AdoCbl-dependent Enzymes Research Articles

Functional genomic, biochemical, and genetic characterization of the Salmonella pduO gene, an ATP:cob(I)alamin adenosyltransferase gene.

Salmonella enterica degrades 1,2-propanediol by a pathway dependent on coenzyme B12 (adenosylcobalamin [AdoCb1]). Previous studies showed that 1,2-propanediol utilization (pdu) genes include those for the conversion of inactive cobalamins, such as vitamin B12, to AdoCbl. However, the specific genes involved were not identified. Here we show that the pduO gene encodes a protein with ATP:cob(I)alamin adenosyltransferase activity. The main role of this protein is apparently the conversion of inactive cobalamins to AdoCbl for 1,2-propanediol degradation. Genetic tests showed that the function of the pduO gene was partially replaced by the cobA gene (a known ATP:corrinoid adenosyltransferase) but that optimal growth of S. enterica on 1,2-propanediol required a functional pduO gene. Growth studies showed that cobA pduO double mutants were unable to grow on 1,2-propanediol minimal medium supplemented with vitamin B(12) but were capable of growth on similar medium supplemented with AdoCbl. The pduO gene was cloned into a T7 expression vector. The PduO protein was overexpressed, partially purified, and, using an improved assay procedure, shown to have cob(I)alamin adenosyltransferase activity. Analysis of the genomic context of genes encoding PduO and related proteins indicated that particular adenosyltransferases tend to be specialized for particular AdoCbl-dependent enzymes or for the de novo synthesis of AdoCbl. Such analyses also indicated that PduO is a bifunctional enzyme. The possibility that genes of unknown function proximal to adenosyltransferase homologues represent previously unidentified AdoCbl-dependent enzymes is discussed.

Molecular modeling of the mechanochemical triggering mechanism for catalysis of carbon–cobalt bond homolysis in coenzyme B12

The possible contributions of the mechanochemical triggering effect to the enzymatic activation of the carbon–cobalt bond of coenzyme B12 (5′-deoxyadenosylcobalamin, AdoCbl) for homolytic cleavage have been studied by molecular modeling and semi-empirical molecular orbital calculations. Classically, this effect has envisioned enzymatic compression of the axial Co–N bond in the ground state to cause upward folding of the corrin ring and subsequent sterically induced distortion of the Co–C bond leading to its destabilization. The models of this process show that in both methylcobalamin (CH3Cbl) and AdoCbl, compression of the axial Co–N bond does engender upward folding of the corrin ring, and that the extent of such upward folding is smaller in an analog in which the normal 5,6-dimethylbenzimidazole axial ligand is replaced by the sterically smaller ligand, imidazole (CH3(Im)Cbl and Ado(Im)Cbl). Furthermore, in AdoCbl, this upward folding of the corrin is accompanied by increases in the carbon–cobalt bond length and in the Co–C–C bond angle (which are also less pronounced in Ado(Im)Cbl), and which indicate that the Co–C bond is indeed destabilized by this mechanism. However, these effects on the Co–C bond are small, and destabilization of this bond by this mechanism is unlikely to contribute more than ca. 3 kcal mol−1 towards the enzymatic catalysis of Co–C bond homolysis, far short of the observed ca. 14 kcal mol−1. A second version of mechanochemical triggering, in which compression of the axial Co–N bond in the transition state for Co–C bond homolysis stabilizes the transition state by increased Co–N orbital overlap, has also been investigated. Stretching the Co–C bond to simulate the approach to the transition state was found to result in an upward folding of the corrin ring, a slight decrease in the axial Co–N bond length, a slight displacement of the metal atom from the plane of the equatorial nitrogens towards the “lower” axial ligand, and a decrease in strain energy amounting to about 8 kcal mol−1 for both AdoCbl and Ado(Im)Cbl. In such modeled transition states, compression of the axial Co–N bond to just below 2.0 Å (the distance subsequently found to provide maximal stabilization of the transition state by increased orbital overlap) required about 4 kcal mol−1 for AdoCbl, and about 2.5 kcal mol−1 for Ado(Im)Cbl. ZINDO/1 calculations on slightly simplified structures showed that maximal electronic stabilization of the transition state by about 10 kcal mol−1 occurred at an axial Co–N bond distance of 1.96 Å for both AdoCbl and Ado(Im)Cbl. The net result is that this type of transition state mechanochemical triggering can provide 14 kcal mol−1 of transition state stabilization for AdoCbl, and about 15.5 kcal mol−1 for the Ado(Im)Cbl, enough to completely explain the observed enzymatic catalysis. These results are discussed in the light of current knowledge about class I AdoCbl-dependent enzymes, in which the coenzyme is bound in its “base-off” conformation, with the lower axial ligand position occupied by the imidazole moiety of an active site histidine residue, and the class II enzymes, in which AdoCbl binds to the enzyme in its “base-on” conformation, and the pendent 5,6-dimethylbenzimidazole base remains coordinated to the metal during Co–C bond activation.

