Microbiological synthesis of tetrapyrrole compounds

This paper reviews publications on the biosynthesis of functional tetrapyrroles by microorganisms. Emphasis is given to the structure of uroporphyrin III methylated derivatives termed corriphyrins and their involvement in the formation of two groups of tetrapyrrole pigments--corrinoids and siroheme. Current concepts concerning the final stages of the formation of the corrine ring and potential cobalt-free precursors of vitamin B12 are discussed. It is indicated that the data available may help elucidate evolutionary and biogenetic patterns in the emergence and interaction of tetrapyrrole compounds and formulate problems of practical importance.     

A gene, cobA + hemD, from Selenomonas ruminantium encodes a bifunctional enzyme involved in the synthesis of vitamin B12.

Coenzymes derived from vitamin B12 (cyanocobalamin) are particularly important for core metabolism in ruminant animals. Selenomonas ruminantium, a Gram-positive obligate anaerobe isolated from cattle, is the main contributor of vitamin B12 to such ruminant animals. In nature, there are both aerobic and anaerobic pathways for B12 synthesis - the latter is only partly elucidated. Until now, there has been no investigation of B12 synthesis in S. ruminantium, which must use an anaerobic pathway. This paper reports the cloning of the chromosomal operon from S. ruminantium which is responsible for the first committed steps in corrinoid synthesis. Five open reading frames were found in the cloned fragment. All deduced amino acid sequences had similarity to defined proteins in the databases that are involved in porphyrin and corrin synthesis. Of particular interest is the gene designated cobA + hemD, which encodes a single polypeptide possessing two catalytic functions - uroporphyrinogen III synthase and uroporphyrinogen III 2,7-methyltransferase. This enzyme converts hydroxymethylbilane to precorrin-2. The functions of the protein coded by cobA + hemD were established by heterologous expression in Escherichia coli. The CobA activity has been demonstrated for three distinct types of proteins - monofunctional, bifunctional with siroheme formation and, this report, bifunctional with uroporphyrinogen III synthesis. The type found in S. ruminantium (cobA + hemD) is probably restricted to obligately anaerobic fermentative bacteria.     

Genetic engineering of Escherichia coli for the production of precorrin-3 in vivo and in vitro

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.     

Identification and characterization of a novel vitamin B12 (cobalamin) biosynthetic enzyme (CobZ) from Rhodobacter capsulatus, containing flavin, heme, and Fe-S cofactors

One of the most intriguing steps during cobalamin (vitamin B12) biosynthesis is the ring contraction process that leads to the extrusion of one of the integral macrocyclic carbon atoms from the tetrapyrrole-derived framework. The aerobic cobalamin pathway requires the action of a monooxygenase called CobG (precorrin-3B synthase), which generates a hydroxylactone intermediate that is subsequently ring-contracted by CobJ. However, in the photosynthetic bacterium Rhodobacter capsulatus, which harbors an aerobic-like pathway, there is no cobG in the main cobalamin biosynthetic operon although it does contain an additional uncharacterized gene called orf663. To demonstrate the involvement of Orf663 in cobalamin synthesis, the first dedicated 10 genes of the B12 pathway (including orf663), encoding enzymes for the transformation of uroporphyrinogen III into hydrogenobyrinic acid (HBA), were sequentially cloned into a plasmid to generate an artificial operon, which, when transformed into Escherichia coli, endowed the host with the ability to make HBA. Deletion of orf663 from this operon prevented HBA synthesis, demonstrating that it was essential for corrin construction. HBA synthesis was restored to this recombinant strain either by returning orf663 or by substituting it with cobG. Recombinant overproduction of Orf663, now renamed CobZ, allowed the characterization of a novel cofactor-rich protein, housing two Fe-S centers, a flavin, and a heme group, which like B12 itself is a modified tetrapyrrole. A mechanism for Orf663 (CobZ) in cobalamin biosynthesis is proposed.     

