The activity of CN-B_<12> for Escherichia coli 215 was lost by incubating the compound with the cultured broth of Streptomyces nitrosporeus. UV absorption spectra of inactivated corrinoid (SA) were very similar to that of CN-B CN-B_<12> With the only exception of an absorption peak at 1,725 cm^<-1>. SA was a mono-carboxylic acid of CN-B CN-B_<12>, but its behaviors in PPC, PEP and TLC did not agree with those of the known CN-B CN-B_<12> monocarboxylic acids. When SA was hydrolyzed with 0.lN HCl at 370℃ for 64 h, a tetracarboxylic acid containing the lower ligand was obtained. From the above results and elementary analysis, it was concluded that SA was a new CN-B CN-B_<12> monocarboxylic acid possessing an acetic acid group in place of an acetamide group which had never been obtained.
A new cyanocobalamin monocarboxylic acid (SA) possessing an acetic acid group in place of an acetamide group prepared from cyanocobalamin (CN-B_<12>) with an enzyme in Streptomyces nitrosporeus was active as a vitamin B_<12> antagonist for E. coli 215, a vitamin B_<12> by auxotroph. The compound was amidated by chemical and biological methods. SA was converted to CN-B_<12> by amidation with dry ammonia under the anhydrous condition, and when incubated with the cells of Pr. shermanii, three forms of corrinoids, SA, adenosyl-SA and DBCC, were found in the cells. However, CN-B_<12> was not observed. It was thus confirmed that this nucleotide-containing monocarboxylic acid of corrinoid could be utilized as a precursor of vitamin B12 biosynthesis by Pr. shermanii.
Pyridoxal kinase was purified from Escherichia coli KG 980 by ammonium sulfate fractionation, and successive chromatographies on DEAE-cellulose, Sephadex G-150 and pyridoxamine (PM)-Sepharose. The overall purification was about 2,500-fold over the crude extract with an yield of about 23%. Polyacrylamide gel electrophoresis indicated that the final product was 70 to 75% homogeneous. The molecular weight of the enzyme was estimated to be 47,000 by Sephadex G-100 gel filtration and 58,000 by SDS polyacrylamide gel electrophoresis. From isoelectric gel electrophoresis, an isoelectric point of 4.8 was estimated. The K_m values for pyridoxine (PN), pyridoxal (PL) and PM were 9.1 × 10^<-6>M, 1.3 × l0^<-4>M and 4.2 × 10^<-4>M, respectively. The relative V_<max> values obtained for PN, PL and PM were 100%, 70% and 170%, respectively. The pH optimum was 6.0 with PN or PL and 7.0 with PM as the substrate. PM, 4-deoxypyridoxine (dPN) and 5'-dPN showed competitive inhibition for phosphorylation of PN with Ki values of 4.4 × l0^<-4>M, 2.5 × l0^<-5>M and 2.9 × l0^<-4>M, respectively. Pyridoxal 5'-phosphate showed noncompetitive inhibition with Ki Value of 9.1 × 10^<-4>M. PL and 5'-deoxypyridoxal (dPL) were both competi-tive and noncompetitive inhibitors for PN. The K_i Values of PL was 1.4 × 10^<-4>M (as a competitive inhibitor) and 5.9 × l0^<-5>M (as a noncompetitive inhibitor), and the K_i Values of 5'-dPL was 2.5 × l0^<-4>M (as a competitive inhibitor) and 3.8 × 10^<-5>M (as a noncompetitive inhibitor).
Pyridoxal kinase from Escherichia. coli KG 980 was inactivated by pyridoxal (PL) as follows : 1) The inactivation of the enzyme was reversible. The inactivation showed an equilibrium reaction pattern with K_<eq> values of 37 to 31 mM^<-1>. 2) The inactivation followed pseudo-first order kinetics with respect to time, and then maximal inactivation was achieved by incubation for 15〜20 min. 3) The results were consistent with a one-step inactivation mechanism. 4) The inactivation was due to the specific binding of PL to one crucial PL binding site, by the formation of Schiff base. 5) The substrate (or analog) did not protect against inactivation with PL. 6) The binding of PL was accompanied by the complete loss of enzyme activity.