Ribonucleoside Triphosphate Reductase from Lactobacillus leichmannii: Kinetic Evaluation of a Series of Adenosylcobalamin Competitive Inhibitors, [ω-(Adenosin-5′-O-yl)alkyl]cobalamins, Which Mimic the Post Co-C Homolysis Intermediate

A series of [ω-(adenosin-5′-O-yl)alkyl]cobalamins were examined for their inhibitory properties of ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii in the presence of 5′-deoxyadenosylcobalamin (AdoCbl, Coenzyme B12). These AdoCbl analogs, in which oligomethylene chains (C3-C7) were inserted between the corrin Co-atom and a 5′-O-atom of the adenosine moiety, were designed to probe the Co-C bond posthomolysis state in AdoCbl-dependent enzymes, a state in which the Co and 5′-C distance is believed to be significantly increased. Experimentally, all five analogs were competitive inhibitors, with Ki in the range of 8–56 μM. The [ω-(adenosin-5′-O-yl)alkyl]cobalamin analog with C5 methylene carbons was the strongest inhibitor. This same pattern of inhibition, in which the C5-analog is the strongest inhibitor, was previously observed in the AdoCbl-dependent eliminase enzyme systems, diol dehydratase and glycerol dehydratase. However, in methylmalonyl CoA mutase, the strongest inhibition is by the C6-analog. This supports the hypothesis that the cobalamin posthomolysis intermediate in the eliminase enzymes differs from that in the mutase enzymes. These findings led, in turn, to an examination of the visible spectra of enzyme-bound cob(II)alamin in these two subclasses of AdoCbl-dependent enzymes. The results reveal an additional insight into the difference between the two classes: in the eliminases, the γ-band of bound cob(II)alamin is shifted from the 473 nm for free cob(II)alamin to longer wavelengths, 475–480 nm. However, in mutases, the γ-band of bound cob(II)alamin is shifted to shorter wavelengths, 465–470 nm. Overall, the results (a) provide strong evidence that two subclasses of AdoCbl-dependent enzymes exist, (b) give insights into the probable posthomolysis state in RTPR and other eliminases, and (c) identifies the C5-analog as the tightest-binding analog for crystallization and other biophysical studies.

Binding of Cob(II)alamin to the Adenosylcobalamin-dependent Ribonucleotide Reductase fromLactobacillus leichmannii: IDENTIFICATION OF DIMETHYLBENZIMIDAZOLE AS THE AXIAL LIGAND

The ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii catalyzes the reduction of nucleoside 5'-triphosphates to 2'-deoxynucleoside 5'-triphosphates and uses coenzyme B12, adenosylcobalamin (AdoCbl), as a cofactor. Use of a mechanism-based inhibitor, 2'-deoxy-2'-methylenecytidine 5'-triphosphate, and isotopically labeled RTPR and AdoCbl in conjunction with EPR spectroscopy has allowed identification of the lower axial ligand of cob(II)alamin when bound to RTPR. In common with the AdoCbl-dependent enzymes catalyzing irreversible heteroatom migrations and in contrast to the enzymes catalyzing reversible carbon skeleton rearrangements, the dimethylbenzimidazole moiety of the cofactor is not displaced by a protein histidine upon binding to RTPR.

