Biosynthesis of cobalamin (vitamin B(12)).

The biosynthesis of vitamin B(12) is summarized, emphasizing the differences observed between the aerobic and anaerobic pathways. The biosynthetic route to adenosylcobalamin from its five-carbon precursor, 5-aminolaevulinic acid, can be divided into three sections: (1) the biosynthesis of uroporphyrinogen III from 5-aminolaevulinic acid, which is common to both pathways; (2) the conversion of uroporphyrinogen III into the ring-contracted, deacylated intermediate precorrin 6 or cobalt-precorrin 6, which includes the primary differences between the two pathways; and (3) the transformation of this intermediate to form adenosylcobalamin.

Engineering Escherichia coli for the synthesis of taxadiene, a key intermediate in the biosynthesis of taxol.

Taxadiene, the key intermediate of paclitaxel (Taxol) biosynthesis, has been prepared enzymatically from isopentenyl diphosphate in cell-free extracts of Escherichia coli by overexpressing genes encoding isopentenyl diphosphate isomerase, geranylgeranyl diphosphate synthase and taxadiene synthase. In addition, by the expression of three genes encoding four enzymes on the terpene biosynthetic pathway in a single strain of E. coli, taxadiene can be conveniently synthesized in vivo, at the unoptimized yield of 1.3mg per liter of cell culture. The success of both in vitro and in vivo synthesis of taxadiene bodes well for the future production of taxoids by non-paclitaxel producing organisms through pathway engineering.

Crystal structure of precorrin-8x methyl mutase.

BACKGROUND: The crystal structure of precorrin-8x methyl mutase (CobH), an enzyme of the aerobic pathway to vitamin B12, provides evidence that the mechanism for methyl migration can plausibly be regarded as an allowed [1,5]-sigmatropic shift of a methyl group from C-11 to C-12 at the C ring of precorrin-8x to afford hydrogenobyrinic acid. RESULTS: The dimeric structure of CobH creates a set of shared active sites that readily discriminate between different tautomers of precorrin-8x and select a discrete tautomer for sigmatropic rearrangement. The active site contains a strictly conserved histidine residue close to the site of methyl migration in ring C of the substrate. CONCLUSION: Analysis of the structure with bound product suggests that the [1,5]-sigmatropic shift proceeds by protonation of the ring C nitrogen, leading to subsequent methyl migration.

Multiple biosynthetic pathways for vitamin B12: variations on a central theme.

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.

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.

How corrinoids are synthesized without oxygen: nature's first pathway to vitamin B12.

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.

Enzymatic synthesis of S-adenosyl-L-methionine on the preparative scale.

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.

Achieving natural product synthesis and diversity via catalytic networking ex vivo.

Recent studies on ex vivo synthesis of natural products reveal that even complex multistep pathways can be successfully reconstructed. Genetic engineering of such reconstituted pathways has already been used to generate 'unnatural' natural products related to the original compound. In the future, it may be possible to use these approaches to make natural products that are currently inaccessible to conventional synthesis.

Genetically engineered synthesis of natural products: from alkaloids to corrins.

Because many natural products are of biological and medicinal importance, methods are continually being sought for studying their biosynthetic pathways, which may eventually result in increased production and the generation of novel compounds. Advances in genetic engineering have enabled the homologous or heterologous expression of many natural product biosynthetic genes from divergent sources, resulting in a supply of enzymes not readily available by isolation from the producing organism. Mixing and matching of these enzymes in cell-free reactions can provide information, not available by any other means, about enzyme mechanisms, pathway intermediates, and possible variations in the structure of the final product.

Fluorescence-based method for selection of recombinant plasmids.

A fluorescence-based method for the selection of recombinant plasmids has been developed. Escherichia coli strains bearing plasmids for the overexpression of the gene encoding uroporphyrinogen III methyltransferase (cobA or cysG gene) accumulate fluorescent porphyrinoid compounds. When illuminated with ultraviolet light, the cells fluoresce with a bright red color. Replacement or disruption of the gene with other fragments of DNA results in loss of enzymatic activity and nonfluorescent cells. The detection of the recombinant plasmids can be accomplished without special host strains or expensive chromogenic reagents required for the blue-white screening technique.

Evidence for conformational changes in Escherichia coli porphobilinogen deaminase during stepwise pyrrole chain elongation monitored by increased reactivity of cysteine-134 to alkylation by N-ethylmaleimide.

Porphobilinogen deaminase from Escherichia coli becomes progressively more susceptible to inactivation by the thiophilic reagent N-ethylmaleimide (NEM) as the catalytic cycle proceeds through the enzyme-intermediate complexes ES, ES2, ES3, and ES4. Site-directed mutagenesis of potentially reactive cysteines has been used to identify cysteine-134 as the key residue that becomes modified by the reagent and leads to inactivation. Since cysteine-134 is buried at the interface between domains 2 and 3 of the E. coli deaminase molecule, the observations suggest that a stepwise conformational change occurs between these domains during each stage of tetrapyrrole assembly. Interestingly, mutation of the invariant active-site cysteine-242 to serine leads to an enzyme with up to a third of the catalytic activity found in the wild-type enzyme. Electrospray mass spectrometry indicates that serine can substitute for cysteine as the dipyrromethane cofactor attachment site.

Overexpression in Escherichia coli of 12 vitamin B12 biosynthetic enzymes.

The first 12 enzymes involved in the biosynthesis of vitamin B12 from its five-carbon precursor, aminolevulinic acid, have been overexpressed in recombinant strains of Escherichia coli. The activity of each enzyme has been demonstrated by the biosynthesis of hydrogenobyrinic acid from aminolevulinic acid.

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.

Genetically engineered synthesis of precorrin-6x and the complete corrinoid, hydrogenobyrinic acid, an advanced precursor of vitamin B12.

