Conversion of cobalt-free corrinoid to vitamin B12 with the resting cells of Rhodobacter sphaeroides

A cobalt-free corrinoid (CFC) isolated from Rhodobacter sphaeroides was converted to cobalamin intracellularly with the resting cells prepared from an aerobic culture on a cobalt-containing medium. The maximum conversion activity was observed with the cells harvested at early exponential growth phase. The CFC might be used as a peculiar precursor in cobalamin biosynthesis.     

Human plasma R-type vitamin B12-binding proteins. II. The role of transcobalamin I, transcobalamin III, and the normal granulocyte vitamin B12-binding protein in the plasma transport of vitamin B12.

The normal human granulocyte vitamin B12-binding protein, transcobalamin I, and transcobalamin III, have been labeled with 125I-labeled N-succinimidyl 3-(4-hydroxyphenyl)propionate and utilized for plasma clearance studies performed with rabbits. Both moieties of 125I-labeled granulocyte vitamin B12-binding protein-[57Co]vitamin B12 were cleared rapidly from the plasma (is less than 90% by 5 min) by the liver. After 30 min, the bulk of the 125I reappeared in the plasma in small molecular weight (less than 1000) form and was rapidly excreted in the urine. After 60 min the bulk of the [57Co]vitamin B12 reappeared in the plasma bound to rabbit transcobalamin II and was subsequently taken up by a variety of tissues. Approximately 15% of the 125I-labeled granulocyte vitamin B12-binding protein-[57Co-a1vitamin B12 was excreted intact into the bile during the period from 10 to 80 min after injection. The hepatic uptake of the protein-vitamin B12 complex was blocked by the prior injection of desialyzed fetuin but not by native fetuin. Similar results were obtained with 125I-labeled transcobalamin III-[57Co]vitamin B12. Approximately 90% of both moieties of 125I-labeled transcobalamin I-[57Co]vitamin B12 had prolonged plasma survivals similar to that of 125I-labeled bovine serum albumin. After treatment with neuraminadase, both moieties of the 125I-labeled transcobalamin I-[57Co]vitamin B12 complex were cleared rapidly from the plasma by the liver in a manner that was indistinguishable from that observed in the case of untreated granulocyte vitamin B12-binding protein and transcobalamin III. These observations indicate that desialyzed transcobalamin I and the native forms of the granulocyte vitamin B12-binding protein and transcobalamin III are cleared from plasma by the mechanism elucidated by Ashwell and Morell (Ashwell, G., and Morell A. G. (1974) Adv. Enzymol. 41, 99-128) that is capable of clearing a wide variety of asialoglycoproteins. These observations have implications concerning the function of the human R-type vitamin B12-binding proteins, the nature of the enterohepatic circulation of vitamin B12, the biological significance of the mechanism described by Ashwell and Morell, and the etiology of the increased plasma concentration of human R-type protein that occurs frequently in chronic myelogenous leukemia and occasionally in hepatocellular carcinoma and other solid tumors.     

Resonance Raman spectra of vitamin B12 and dicyanocobalamin

The Raman spectra of cyanocobalamin (vitamin B12) and dicyanocobinamide have been obtained from aqueous solutions at concentrations of approx. 1 10⁻⁴ M. The spectra were excited by laser radiation coincident in wavelength with the visible absorption, this resulting in selective enhancement of some vibrational modes through the rigorous resonance Raman effect. In spite of substantial chemical differences, cyanocobalamin and dicyanocobinamide give essentially identical spectra, indicating that only those modes associated with the common corrin ring system are resonance enhanced.     

The interaction between vitamin B-12 and micelles in aqueous solution

The apparent pK for benzimidazole displacement of a number of cobalamins is markedly affected by the presence of sodium lauryl sulfate micelles. However, micelles of cetyltrimethylammonium bromide or Triton X have little or no effect on the pK. By measuring the apparent pK as a function of sodium lauryl sulfate concentration, the association constants between the micelles and both base on and base off methylcobalamin were calculated. This calculation indicates that the base off form is strongly associated with the micelle while the base on form is not.     

Identification of structural markers for vitamin B12 and other corrinoid derivatives in solution using FTIR spectroscopy

The identification of structural markers for B12/protein interactions is crucial to a complete understanding of vitamin B12 transport and metabolic reaction mechanisms of B12 coenzymes. Fourier transform infrared spectroscopy can provide direct measurements of changes in the side chains and corrin ring resulting from B12/protein interactions. Using FTIR spectroscopy in various solvent systems, we have identified structural markers for corrinoids in the physiological state. We assign the major band (denoted B), which occurs at ca. 1630 cm-1 in D2O and ca. 1675 cm-1 in ethanol, to the amide I C=O stretching mode of the propionamide side chains of the corrin ring. The lower frequency of band B in D2O versus ethanol is due to the greater hydrogen-bonding properties of D2O that stabilize the charged amide resonance form. Since the propionamides are known to be important in protein binding, band B is a suitable marker for monitoring the interaction of these side chains with proteins. We assign bands at ca. 1575 and 1545 cm-1 (denoted C and D) as breathing modes of the corrin ring on the basis of the bands' solvent independence and their sensitivity to changes in axial ligation. As the sigma-donating strength of the axial ligands increases, the frequencies of bands C and D decrease, possibly indicating a lengthening of the corrin conjugated system. Band A, the known cyanide stretching frequency at ca. 2130 cm-1, probes the cobalt-carbon distance in cyanocorrinoids. As the frequency of band A increases, the cobalt-carbon bond strength should decrease.     

The interaction between vitamin B-12 and micelles in aqueous solution.

The apparent pK for benzimidazole displacement of a number of cobalamins is markedly affected by the presence of sodium lauryl sulfate micelles. However, micelles of cetyltrimethylammonium bromide or Triton X have little or no effect on the pK. By measuring the apparent pK as a function of sodium lauryl sulfate concentration, the association constants between the micelles and both base on and base off methylcobalamin were calculated. This calculation indicates that the base off form is strongly associated with the micelle while the base on form is not.     

Characterization of riboflavin (vitamin B2) transport proteins from Bacillus subtilis and Corynebacterium glutamicum

Riboflavin (vitamin B(2)) is the direct precursor of the flavin cofactors flavin mononucleotide and flavin adenine dinucleotide, essential components of cellular biochemistry. In this work we investigated the unrelated proteins YpaA from Bacillus subtilis and PnuX from Corynebacterium glutamicum for a role in riboflavin uptake. Based on the regulation of the corresponding genes by a riboswitch mechanism, both proteins have been predicted to be involved in flavin metabolism. Moreover, their primary structures suggested that these proteins integrate into the cytoplasmic membrane. We provide experimental evidence that YpaA is a plasma membrane protein with five transmembrane domains and a cytoplasmic C terminus. In B. subtilis, riboflavin uptake was increased when ypaA was overexpressed and abolished when ypaA was deleted. Riboflavin uptake activity and the abundance of the YpaA protein were also increased when riboflavin auxotrophic mutants were grown in limiting amounts of riboflavin. YpaA-mediated riboflavin uptake was sensitive to protonophors and reduced in the absence of glucose, demonstrating that the protein requires metabolic energy for substrate translocation. In addition, we demonstrate that PnuX from C. glutamicum also is a riboflavin transporter. Transport by PnuX was not energy dependent and had high apparent affinity for riboflavin (K(m) 11 microM). Roseoflavin, a toxic riboflavin analog, appears to be a substrate of PnuX and YpaA. We propose to designate the gene names ribU for ypaA and ribM for pnuX to reflect that the encoded proteins function in riboflavin uptake and that the genes have different phylogenetic origins.