Evolution of coenzyme B12 synthesis among enteric …

Enzymatic synthesis of A B12 coenzyme - ScienceDirect

Biochemistry of B12-Cofactors in Human Metabolism

Folate and B12 coenzymes are involved in the biosynthesis of purines, pyrimidines and amino acids and thus play an important role in cell replication. Regulation of folate- and B12-dependent metabolism, and the possible exploitation of such regulation in cancer chemotherapy, can be achieved at several different levels: (a) transport of the vitamins into cells; (b) conversion of these vitamins to their coenzyme forms; and (c) reactions catalyzed by coenzyme-dependent systems. Transport of folate compounds into mammalian cells is an energy-dependent, carrier-mediated process. Three separate lines of evidence (substrate competition, mutant sublines and sulfhydryl inhibitors) indicate that L1210 cells utilize two separate carrier systems for folate compounds. One is specific for folate, while the other is specific for 5-methyl tetrahydrofolate; the latter system also transports tetrahydrofolate, 5-formyl tetrahydrofolate (folinate) and amethopterin (Methotrexate). In contrast, Lactobacillus casei utilizes a single carrier system for all of the folate compounds. The intracellular conversion of folate to the coenzyme, tetrahydrofolate, is mediated by the TPNH-dependent enzyme, dihydrofolate reductase. This enzyme is the target for folate antagonists such as amethopterin. Interaction with the drug, however, is markedly dependent upon prior binding of TPNH to the enzyme. Treatment of cells with amethopterin generally causes the level of dihydrofolate reductase to rise in subsequent generations. This has been attributed to a decreased rate of degradation of the enzyme (stabilized by complex formation with amethopterin) rather than to an increased rate of synthesis. Polyacrylamide gel electrophoresis and absorbance spectra have been used to study the interaction of dihydrofolate reductases from L1210 cells and L. casei with various substrates and inhibitors. Transport of vitamin B12 is an energy-dependent, carrier-mediated process. In mammalian cells transport requires, in addition to the membrane carrier system, an external protein, transcobalamin-II. Analogs of adenosyl cobalamin inhibit this process. Conversion of B12 to its coenzyme forms (adenosyl- and methyl cobalamin) is accomplished by the two-step reduction of B12a (CoIII) to B12r (CoII) to B12s (CoI), followed by reaction of the latter with ATP or adenosyl methionine. A variant of this system has been encountered in the activation of the B12-containing methionine synthetase by TPNH and two flavoproteins.

In contrast, vitamin B12r, the exclusiveproduct obtained from hydroxocobalamin and chromous acetate at pH 5, isquite unreactive.

Cobalamin (coenzyme B12): Synthesis and biological significance …

N2 - Adenosyltransferases (ATRs) catalyze the synthesis of the reactive cobalt-carbon bond found in coenzyme B12 or 5′- deoxyadenosylcobalamin (AdoCbl), which serves as a cofactor for a number of isomerases. The reaction involves a reductive adenosylation of cob(II)alamin in which an electron delivered by a reductase reduces cob(II)alamin to cob(I)alamin, which attacks the 5′-carbon of ATP to form AdoCbl and inorganic triphosphate. Of the three classes of ATRs found in nature, the PduO type, which is also the only one found in mammals, is the most extensively studied. The crystal structures of a number of PduO-type ATRs are available and reveal a trimeric organization with the active sites located at the subunit interfaces. We have previously demonstrated that the ATR from Methylobacterium extorquens, which supports methylmalonyl-CoA mutase activity, serves dual functions; i.e., it tailors the active AdoCbl form of the cofactor and then transfers it directly to the dependent mutase (Padovani et al. (2008) Nat. Chem. Biol. 4, 194). Only two of the three active sites in ATR are simultaneously occupied by AdoCbl. In this study, we demonstrate that binding of the substrate ATP to ATR that is fully loaded with AdoCbl leads to the ejection of 1 equivalent of the cofactor into solution. In the presence of methylmalonyl-CoA mutase and ATP, AdoCbl is transferred from ATR to the acceptor protein in a process that exhibits an ∼3.5-fold lower Kact for ATP compared to the one in which cofactor is released into solution. Furthermore, ATP favorably influences cofactor transfer in the forward direction by reducing the ratio of apo-methylmalonyl-CoA mutase/holo-ATR required for delivery of 1 equivalent of AdoCbl, from 4 to 1. These results lead us to propose a rotary mechanism for ATR function in which, at any given time, only two of its active sites are used for AdoCbl synthesis and where binding of ATP to the vacant site leads to the transfer of the high value AdoCbl product to the acceptor mutase.