BACKGROUND: Genetically engineered synthesis, in which the gene products, cofactors, and substrates of a complete pathway are combined in vitro in a single flask to give the target, can be a viable alternative to conventional chemical construction of molecules of complex structure and stereochemistry. We chose to attempt to synthesize the metal-free corrinoid hydrogenobyrinic acid, an advanced precursor of vitamin B12. RESULTS: Cloning and overexpression of the genes necessary for the S-adenosyl methionine dependent conversion of 5-aminolevulinic acid (ALA) to precorrin-3 and those required for the synthesis of hydrogenobyrinic acid from precorrin-3 completed the repertoire of the 12 biosynthetic enzymes involved in corrin synthesis. Using these enzymes and the necessary cofactors, the multi-enzyme synthesis of hydrogenobyrinic acid from ALA can be achieved in 20% overall yield in a single reaction vessel, corresponding to an average of at least 90% conversion for each of the 17 steps involved. CONCLUSIONS: By replacing the cell wall with glass, and by mixing the soluble biosynthetic enzymes and necessary cofactors, the major segment of the physiological synthesis of vitamin B12 has been accomplished. Since only those enzymes necessary for the synthesis of hydrogenobyrinic acid from ALA are supplied, none of the intermediates is deflected from the direct pathway. This results in an efficiency which in fact surpasses that of nature.

Gene dissection demonstrates that the Escherichia coli cysG gene encodes a multifunctional protein.

The C-terminus of the Escherichia coli CysG protein, consisting of amino acids 202-457, was expressed as a recombinant protein using gene dissection methodology. Analysis of the activity of this truncated protein, termed CysGA, revealed that it was able to methylate uroporphyrinogen III in the same S-adenosyl-L-methionine (SAM)-dependent manner as the complete CysG protein. However, this truncated protein was not able to complement E. coli cysG cells, thereby suggesting that the first 201 amino acids of the CysG protein had an enzymic activity associated with the conversion of dihydrosirohydrochlorin into sirohaem. Analysis of the N-terminus of the CysG protein revealed the presence of a putative pyridine dinucleotide binding site. When the purified CysG protein was incubated with NADP+, uroporphyrinogen III and SAM the enzyme was found to catalyse a coenzyme-mediated dehydrogenation to form sirohydrochlorin. The CysGA protein on the other hand showed no such coenzyme-dependent activity. Analysis of the porphyrinoid material isolated from strains harbouring plasmids containing the complete and truncated cysG genes suggested that the CysG protein was also involved in ferrochelation. The evidence presented in this paper suggests that the CysG protein is a multifunctional protein involved in SAM-dependent methylation, pyridine dinucleotide dependent dehydrogenation and ferrochelation.

The Escherichia coli cysG gene encodes the multifunctional protein, siroheme synthase.

Previously, the E. coli cysG gene product had been shown to sequentially methylate uro'gen III to produce precorrin-2, hence it was given the trivial name uro'gen III methylase. We now report that in addition to methylase activity, the CysG protein catalyses both the NAD+ dependent oxidation of precorrin-2 to sirohydrochlorin, but also the insertion of iron into this oxidized intermediate, thereby producing siroheme. Thus CysG is a multifunctional protein solely responsible for siroheme synthesis from uro'gen III in E. coli, and accordingly is renamed siroheme synthase.

Biosynthesis of vitamin B12. Discovery of the enzymes for oxidative ring contraction and insertion of the fourth methyl group.

In the vitamin B12 biosynthetic pathway the enzymes responsible for the conversion of precorrin-3 to precorrin-4 have been identified as the gene products of cobG and cobJ from Pseudomonas denitrificans. CobG catalyzes the oxidation of precorrin-3 to precorrin-3x (a hydroxy lactone) whereas CobJ is a SAM-dependent C-17 methyl transferase and is necessary for ring contraction. A mechanism for ring contraction is proposed.

Sequence of the Candida albicans erg7 gene.

The nucleotide sequence of the Candida albicans erg7 gene, which complements erg7 mutants of Saccharomyces cerevisiae and restores oxidosqualene cyclase activity, was determined. The gene encodes a 728-aa protein that displays homology with squalene-hopene cyclase, providing further evidence that erg7 is the gene encoding 2,3-oxidosqualene cyclase.

Purification of an indole alkaloid biosynthetic enzyme, strictosidine synthase, from a recombinant strain of Escherichia coli.

The gene for the indole alkaloid biosynthetic enzyme, strictosidine synthase, of Catharanthus roseus has been cloned into an inducible Escherichia coli expression vector using an expression cassette polymerase chain reaction technique. Induction of the gene resulted in overexpression of the enzyme which accumulated mainly as insoluble inclusion bodies. Denaturation and refolding of the insoluble protein resulted in the ability to purify up to 6 mg of active enzyme from a single liter of cell culture. The recombinant enzyme has good activity (approximately 30 nkat/mg).

Expression of 9 Salmonella typhimurium enzymes for cobinamide synthesis. Identification of the 11-methyl and 20-methyl transferases of corrin biosynthesis.

Nine of the cbi genes from the 17.5 kb cob operon of Salmonella typhimurium previously shown by genetic studies to be involved in the biosynthesis of cobinamide from precorrin-2, have been subcloned and expressed in Escherichia coli. Seven of the gene products were found in the soluble fraction of cell lysates and have been purified. The gene products corresponding to cbi E, F, H and L were shown by SAM binding and by homology with other SAM-binding proteins to be candidates for the methyltransferases of vitamin B12 biosynthesis. The enzymatic functions of the gene products of cbiL and cbiF are associated with C-methylation at C-20 of precorrin-2 and C-11 of precorrin-3.