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.

Chemoenzymatic synthesis of an unnatural tetramethyl cobalt corphinoid.

The chemoenzymatic synthesis and structural characterization by 13C NMR of a tetramethyl cobalt-corphinoid produced by methylation of cobalt-precorrin-3 using CbiF are described.

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.

Biosynthesis of vitamin B12: concerning the identity of the two-carbon fragment eliminated during anaerobic formation of cobyrinic acid.

It has been proved that, during anaerobic biosynthesis of the corrin macrocycle, the two-carbon fragment excised from the precursor, precorrin-3, is acetaldehyde, which originates from C-20 and its attached methyl group. This apparently contradictory finding is rationalized in terms of the subsequent enzymatic oxidation of acetaldehyde to acetic acid, which was previously regarded as the volatile fragment released by the action of the biosynthetic enzymes of Propionibacterium shermanii. The observation that acetaldehyde (rather than acetic acid) is extruded during anaerobic B12 synthesis is in full accord with the structure of factor IV, a new intermediate on the pathway.

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.

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.

Enzymatic synthesis and structure of precorrin-3, a trimethyldipyrrocorphin intermediate in vitamin B12 biosynthesis.

The trimethylated intermediate of vitamin B12 (corrin) biosynthesis, precorrin-3, was produced from various 13C-enriched isotopomers of 5-aminolevulinic acid (ALA), using a multiple-enzyme system containing ALA dehydratase, porphobilinogen deaminase, uro'gen III synthetase, and the S-adenosyl-L-methionine-(SAM)-dependent uro'gen III methyltransferase (M-1) and precorrin-2 methyltransferase (M-2) in the presence of [13C]SAM. Structural analysis of the resulting product, precorrin-3, reveals a close similarity to precorrin-2 but with several subtle differences in the conjugated array of C = C and C = N bonds which reflect the presence of the new C-methyl group at C20 and its influence on the electronic distribution in the dipyrrocorphin chromophore. The implications of this structure for corrin biosynthesis are discussed.

Bacteriochlorophyll c formation via the C5 pathway of 5-aminolevulinic acid synthesis in Chloroflexus aurantiacus.

Biosynthesis of 5-aminolevulinic acid (ALA) in Chloroflexus aurantiacus, a thermophilic bacterium forming bacteriochlorophyll c, is shown to proceed via the C5 pathway by demonstrating (1) the specific labeling of its chlorin ring with [1 - 13C]glutamate and (2) the enzyme activity to produce ALA from glutamate in a cell-free extract. From the phylogenetic distribution it is suggested that ALA synthetase distributed in some aerobic eubacteria could be monophyletic in origin.

The Escherichia coli cysG gene encodes S-adenosylmethionine-dependent uroporphyrinogen III methylase.

The Escherichia coli cysG gene was successfully subcloned and over-expressed to produce a 52 kDa protein that was purified to homogeneity. This protein was shown to catalyse the S-adenosylmethionine-dependent methylation of uroporphyrinogen III to give a product identified as sirohydrochlorin on the basis of its absorption spectra, incorporation of 14C label from S-adenosyl[Me-14C]methionine and mass and 1H-n.m.r. spectra of its octamethyl ester. Further confirmation of the structure was obtained from a 14C-n.m.r. spectrum of the methyl ester produced by incubation of the methylase with uroporphyrinogen III, derived from [4.6-13C2]porphobilinogen, and S-adenosyl[Me-13C]methionine.

Enzymatic synthesis of dihydrosirohydrochlorin (precorrin-2) and of a novel pyrrocorphin by uroporphyrinogen III methylase.

Uroporphyrinogen III methylase was purified from a recombinant hemB-strain of E. coli harbouring a plasmid containing the cysG gene. N-terminal analysis of this purified protein gave an amino acid sequence corresponding to that predicted from the genetic code. From the u.v./visible spectrum of the reaction catalysed by this SAM dependent methylase it was possible to observe the sequential appearance of the chromophores of a dipyrrocorphin and subsequently of a pyrrocorphin. Confirmation of this transformation was obtained from 13C-NMR studies when it was demonstrated, for the first time directly, that uroporphyrinogen is initially converted into dihydrosirohydrochlorin (precorrin-2) and then, by further methylation, into a novel trimethylpyrrocorphin.

Reconstitution of apo-porphobilinogen deaminase: structural changes induced by cofactor binding.

Expression of porphobilinogen deaminase in a hemB- strain of E. coli has permitted the isolation of the apoenzyme, i.e. deaminase lacking the porphobilinogen-derived dipyrromethane cofactor. Incubation of purified apoenzyme with porphobilinogen resulted in reconstitution of the covalently attached dipyrromethane cofactor, indicating no additional cofactors or enzymes are required for biosynthesis of holoenzyme. Electrophoretic and 13C-NMR spectroscopic analyses demonstrate that the apoenzyme exists in a conformationally unstable form which is converted to a highly stable tertiary structure on covalent attachment of the dipyrromethane cofactor.

Biosynthesis of vitamin B12: concerning the origin of the methine protons of the corrin nucleus.

13C NMR spectroscopy has been used to locate six deuterium atoms incorporated biosynthetically on the periphery of the corrin nucleus of vitamin B12 (cyanocobalamin) derived from cells of Propionibacterium shermanii grown in a medium containing 50% 2H2O and 13C-enriched delta-aminolevulinic acid. The implications of these results for the mechanism of vitamin B12 biosynthesis are discussed, and it is concluded that the same oxidation level of the intermediates is maintained throughout the biosynthetic pathway, from delta-aminolevulinic acid to corrin.