AB - Adenosyltransferases (ATRs) catalyze the synthesis of the reactive cobalt-carbon bond found in coenzyme B12 or 5′- deoxyadenosylcobalamin (AdoCbl), which serves as a cofactor for a number of isomerases. The reaction involves a reductive adenosylation of cob(II)alamin in which an electron delivered by a reductase reduces cob(II)alamin to cob(I)alamin, which attacks the 5′-carbon of ATP to form AdoCbl and inorganic triphosphate. Of the three classes of ATRs found in nature, the PduO type, which is also the only one found in mammals, is the most extensively studied. The crystal structures of a number of PduO-type ATRs are available and reveal a trimeric organization with the active sites located at the subunit interfaces. We have previously demonstrated that the ATR from Methylobacterium extorquens, which supports methylmalonyl-CoA mutase activity, serves dual functions; i.e., it tailors the active AdoCbl form of the cofactor and then transfers it directly to the dependent mutase (Padovani et al. (2008) Nat. Chem. Biol. 4, 194). Only two of the three active sites in ATR are simultaneously occupied by AdoCbl. In this study, we demonstrate that binding of the substrate ATP to ATR that is fully loaded with AdoCbl leads to the ejection of 1 equivalent of the cofactor into solution. In the presence of methylmalonyl-CoA mutase and ATP, AdoCbl is transferred from ATR to the acceptor protein in a process that exhibits an ∼3.5-fold lower Kact for ATP compared to the one in which cofactor is released into solution. Furthermore, ATP favorably influences cofactor transfer in the forward direction by reducing the ratio of apo-methylmalonyl-CoA mutase/holo-ATR required for delivery of 1 equivalent of AdoCbl, from 4 to 1. These results lead us to propose a rotary mechanism for ATR function in which, at any given time, only two of its active sites are used for AdoCbl synthesis and where binding of ATP to the vacant site leads to the transfer of the high value AdoCbl product to the acceptor mutase.


785. A partial synthesis of the vitamin B12 coenzyme …

Adenosyltransferases (ATRs) catalyze the synthesis of the reactive cobalt-carbon bond found in coenzyme B12 or 5′- deoxyadenosylcobalamin (AdoCbl), which serves as a cofactor for a number of isomerases. The reaction involves a reductive adenosylation of cob(II)alamin in which an electron delivered by a reductase reduces cob(II)alamin to cob(I)alamin, which attacks the 5′-carbon of ATP to form AdoCbl and inorganic triphosphate. Of the three classes of ATRs found in nature, the PduO type, which is also the only one found in mammals, is the most extensively studied. The crystal structures of a number of PduO-type ATRs are available and reveal a trimeric organization with the active sites located at the subunit interfaces. We have previously demonstrated that the ATR from Methylobacterium extorquens, which supports methylmalonyl-CoA mutase activity, serves dual functions; i.e., it tailors the active AdoCbl form of the cofactor and then transfers it directly to the dependent mutase (Padovani et al. (2008) Nat. Chem. Biol. 4, 194). Only two of the three active sites in ATR are simultaneously occupied by AdoCbl. In this study, we demonstrate that binding of the substrate ATP to ATR that is fully loaded with AdoCbl leads to the ejection of 1 equivalent of the cofactor into solution. In the presence of methylmalonyl-CoA mutase and ATP, AdoCbl is transferred from ATR to the acceptor protein in a process that exhibits an ∼3.5-fold lower Kact for ATP compared to the one in which cofactor is released into solution. Furthermore, ATP favorably influences cofactor transfer in the forward direction by reducing the ratio of apo-methylmalonyl-CoA mutase/holo-ATR required for delivery of 1 equivalent of AdoCbl, from 4 to 1. These results lead us to propose a rotary mechanism for ATR function in which, at any given time, only two of its active sites are used for AdoCbl synthesis and where binding of ATP to the vacant site leads to the transfer of the high value AdoCbl product to the acceptor mutase.