Cobalamin Reductase Research Articles

Diol Dehydratase-Reactivase Is Essential for Recycling of Coenzyme B12 in Diol Dehydratase.

Holoenzymes of adenosylcobalamin-dependent diol and glycerol dehydratases undergo mechanism-based inactivation by glycerol and O2 inactivation in the absence of substrate, which accompanies irreversible cleavage of the coenzyme Co-C bond. The inactivated holodiol dehydratase and the inactive enzyme·cyanocobalamin complex were (re)activated by incubation with NADH, ATP, and Mg(2+) (or Mn(2+)) in crude extracts of Klebsiella oxytoca, suggesting the presence of a reactivating system in the extract. The reducing system with NADH could be replaced by FMNH2. When inactivated holoenzyme or the enzyme·cyanocobalamin complex, a model of inactivated holoenzyme, was incubated with purified recombinant diol dehydratase-reactivase (DD-R) and an ATP:cob(I)alamin adenosyltransferase in the presence of FMNH2, ATP, and Mg(2+), diol dehydratase activity was restored. Among the three adenosyltransferases (PduO, EutT, and CobA) of this bacterium, PduO and CobA were much more efficient for the reactivation than EutT, although PduO showed the lowest adenosyltransfease activity toward free cob(I)alamin. These results suggest that (1) diol dehydratase activity is maintained through coenzyme recycling by a reactivating system for diol dehydratase composed of DD-R, PduO adenosyltransferase, and a reducing system, (2) the releasing factor DD-R is essential for the recycling of adenosycobalamin, a tightly bound, prosthetic group-type coenzyme, and (3) PduO is a specific adenosylating enzyme for the DD reactivation, whereas CobA and EutT exert their effects through free synthesized coenzyme. Although FMNH2 was mainly used as a reductant in this study, a natural reducing system might consist of PduS cobalamin reductase and NADH.

Characterisation of PduS, the pdu Metabolosome Corrin Reductase, and Evidence of Substructural Organisation within the Bacterial Microcompartment

PduS is a corrin reductase and is required for the reactivation of the cobalamin-dependent diol dehydratase. It is one component encoded within the large propanediol utilisation (pdu) operon, which is responsible for the catabolism of 1,2-propanediol within a self-assembled proteinaceous bacterial microcompartment. The enzyme is responsible for the reactivation of the cobalamin coenzyme required by the diol dehydratase. The gene for the cobalamin reductase from Citrobacter freundii (pduS) has been cloned to allow the protein to be overproduced recombinantly in E. coli with an N-terminal His-tag. Purified recombinant PduS is shown to be a flavoprotein with a non-covalently bound FMN that also contains two coupled [4Fe-4S] centres. It is an NADH-dependent flavin reductase that is able to mediate the one-electron reductions of cob(III)alamin to cob(II)alamin and cob(II)alamin to cob(I)alamin. The [4Fe-4S] centres are labile to oxygen and their presence affects the midpoint redox potential of flavin. Evidence is presented that PduS is able to bind cobalamin, which is inconsistent with the view that PduS is merely a flavin reductase. PduS is also shown to interact with one of the shell proteins of the metabolosome, PduT, which is also thought to contain an [Fe-S] cluster. PduS is shown to act as a corrin reductase and its interaction with a shell protein could allow for electron passage out of the bacterial microcompartment.

Characterization of the PduS Cobalamin Reductase of Salmonella enterica and Its Role in the Pdu Microcompartment

Salmonella enterica degrades 1,2-propanediol (1,2-PD) in a coenzyme B12 (adenosylcobalamin, AdoCbl)-dependent fashion. Salmonella obtains AdoCbl by assimilation of complex precursors, such as vitamin B12 and hydroxocobalamin. Assimilation of these compounds requires reduction of their central cobalt atom from Co3+ to Co2+ to Co+, followed by adenosylation to AdoCbl. In this work, the His6-tagged PduS cobalamin reductase from S. enterica was produced at high levels in Escherichia coli, purified, and characterized. The anaerobically purified enzyme reduced cob(III)alamin to cob(II)alamin at a rate of 42.3±3.2 μmol min(-1) mg(-1), and it reduced cob(II)alamin to cob(I)alamin at a rate of 54.5±4.2 nmol min(-1) mg(-1) protein. The apparent Km values of PduS-His6 were 10.1±0.7 μM for NADH and 67.5±8.2 μM for hydroxocobalamin in cob(III)alamin reduction. The apparent Km values for cob(II)alamin reduction were 27.5±2.4 μM with NADH as the substrate and 72.4±9.5 μM with cob(II)alamin as the substrate. High-performance liquid chromatography (HPLC) and mass spectrometry (MS) indicated that each monomer of PduS contained one molecule of noncovalently bound flavin mononucleotide (FMN). Genetic studies showed that a pduS deletion decreased the growth rate of Salmonella on 1,2-PD, supporting a role in cobalamin reduction in vivo. Further studies demonstrated that the PduS protein is a component of the Pdu microcompartments (MCPs) used for 1,2-PD degradation and that it interacts with the PduO adenosyltransferase, which catalyzes the terminal step of AdoCbl synthesis. These studies further characterize PduS, an unusual MCP-associated cobalamin reductase, and, in conjunction with prior results, indicate that the Pdu MCP encapsulates a complete cobalamin assimilation system.