Effect of nucleotide containing corrinoids on vitamin B 12 synthesis in Propionibacterium shermanii]

The influence of B12-CN, B12-OH, coenzyme B12, factor III and factor B on the synthesis of vitamin B12 and porphyrins by different strains of P. shermanii was investigated. Neither compound inhibited the development of propionic bacteria or suppressed porphyrin formation. All nucleotide containing analogues of vitamin B12 produced a strong repressive effect on the synthesis of corrinoid compounds regardless of the modifications in the upper and lower cobalt ligands. Factor B containing no nucleotide moiety did not show this effect. It is suggested that the nucleotide moiety of the vitamin B12 molecule is responsible for the binding of vitamin to protein aporepressor.     

A Clostridium perfringens hem gene cluster contains a cysG(B) homologue that is involved in cobalamin biosynthesis

The hem gene cluster, which consists of hemA, cysG(B), hemC, hemD, hemB, and hemL genes, and encodes enzymes involved in the biosynthetic pathway from glutamyl-tRNA to uroporphyrinogen III, has been identified by the cloning and sequencing of two overlapping DNA fragments from Clostridium perfringens NCTC8237. The deduced amino acid sequence of the N-terminal region of C. perfringens HemD is homologous to those reported for the C-terminal region of Salmonella typhimurium CysG and Clostridium josui HemD. C. perfringens CysG(B) is a predicted 220-residue protein which shows homology to the N-terminal region of S. typhimurium CysG. Disruption of the cysG(B) gene in C. perfringens strain 13 by homologous recombination reduced cobalamin (vitamin B12) levels by a factor of 200. When grown in vitamin B12-deficient medium, the mutant strain showed a four-fold increase in its doubling time compared with that of the wild-type strain, and this effect was counteracted by supplementing the medium with vitamin B12. These results suggest that C. perfringens CysG(B) is involved in the chelation of cobalt to precorrin II as suggested for the CysG(B) domain of S. typhimurium CysG, enabling the synthesis of cobalamin.     

Recent advances in microbial production of δ-aminolevulinic acid and vitamin B12

δ-aminolevulinate (ALA) is an important intermediate involved in tetrapyrrole synthesis (precursor for vitamin B₁₂, chlorophyll and heme) in vivo. It has been widely applied in agriculture and medicine. On account of many disadvantages of its chemical synthesis, microbial production of ALA has been received much attention as an alternative because of less expensive raw materials, low pollution, and high productivity. Vitamin B₁₂, one of ALA derivatives, which plays a vital role in prevention of anaemia has also attracted intensive works. In this review, recent advances on the production of ALA and vitamin B₁₂ with novel approaches such as whole-cell enzyme-transformation and metabolic engineering are described. Furthermore, the direction for future research and perspective are also summarized.     

Dependence of betaine stimulation of vitamin b(12) overproduction on protein synthesis

The betaine-stimulated differential synthesis of vitamin B(12), i.e., the increase in B(12) per increase in dry cell weight, by Pseudomonas denitrificans was inhibited by rifampin and chloramphenicol but not by benzylpenicillin and carbenicillin at concentrations of antibiotic that inhibit growth. The level of the first enzyme of corrin (and porphyrin) biosynthesis, delta-aminolevulinic acid synthetase, was decreased to a much greater degree by rifampin and chloramphenicol than by the penicillins. These data support the concept that betaine stimulation of B(12) synthesis is a result of its stimulation of synthesis of delta-aminolevulinic acid synthetase, a labile and presumably rate-limiting enzyme of corrin formation requiring continuous induction. In further support of this hypothesis, it was found that chloramphenicol immediately interfered with both vitamin B(12) and delta-aminolevulinic acid synthetase formation, no matter when it was added to the system.     

Role of cobalt in the biosynthesis of the components of the gentamicin complex

It was shown that addition of cobalt ions or vitamin B12 to the fermentation medium resulted in an increase in the level of gentamicin accumulation, the relative content of the most methylated components C1 and C2 in the gentamicin complex being increased, while the content of the least methylated components CIa and "minors" decreased. Addition of sulphodimezine lowered the gentamicin biosynthesis rate and the relative content of gentamicins C1 and C2. It was supposed that cobalt stimulated the B12-dependent synthesis of methionine, being the source of the methyl groups for biosynthesis of the methylated components of gentamicin complex.     

