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.

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.

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.

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.

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.

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.

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.

Site-directed mutagenesis and high-resolution NMR spectroscopy of the active site of porphobilinogen deaminase.

The active site of porphobilinogen (PBG)1 deaminase (EC 4.3.1.8) from Escherichia coli has been found to contain an unusual dipyrromethane derived from four molecules of 5-aminolevulinic acid (ALA) covalently linked to Cys-224, one of the two cysteine residues conserved in E. coli and human deaminase. By use of a hemA- strain of E. coli the enzyme was enriched from [5-13C]ALA and examined by 1H-detected multiple quantum coherence spectroscopy, which revealed all of the salient features of a dipyrromethane composed of two PBG units linked head to tail and terminating in a CH2-S bond to a cysteine residue. Site-specific mutagenesis of Cys-99 and Cys-242, respectively, has shown that substitution of Ser for Cys-99 does not affect the enzymatic activity, whereas substitution of Ser for Cys-242 removes essentially all of the catalytic activity as measured by the conversion of the substrate PBG to uro'gen I. The NMR spectrum of the covalent complex of deaminase with the suicide inhibitor 2-bromo-[2,11-13C2]PBG reveals that the aninomethyl terminus of the inhibitor reacts with the enzyme's cofactor at the alpha-free pyrrole. NMR spectroscopy of the ES2 complex confirmed a PBG-derived head-to-tail dipyrromethane attached to the alpha-free pyrrole position of the enzyme. A mechanistic rationale for deaminase is presented.

Identification of a cysteine residue as the binding site for the dipyrromethane cofactor at the active site of Escherichia coli porphobilinogen deaminase.

The dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase was specifically labelled with 13C by growth of the bacteria in the presence of 5-amino[5-13C]levulinic acid. Using 13C-NMR spectroscopy, the structure of the cofactor was confirmed as a dipyrromethane made up of two linked pyrrole rings each derived from porphobilinogen. The chemical shift data indicate that one of the pyrrole rings of the cofactor is covalently linked to the deaminase enzyme through a cysteine residue. Evidence from protein chemistry studies suggest that cysteine-242 is the covalent binding site for the cofactor.

Formation of transmembrane channels in liposomes during injection of lambda DNA.

Bacteriophage lambda binds to unilamellar liposomes bearing its receptor protein, LamB, and the lambda DNA can be injected into the internal aqueous space. During this process, transmembrane channels are formed in the liposomes which permit the entry and escape of small molecules, but not proteins. The channels are stable and persist after DNA injection.

Proteinase sensitivity of bacteriophage lambda tail proteins gpJ and pH in complexes with the lambda receptor.

Previous studies have shown that bacteriophage lambda initially binds to liposomes bearing its receptor protein by the tip of the tail fiber (type 1 complex). It then associates more directly so that the hollow tail tube is in direct contact with the membrane (type 2 complex). DNA can be injected across the lipid bilayer into the liposome from type 2 complexes. We show here that gpJ, the tail fiber protein, becomes more sensitive to proteolytic degradation in type 2 complexes, indicating that the tail fiber does not pass into the liposome and that the tail fiber may undergo a conformational change in type 2 complexes. Another bacteriophage protein, pH, is sensitive to proteolytic degradation in free bacteriophage, type 1 complexes, or type 2 complexes formed with free receptor, but is resistant to proteinases in type 2 complexes formed with liposomes. This finding suggests that pH associates with the membrane. We suggest that this association is part of the mechanism by which a transmembrane hole for DNA entry is formed.