Mechanism based inactivation of the adenosylcobalamin-dependent ribonucleotide reductase from L. leichmannii by 2′-ara-2′-azido-2′-deoxy adenosine-5′-triphosphate: Observation of paramagnetic intermediates

Ribonucleotide reductases catalyze the reduction of nucleotides to deoxynucleotides in all organisms. 2′-Ara-2′-azido-2′-deoxyadenosin-5′-triphosphate (araN 3ATP) is a potent mechanism based inhibitor of the AdoCbl-dependent enzyme from Lactobacillus leichmannii (RTPR). Incubation of RTPR with [3′- 3H]- or [β- 32P]- or [8- 3H]-araN 3ATP reveals that inactivation is accompanied by covalent labeling, formation of cob(II)alamin and 5′-deoxyadenosine in a ratio of 0.4:0.4:0.4 equivalent/equivalent of RTPR. During this inactivation at least three paramagnetic species are detected by EPR spectroscopy. RTPR inactivation by araN 3ATP, in contrast to all other mechanism based inhibitors, does NOT appear to involve 3′-carbon-hydrogen bond cleavage.

Electron spin-echo studies of the composition of the paramagnetic intermediate formed during the deamination of propanolamine by ethanolamine ammonia-lyase, and AdoCbl-dependent enzyme.

During the deamination of S-2-aminopropanol by the AdoCbl-dependent ethanolamine ammonia-lyase of Clostridia sp., a catalytic intermediate accumulates whose active site contains two paramagnetic species: cob(II)alamin and a free radical derived from the substrate molecule. Spin-echo spectroscopy has revealed that the unpaired electron on the substrate-derived radical is delocalized over a nitrogen atom that from its quadrupole splittings is probably a component of a secondary amide group. Experiments with 15N- and deuterium-labeled propanolamine gave no evidence of an interaction between this unpaired electron and the nitrogen originally attached to the substrate molecule. These results strongly suggest that the substrate-derived radical in this intermediate has already lost its nitrogen, and that this radical is stabilized by delocalization of the unpaired electron onto a nitrogen most likely situated in one of the peptide bonds of the enzyme backbone.

7] Ribosome-associated vitamin B12s adenosylating enzyme of Lactobacillus leichmannii

This chapter describes the partial purification of ribosome-associated vitamin B 12 adenosylating enzyme of Lactobacillus leichrnannii and the properties of the enzyme. Adenosylcobalamin (AdoCbl) synthesized in an initial incubation containing adenosylating enzyme, vitamin B 12r , ATP, and MnCl 2 is assayed in a second incubation by means of an AdoCbl-dependent enzyme reaction, the rate behavior of which is a function of AdoCbl concentration. The initial incubation of the adenosylating enzyme assay is conducted in small Thunberg tubes initially, containing the ingredients: cyanocobalamin (or hydroxocobalamin), 15 nmol; ATP, 0.088/μmol; MnCI 2 , 0.13/μmol; and Tris–HC1, pH 8.0, 20 μmol, in a total volume of 0.4 ml. Tubes are placed in ice, evacuated, and flushed with nitrogen. Potassium borohydride, 1 mg, is added to each tube under a stream of nitrogen, followed by enzyme-containing samples. Tubes are again evacuated, flushed with nitrogen, and kept chilled until reduction of vitamin B 12a is complete. The tube is incubated for 45 min in the dark at 37°, then cooled to 0°, a 10- to 50-μl aliquot is assayed for AdoCbl. Ribosomes from large-scale cultures of L. leichmannii are washed in TSM buffer and stored at –20° until use. All purification steps are performed at 0–4°.