Studies on the biosynthesis of corrinoids and porphyrinoids. I. The labeling of oxygen of vitamin B12

Recently the amide-oxygen has been suggested to participate in the formation of the corrin ring of vitamin B12. To confirm this hypothesis, 17O-labeled aminolevulinic acid (ALA) was prepared and administered to Propionibacterium shermanii. The isolated vitamin B12 showed only broad 17O signals in the oxygen-17 nuclear magnetic resonance (17O-NMR) spectrum. However, distinct isotope-shifted peaks were observed in the 13C-NMR spectrum of vitamin B12 isolated after incorporation of [1-13C:1,4-18O2]ALA. Of these shifted peaks, one peak (C27) showed very low intensity. This indicates that dilution of 18O occurred at the acetyl chain of the A ring of vitamin B12. This result supports the assumption that the lactone formation of the A ring promotes the ring contraction, as proposed by Eschenmoser.     

Studies on the biosynthesis of corrinoids and porphyrinoids. II. The origin of nitrogen of vitamin B12

To clarify the origin of nitrogen of vitamin B12, 15N-labeled aminolevulinic acid (ALA) was prepared and administered to Propionibacterium shermanii. Vitamin B12 thus isolated showed four signals in the nitrogen-15 nuclear magnetic resonance (15N-NMR) spectrum. The nitrogen of [5-15N]riboflavine was incorporated into the benzimidazole part of vitamin B12. Hydroxycobalamin was transformed into cyanocobalamin by treatment with [15N]potassium cyanide, and the 15N-NMR spectrum was measured. The results of these experiments revealed the origin of the nitrogen atoms of vitamin B12, and allowed the 15N-NMR signals to be assigned.     

Studies on the biosynthesis of corrinoids and porphyrinoids. III. The origin of amide nitrogen of vitamin B12

To clarify the origin of amide-nitrogen of vitamin B12, [1-13C]aminolevulinic acid (ALA) and L-[amide-15N]glutamine were administered to P. shermanii. The 13C-nuclear magnetic resonance spectrum of the vitamin B12 subsequently isolated showed distinct 13C-15N coupling and isotope shift at six amide carbons. However, the C-57 amide carbon showed neither coupling, nor shift. Thus, it was concluded that the nitrogens of 6 amides of the side chain were derived from glutamine and the C-57 amide nitrogen was from threonine.     

Biosynthesis of vitamin B12: the multi-enzyme synthesis of precorrin-4 and factor IV

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.     

Hydrophobic vitamin B12. Part 19: electroorganic reaction of DDT mediated by hydrophobic vitamin B12

The controlled-potential electrolysis of 1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane (DDT) was carried out at -1.4 V vs. Ag-AgCl in the presence of a hydrophobic vitamin B12, heptamethyl cobyrinate perchlorate. DDT was dechlorinated to form 1,1-bis(4-chlorophenyl)-2,2-dichloroethane (DDD), 1,1-bis(4-chlorophenyl)-2,2-dichloroethylene (DDE), 1-chloro-2,2-bis(4-chlorophenyl)ethylene (DDMU) and 1,1,4,4-tetrakis(4-chlorophenyl)-2,3-dichloro-2-butene (TTDB)(E/Z), and quantitative recovery of the catalyst after the electrolysis was confirmed by electronic spectroscopy. A photo-sensitive intermediate having a cobalt-carbon bond formed during the electrolysis was characterized by electronic spectroscopy. A mechanism for the formation of various dechlorinated products was investigated by using deuterium solvents and various spectroscopic measurements such as UV-VIS and the EPR spin-trapping technique.     

Immobilised vitamin B12 as a biomimetic model for base-off/histidine-on coordination

Immobilisation of vitamin B12 allows synthesising a biomimetic base-off/histidine-on complex that resembles structural features of cobalamin dependent enzyme active sites.     

Cyanide-bridged vitamin B12-cisplatin conjugates

cis-[PtCl(OH2)(NH3)2]+, the monoactivated form of cisplatin, reacts with the cyano ligand of cobalt in vitamin B12 (cyanocobalamin) to form a Co-C[triple chemical bond]N-Pt conjugate (1). Compound 1 is prepared in good yield directly in aqueous solution. The remaining chloride ligand of Pt(II) is labile. It hydrolyzes slowly in aqueous solution and can be exchanged by stronger coordinating ligands, such as 9-methylguanine or 2'-deoxyguanosine, to yield vitamin B12-nucleobase conjugates. X-ray structures of the vitamin B12-cisplatin conjugate 1 as well as of the product with coordinated 9-methylguanine (2) are presented. The coordination geometry at Pt(II) is almost perfectly square-planar. The structure of the cobalamin compound remains essentially unchanged when compared with the original B(12) structure. The guanine moiety of compound 2 binds in a 45 degrees angle to the cisplatin molecule and interacts with neighboring molecules by means of pi stacking and hydrogen bonds.     

Radiometric microbiological estimation of vitamin B12

Estimation of vitamin B₁₂ in blood is very important to determine in decifiency and diagnosis of anemic patients. Vitamin B₁₂ in blood can be estimated by spectrochemical, enzymatic, radioisotopic and microbiological methods. In the present study vitamin B₁₂ was determined in 48 normal subjects of Rawalpindi/Islamabad by radiometric microbiological assay (RMA) technique using a very rapid, sensitive and automated instrument Bactec 460. In this procedure¹⁴C-glucose media and microorganismsLactobacillus leichmannii were used. The sensitivity of the method for vitamin B₁₂ is 1 pg/ml and the vitamin B₁₂ found in normal subjects was in the range of 105–535 pg/ml with a median value of 246±6 pg/ml.     

A cyclodecapeptide ligand to vitamin B12

Libraries of cyclic decapeptides were screened with vitamin B(12) derivatives to give cyclic peptide ligands incorporating histidine and cysteine as coordinating residues and negatively charged amino acids. Two hits, cyclo-(HisAspGluProGlyIleAlaThrProdGln) and cyclo-(ValAspGluProGlyGluAspCysProdGln) were resynthesized in good yields for solution experiments. The peptides bind aquocobalamin with coordination of His or Cys to the cobalt with high affinities (K(a) approximately 10(5) M(-1)). Additional interactions between the peptide side chains and the vitamin B(12) corrin moiety were determined by studying the (1)H NMR solution structure. The cyclopeptide-cobalamin complex with the histidine residue showed enhanced stability towards cyanide exchange, demonstrating the shielding effect of the ligand on the metal center.     

Biosynthesis of vitamin B(6) in yeast: incorporation pattern of glucose.

Two yeasts, Saccharomyces cerevisiae ATCC 7752 and Candida utilis ATCC 9256, were incubated in the presence of variously multiply (13)C-labeled samples of D-glucose. The (13)C incorporation pattern within pyridoxamine dihydrochloride, established by (13)C NMR spectroscopy, differed from that which had previously been found within pyridoxine, isolated from Escherichia coli. Thus, the origin of the carbon skeleton of vitamin B(6) in yeast differs substantially from its origin in E. coli. In particular, in yeast the distribution of (13)C within the C(5) chain C-2',2,3,4,4' of pyridoxamine corresponds to the distribution of (13)C within the C(5) chain C-1,2,3,4,5 of the ribose component of cytidine. It follows that the C(5) chains of pyridoxamine and of ribose originate from a common glucose-derived pentulose or pentose intermediate. By contrast, in E. coli the C(5) chain of pyridoxine is derived from 1-deoxy-D-xylulose 5-phosphate which, in turn, originates by condensation of pyruvic acid with glyceraldehyde 3-phosphate.