experiments or isolated as their didehydro octamethyl esters in > 40% overall yield. The enzyme system which produces precorrin-3A is sufficiently stable to allow long incubations on a large scale, affording substantial quantities (15-20 mg) of product.


Assuntos
Uroporfirinas/biossíntese , Vitamina B 12/biossíntese , Bacillus subtilis/enzimologia , Sequência de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Hidroximetilbilano Sintase/genética , Hidroximetilbilano Sintase/metabolismo , Dados de Sequência Molecular , Sintase do Porfobilinogênio/genética , Sintase do Porfobilinogênio/metabolismo , Uroporfirinogênio III Sintetase/genética , Uroporfirinogênio III Sintetase/metabolismo , Uroporfirinogênios/metabolismo
19.
Chin Med J (Engl) ; 108(4): 286-90, 1995 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-7789217

RESUMO

After intraperitoneal (IP) injection of delta-aminolevulinic acid (ALA), the endogenous porphyrins in murine skin and tumor tissues were determined by a method involving solvent and acid extractions. The results showed that the total amount of porphyrins in the tumor tissues after ALA injection was much higher than that in the skin from the same mice, although the amount of porphyrins in the skin from the ALA-injected mice was higher than that from the saline-injected (control) mice. The porphyrins in the tumor were mostly protoporphyrin and coproporphyrin, with only a small amount of uroporphyrin. The optimum period for porphyrin accumulation in the tumor as well as in the skin was 1 hour after the injection of ALA. As the period was extended to 3 and 6 hours, the amount of porphyrins in these tissues decreased considerably. These findings could be valuable for further application of ALA in the photodynamic therapy of skin cancer.


Assuntos
Ácido Aminolevulínico/farmacologia , Carcinoma de Células Escamosas/metabolismo , Porfirinas/biossíntese , Pele/metabolismo , Animais , Carcinoma de Células Escamosas/patologia , Coproporfirinas/biossíntese , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Fotoquimioterapia , Protoporfirinas/biossíntese , Células Tumorais Cultivadas , Uroporfirinas/biossíntese
20.
Protein Expr Purif ; 6(2): 155-63, 1995 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-7606163
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