Altered cobalamin metabolism in Escherichia coli btuR mutants affects btuB gene regulation.

Synthesis of the Escherichia coli outer membrane protein BtuB, which mediates the binding and transport of vitamin B12, is repressed when cells are grown in the presence of vitamin B12. Expression of btuB-lacZ fusions was also found to be repressed, and selection for constitutive production of beta-galactosidase in the presence of vitamin B12 yielded mutations at btuR. The btuR locus, at 27.9 min on the chromosome map, was isolated on a 952-base-pair EcoRV fragment, and its nucleotide sequence was determined. The BtuR protein was identified in maxicells as a 22,000-dalton polypeptide, as predicted from the nucleotide sequence. Strains mutant at btuR had negligible pools of adenosylcobalamin but did convert vitamin B12 into other derivatives. Although btuB expression in a btuR strain could not be repressed by cyano- or methylcobalamin, it was repressed by adenosylcobalamin. Growth on ethanolamine as the sole nitrogen source requires adenosylcobalamin. btuR mutants grew on ethanolamine but were affected in the length of the lag period before initiation of growth, which suggested that an alternative route for adenosylcobalamin synthesis might exist. No mutations were found that conferred constitutive btuB expression in the presence of adenosylcobalamin. Other genes near btuR may also be involved in cobalamin metabolism, as suggested from the complementation behavior of strains generated by excision of the Tn10 element in btuR. These results indicated that the btuR product is involved in the metabolism of adenosylcobalamin and that this cofactor, or some derivative, controls btuB expression.     

Biosynthesis of corrinoids and other tetrapyrrole compounds by an acetogenic Clostridium

Biosynthesis of corrinoids and other tetrapyrrole pigments by the pure culture of the acetogenic Clostridium 99 was studied. When growing on media containing glucose or methanol, the physiological and biochemical characteristics of Clostridium 99 are very close to those of C. thermoautotrophicum. Methanol was shown to stimulate the corrinoid accumulation with the yield increasing from 154 micrograms/g dry biomass (glucose medium) up to 2250 micrograms/g dry biomass (methanol medium). According to the paper chromatography the corrinoid accumulated in Clostridium 99 cells differed both from vitamin B12 and Factor III. A study on the composition of extracellular tetrapyrroles, accumulated when the culture grows on the medium containing glucose and delta-aminolevulinic acid, revealed that they are represented both by uroporphyrin III and sirohydrochlorine-like pigments. The latters differ by a number of properties from sirohydrochlorine (corrifirine-2) of propione acidic bacteria. These pigments appear to be involved as intermediants in biosynthesis of corrinoids and other tetrapyrroles.     

Folic acid fortification: why not vitamin B12 also?

Folic acid fortification of cereal grains was introduced in many countries to prevent neural tube defect occurrence. The metabolism of folic acid and vitamin B12 intersect during the transfer of the methyl group from 5-methyltetrahydrofolate to homocysteine catalyzed by B12-dependent methioine synthase. Regeneration of tetrahydrofolate via this reaction makes it available for synthesis of nucleotide precursors. Thus either folate or vitamin B12 deficiency can result in impaired cell division and anemia. Exposure to extra folic acid through fortification may be detrimental to those with vitamin B12 deficiency. Among participants of National Health And Nutrition Examination Survey with low vitamin B12 status, high serum folate (>59 nmol/L) was associated with higher prevalence of anemia and cognitive impairment when compared with normal serum folate. We also observed an increase in the plasma concentrations of total homocysteine and methylmalonic acid (MMA), two functional indicators of vitamin B12 status, with increase in plasma folate under low vitamin B12 status. These data strongly imply that high plasma folate is associated with the exacerbation of both the biochemical and clinical status of vitamin B12 deficiency. Hence any food fortification policy that includes folic acid should also include vitamin B12.     