Increase in urinary purines and pyrimidines in patients with methylmalonic aciduria combined with homocystinuria

BackgroundMethylmalonic aciduria combined with homocystinuria (MMA–HC) is the biochemical trait of a metabolic disorder resulting from impaired conversion of dietary cobalamin (cbl, or vitamin B12) to its two metabolically active forms. Effects on urinary purine and pyrimidine levels have not been described for this condition. MethodsUrine samples were collected from three patients with methylmalonic aciduria combined with homocystinuria and from 70 healthy subjects. Urinary purine and pyrimidine levels were quantitated by the use of LC/UV–Vis and LC/ESI/MS. ResultsHigher urine levels of pyrimidines were detected with both methods in patients compared to controls. ConclusionMethylmalonic aciduria with homocystinuria is due to deficiency of the enzyme, cobalamin reductase. The enzyme defect leads to altered hepatic metabolism, which appears to modify circulating pyrimidine levels.

Identification, Characterization, and Structure/Function Analysis of a Corrin Reductase Involved in Adenosylcobalamin Biosynthesis

Vitamin B(12), the antipernicious anemia factor, is the cyano derivative of adenosylcobalamin, which is one of nature's most complex coenzymes. Adenosylcobalamin is made along one of two similar yet distinct metabolic pathways, which are referred to as the aerobic and anaerobic routes. The aerobic pathway for cobalamin biosynthesis proceeds via cobalt insertion into a ring-contracted macrocycle, which is closely followed by adenosylation of the cobalt ion. An important prerequisite for adenosylation is the reduction of the centrally chelated metal from Co(II) to a highly nucleophilic Co(I) form. We have cloned a gene, cobR, encoding a biosynthetic enzyme with this co(II)rrin reductase activity from Brucella melitensis. The protein has been overproduced, and the resulting flavoprotein has been purified, characterized, and crystallized and its structure determined to 1.6A resolution. Kinetic and EPR analysis reveals that the enzyme proceeds via a semiquinone form. It is proposed that CobR may interact with the adenosyltransferase to overcome the large thermodynamic barrier required for co(II)rrin reduction.

Biochemical evidence that the pduS gene encodes a bifunctional cobalamin reductase

Salmonella enterica degrades 1,2-propanediol (1,2-PD) by a pathway that requires coenzyme B(12) (adenosylcobalamin; AdoCbl). The genes specifically involved in 1,2-PD utilization (pdu) are found in a large contiguous cluster, the pdu locus. Earlier studies have indicated that this locus includes genes for the conversion of vitamin B(12) (cyanocobalamin; CNCbl) to AdoCbl and that the pduO gene encodes an ATP : cob(I)alamin adenosyltransferase which catalyses the terminal step of this process. Here, in vitro evidence is presented that the pduS gene encodes a bifunctional cobalamin reductase that catalyses two reductive steps needed for the conversion of CNCbl into AdoCbl. The PduS enzyme was produced in high levels in Escherichia coli. Enzyme assays showed that cell extracts from the PduS expression strain reduced cob(III)alamin (hydroxycobalamin) to cob(II)alamin at a rate of 91 nmol min(-1) mg(-1) and cob(II)alamin to cob(I)alamin at a rate of 7.8 nmol min(-1) mg(-1). In contrast, control extracts had only 9.9 nmol min(-1) mg(-1) cob(III)alamin reductase activity and no detectable cob(II)alamin reductase activity. Thus, these results indicated that the PduS enzyme is a bifunctional cobalamin reductase. Enzyme assays also showed that the PduS enzyme reduced cob(II)alamin to cob(I)alamin for conversion into AdoCbl by purified PduO adenosyltransferase. Moreover, studies in which iodoacetate was used as a chemical trap for cob(I)alamin indicated that the PduS and PduO enzymes physically interact and that cob(I)alamin is sequestered during the conversion of cob(II)alamin to AdoCbl by these two enzymes. This is likely to be important physiologically, since cob(I)alamin is extremely reactive and would need to be protected from unproductive by-reactions. Lastly, bioinformatic analyses showed that the PduS enzyme is unrelated in amino acid sequence to enzymes of known function currently present in GenBank. Hence, results indicate that the PduS enzyme represents a new class of cobalamin reductase.

Glutathionylcobalamin as an intermediate in the formation of cobalamin coenzymes

To evaluate the possible role of glutathionylcobalamin (GS-Cbl) in the intracellular metabolism of cobalamin, the following reactions were analyzed using extracts of rabbit spleen: (i) decyanation of cyanocobalamin; (ii) utilization of GS-Cbl by cobalamin reductase; (iii) participation of GS-Cbl in methionine biosynthesis; and (iv) conversion of GS-Cbl to adenosylcobalamin. Decyanation of cyanocobalamin required reduced glutathione which appeared to form a complex with the cobalamin. This complex decomposed during the extraction steps to sulfitocobalamin which was identified by high-performance liquid chromatography. Cobalamin reductase in spleen extract was more active with GS-Cbl than with aquocobalamin or cyanocobalamin as substrates (specific activities: 10.4, 2.8 and 0.93 nmol/mg/min, respectively). Methionine synthase utilized GS-Cbl as cofactor more efficiently than aquocobalamin or cyanocobalamin based on initial rates of enzyme activity. This suggests that GS-Cbl is a more direct precursor of the coenzyme required for methionine synthase. Formation of adenosylcobalamin from GS-Cbl was four times greater than from aquocobalamin alone. Based on these results, we propose that GS-Cbl or a closely related thiol-cobalamin adduct is a proximal precursor in cobalamin coenzyme biosynthesis.