The reaction of HOCl and cyanocobalamin: corrin destruction and the liberation of cyanogen chloride

Overproduction of hypochlorous acid (HOCl) has been associated with the development of a variety of disorders such as inflammation, heart disease, pulmonary fibrosis, and cancer through its ability to modify various biomolecules. HOCl is a potent oxidant generated by the myeloperoxidase-hydrogen peroxide-chloride system. Recently, we have provided evidence to support the important link between higher levels of HOCl and heme destruction and free iron release from hemoglobin and RBCs. Our current findings extend this work and show the ability of HOCl to mediate the destruction of metal-ion derivatives of tetrapyrrole macrocyclic rings, such as cyanocobalamin (Cobl), a common pharmacological form of vitamin B12. Cyanocobalamin is a water-soluble vitamin that plays an essential role as an enzyme cofactor and antioxidant, modulating nucleic acid metabolism and gene regulation. It is widely used as a therapeutic agent and supplement, because of its efficacy and stability. In this report, we demonstrate that although Cobl can be an excellent antioxidant, exposure to high levels of HOCl can overcome the beneficial effects of Cobl and generate proinflammatory reaction products. Our rapid kinetic, HPLC, and mass spectrometric analyses showed that HOCl can mediate corrin ring destruction and liberate cyanogen chloride (CNCl) through a mechanism that initially involves α-axial ligand replacement in Cobl to form a chlorinated derivative, hydrolysis, and cleavage of the phosphonucleotide moiety. Additionally, it can liberate free Co, which can perpetuate metal-ion-induced oxidant stress. Taken together, these results are the first report of the generation of toxic molecular products through the interaction of Cobl with HOCl.     

EFFECT OF THE ANTIBIOTIC,CYCLOHEXIMIDE, ON THE METABOLISM AND GROWTH OF SACCHAROMYCES PASTORIANUS

Coursen, B. W., and H. D. Sisler (U. Maryland, College Park.) Effect of the antibiotic, cycloheximide, on the metabolism and growth of Saccharomyces pastorianus. Amer. Jour. Bot. 47(7): 541–549. Illus. 1960.—Studies were made of the toxicity of cycloheximide and certain of its derivatives to Saccharomyces pastorianus Hansen. The ED₅₀ values for cycloheximide, its semicarbazone derivative and its oxime derivative are 0.018, 0.37, and 12.0.p.p.m., respectively. In auxanographic and liquid culture tests involving 160 organic and biochemicals, only certain methylated ring ketones and vitamin A alcohol or acetate showed appreciable antagonistic activity to the toxicity of cycloheximide. Yeast cells exposed to 3.16 p.p.m. of cycloheximide and incubated for 30 min. with uniformly labeled ¹⁴C glucose remove about 10% less activity from the medium than untreated cells. Measurements of radioactivity in compounds extracted from cells with 80% ethanol showed the presence of appreciable activity in the glutamine from untreated cells but no measurable activity in this compound from treated cells. Activity in glutamic acid from treated cells was reduced while activity in alanine and aspartic acid was increased when compared with the activity in these compounds from untreated cells. There were other differences, also, especially in the levels of activity in organic phosphorus compounds, but, in many cases, the activity in compounds from treated cells was similar to that in the corresponding compounds from untreated cells. It is possible that the antibiotic interferes with the metals involved in the enzymatic reaction leading to the synthesis of glutamic acid and glutamine or it may act as an inhibitory analog in the synthesis of these or similar compounds. The apparent interference of cycloheximide with the formation of a CO-NH bond in the synthesis of glutamine suggests also that peptide bond formation in protein synthesis may be similarly affected. A block of glutamine synthesis by cycloheximide may be sufficient to account for the toxicily of the antibiotic, but the failure of exogenous sources of glutamine to reverse the toxicity indicates that other reactions in cell metabolism may be as sensitive to cycloheximide as the synthesis of glutamine.     

Identification and sequence analysis of genes involved in late steps in cobalamin (vitamin B12) synthesis in Rhodobacter capsulatus.

A 6.4-kb region of a 6.8-kb BamHI fragment carrying Rhodobacter capsulatus genes involved in late steps of cobalamin synthesis has been sequenced. The nucleotide sequence and genetic analysis revealed that this fragment contains eight genes arranged in at least three operons. Five of these eight genes show homology to genes involved in the cobalamin synthesis of Pseudomonas denitrificans and Salmonella typhimurium. The arrangement of these homologous genes differs considerably in the three genera. Upstream of five overlapping genes (named bluFEDCB), a promoter activity could be detected by using lacZ fusions. This promoter shows no regulation by oxygen, vitamin B12 (cobalamin), or cobinamide. Disruption of the bluE gene by a Tn5 insertion (strain AH2) results in reduced expression of the puf and puc operons, which encode pigment-binding proteins of the photosynthetic apparatus. The mutant strain AH2 can be corrected to a wild-type-like phenotype by addition of vitamin B12 or cobinamide dicyanide. Disruption of the bluB gene by an interposon (strain BB1) also disturbs the formation of the photosynthetic apparatus. The mutation of strain BB1 can be corrected by vitamin B12 but not by cobinamide. We propose that a lack of cobalamin results in deregulation and a decreased formation of the photosynthetic apparatus.     

Specificity of the heme requirement for growth of Bacteroides ruminicola

Caldwell, D. R. (U.S. Department of Agriculture, Beltsville, Md.), D. C. White, M. P. Bryant, and R. N. Doetsch. Specificity of the heme requirement for growth of Bacteroides ruminicola. J. Bacteriol. 90:1645-1654. 1965.-Previous studies suggested that most strains of Bacteroides ruminicola subsp. ruminicola require heme for growth. Present studies with heme-requiring strain 23 showed that protoheme was replaced by various porphyrins, uroporphyrinogen, coproporphyrinogen, certain iron-free metalloporphyrins, hemes, and certain heme-proteins containing readily removable hemes. Strain 23 utilized a wider range of tetrapyrroles than hemin-requiring bacteria previously studied. Inactive compounds included porphyrin biosynthesis intermediates preceding the tetrapyrrole stage and related compounds; uroporphyrin, chlorophyll, pheophytin, phycoerythrin, bilirubin, pyrrole, FeSO(4) with or without chelating agents; and representative ferrichrome compounds. Strain 23, two other strains representing predominant biotypes of B. ruminicola subsp. ruminicola, and one closely related strain grew in media containing heme-free protoporphyrin, mesoporphyrin, hematoporphyrin, or deuteroporphyrin, apparently inserting iron into several nonvinyl porphyrins. Porphobilinogen and porphyrin synthesis, apparently via the commonly known heme synthesis pathway, occurred during growth of heme-independent B. ruminicola subsp. brevis strain GA33 in a tetrapyrrole-free medium containing delta-aminolevulinic acid, but delta-aminolevulinic acid metabolism to porphobilinogen or porphyrins could not be detected in cells of heme-requiring strain 23 grown in the same medium with hemin added. Growth of strain 23 with uroporphyrinogen, coproporphyrinogen, or protoporphyrin IX replacing hemin suggests that part of the commonly known heme-biosynthesis pathway is present in this strain, but nutritional and metabolic evidence indicates that some or all of the enzymes synthesizing the tetrapyrrole nucleus from linear molecules are lacking or inactive.     

Synthesis and biological activity of ribose-5'-carbamate derivatives of vitamin B(12)

Twelve biologically active derivatives of vitamin B(12) (cyanocobalamin) have been synthesized in which spacers were attached to the ribose-5'-hydroxyl group of vitamin B(12). Their potential to act as oral delivery agents for proteins, nanospheres, or immunogens using the vitamin B(12) uptake system was evaluated by determining their affinity for intrinsic factor (IF) and non-IF. The ribose-5'-hydroxyl group of vitamin B(12) was activated through the use of 1,1'-carbonyldiimidazole (CDI), 1,1'-carbonyldi(1,2, 4-triazole) (CDT), or di(1-benzotriazolyl) carbonate (DBTC). Subsequent addition of an aminoalkane, diaminoalkane, or alkane diacid dihydrazide gave rise to vitamin B(12) derivatives suitable for attachment to various proteins, peptides, or nanospheres to enable oral delivery utilizing the vitamin B(12) uptake system. The ribose-5'-carbamate derivatives were found to possess similar affinity for intrinsic factor as that of the e-monocarboxylic acid of vitamin B(12). The affinity for non-IF was similar to cyanocobalamin or even higher for some of the smaller derivatives. Polysciences nanoparticles derivatized with vitamin B(12) 5'-carbamate adipic dihydrazide into CaCo-2 cells showed significantly higher levels of transport of the particles, when compared to unmodified particles.     

Involvement of free and enzyme-bound intermediates in the reaction mechanism catalyzed by the bovine liver immobilized porphobilinogen deaminase. Proof that they are substrates for cosynthetase in uroporphyrinogen III biosynthesis

The detection and accumulation of tetrapyrrole intermediates synthesized by the action of bovine liver porphobilinogen deaminase immobilized to Sepharose 4B is reported. Employing Sepharose-deaminase preparations, two phases in uroporphyrinogen I synthesis as a function of time were observed, suggesting the accumulation of free and enzyme-bound intermediates, the concentration and distribution of which were time dependent. The deaminase-bound intermediate behaves as a substrate in uroporphyrinogen I synthesis whereas the free intermediates produce enzyme inhibition. The tetrapyrrole intermediate bound to the Sepharose-enzyme is removed from the protein by the binding of porphobilinogen. Free as well as enzyme-bound intermediates are shown to be substrates for cosynthetase with formation of 80% uroporphyrinogen III.     

CysG structure reveals tetrapyrrole-binding features and novel regulation of siroheme biosynthesis

Sulfur metabolism depends on the iron-containing porphinoid siroheme. In Salmonella enterica, the S-adenosyl-L-methionine (SAM)-dependent bismethyltransferase, dehydrogenase and ferrochelatase, CysG, synthesizes siroheme from uroporphyrinogen III (uro'gen III). The reactions mediated by CysG encompass two branchpoint intermediates in tetrapyrrole biosynthesis, diverting flux first from protoporphyrin IX biosynthesis and then from cobalamin (vitamin B(12)) biosynthesis. We determined the first structure of this multifunctional siroheme synthase by X-ray crystallography. CysG is a homodimeric gene fusion product containing two structurally independent modules: a bismethyltransferase and a dual-function dehydrogenase-chelatase. The methyltransferase active site is a deep groove with a hydrophobic patch surrounded by hydrogen bond donors. This asymmetric arrangement of amino acids may be important in directing substrate binding. Notably, our structure shows that CysG is a phosphoprotein. From mutational analysis of the post-translationally modified serine, we suggest a conserved role for phosphorylation in inhibiting dehydrogenase activity and modulating metabolic flux between siroheme and cobalamin pathways.     

Chloromethane: tetrahydrofolate methyl transfer by two proteins from Methylobacterium chloromethanicum strain CM4.

The cmuA and cmuB genes are required for growth of Methylobacterium chloromethanicum strain CM4 with chloromethane as the sole carbon source. While CmuB was previously shown to possess methylcobalamin:tetrahydrofolate methyltransferase activity, sequence analysis indicated that CmuA represented a novel and so far unique two-domain methyltransferase/corrinoid-binding protein involved in methyl transfer from chloromethane to a corrin moiety. CmuA was purified from wild-type M. chloromethanicum strain CM4 and characterized as a monomeric, cobalt-containing and zinc-containing enzyme of molecular mass 67 kDa with a bound vitamin B12 cofactor. In combination, CmuA and CmuB proteins catalyze the in vitro transfer of the methyl group of chloromethane to tetrahydrofolate, thus affording a direct link between chloromethane dehalogenation and core C1 metabolism of Methylobacterium. Chloromethane dehalogenase activity in vitro is limited by CmuB, as formation of methyltetrahydrofolate from chloromethane displays apparent Michaelis-Menten kinetics with respect to methylated CmuA, with an apparent Km of 0.27 microM and a Vmax of 0.45 U x mg(-1). This contrasts with sequence-related systems for methyl transfer from methanogens, which involve methyltransferase and corrinoid protein components in well-defined stoichiometric ratios.