An unusual light-sensing function for coenzyme B12 in bacterial transcription regulator CarH.

Coenzyme B12 is one of the most complex cofactors found in nature and synthesized de novo by certain groups of bacteria. Although its use in various enzymatic reactions is well characterized, only recently an unusual light-sensing function has been ascribed to coenzyme B12. It has been reported that the coenzyme B12 binding protein CarH, found in the carotenoid biosynthesis pathway of several thermostable bacteria, binds to the promoter region of DNA and suppresses transcription. To overcome the harmful effects of light-induced damage in the cells, CarH releases DNA in the presence of light and promotes transcription and synthesis of carotenoids, thereby working as a photoreceptor. CarH is able to achieve this by exploiting the photosensitive nature of the CoC bond between the adenosyl moiety and the cobalt atom in the coenzyme B12 molecule. Extensive structural and spectroscopy studies provided a mechanistic understanding of the molecular basis of this unique light-sensitive reaction. Most studies on CarH have used the ortholog from the thermostable bacterium Thermus thermophilus, due to the ease with which it can be expressed and purified in high quantities. In this chapter we give an overview of this intriguing class of photoreceptors and report a step-by-step protocol for expression, purification and spectroscopy experiments (both static and time-resolved techniques) employed in our laboratory to study CarH from T. thermophilus. We hope the contents of this chapter will be of interest to the wider coenzyme B12 community and apprise them of the potential and possibilities of using coenzyme B12 as a light-sensing probe in a protein scaffold.

CobT and BzaC catalyze the regiospecific activation and methylation of the 5-hydroxybenzimidazole lower ligand in anaerobic cobamide biosynthesis.

Vitamin B12 and other cobamides are essential cofactors required by many organisms and are synthesized by a subset of prokaryotes via distinct aerobic and anaerobic routes. The anaerobic biosynthesis of 5,6-dimethylbenzimidazole (DMB), the lower ligand of vitamin B12, involves five reactions catalyzed by the bza operon gene products, namely the hydroxybenzimidazole synthase BzaAB/BzaF, phosphoribosyltransferase CobT, and three methyltransferases, BzaC, BzaD, and BzaE, that conduct three distinct methylation steps. Of these, the methyltransferases that contribute to benzimidazole lower ligand diversity in cobamides remain to be characterized, and the precise role of the bza operon protein CobT is unclear. In this study, we used the bza operon from the anaerobic bacterium Moorella thermoacetica (comprising bzaA-bzaB-cobT-bzaC) to examine the role of CobT and investigate the activity of the first methyltransferase, BzaC. We studied the phosphoribosylation catalyzed by MtCobT and found that it regiospecifically activates 5-hydroxybenzimidazole (5-OHBza) to form the 5-OHBza-ribotide (5-OHBza-RP) isomer as the sole product. Next, we characterized the domains of MtBzaC and reconstituted its methyltransferase activity with the predicted substrate 5-OHBza and with two alternative substrates, the MtCobT product 5-OHBza-RP and its riboside derivative 5-OHBza-R. Unexpectedly, we found that 5-OHBza-R is the most favored MtBzaC substrate. Our results collectively explain the long-standing observation that the attachment of the lower ligand in anaerobic cobamide biosynthesis is regiospecific. In conclusion, we validate MtBzaC as a SAM:hydroxybenzimidazole-riboside methyltransferase (HBIR-OMT). Finally, we propose a new pathway for the synthesis and activation of the benzimidazolyl lower ligand in anaerobic cobamide biosynthesis.

The Methanosarcina mazei MM2060 Gene Encodes a Bifunctional Kinase/Decarboxylase Enzyme Involved in Cobamide Biosynthesis.

Cobamides (Cbas) are synthesized by many archaea, but some aspects of Cba biosynthesis in these microorganisms remain unclear. Here, we demonstrate that open reading frame MM2060 in the archaeum Methanosarcina mazei strain Gö1 encodes a bifunctional enzyme with l-threonine- O-3-phosphate (l-Thr-P) decarboxylase (EC 4.1.1.81) and l-Thr kinase activities (EC 2.7.1.177). In Salmonella enterica, where Cba biosynthesis has been extensively studied, the activities mentioned above are encoded by separate genes, namely, cobD and pduX, respectively. The activities associated with the MM2060 protein ( MmCobD) were validated in vitro and in vivo. In vitro, MmCobD used ATP and l-Thr as substrates and generated ADP, l-Thr-P, and ( R)-1-aminopropan-2-ol O-phosphate as products. Notably, MmCobD has a 111-amino acid C-terminal extension of unknown function, which contains a putative metal-binding motif. This C-terminal domain alone did not display activity either in vivo or in vitro. Although the C-terminal MmCobD domain was not required for l-Thr-P decarboxylase or l-Thr kinase activities in vivo, its absence negatively affected both activities. In vitro results suggested that this domain may have a regulatory or substrate-gating role. When purified under anoxic conditions, MmCobD displayed Michaelis-Menten kinetics and had a 1000-fold higher affinity for ATP and a catalytic efficiency 1300-fold higher than that of MmCobD purified under oxic conditions. To the best of our knowledge, MmCobD is the first example of a new class of l-Thr-P decarboxylases that also have l-Thr kinase activity. An archaeal protein with l-Thr kinase activity had not been identified prior to this work.

Characterization of 1,2-Propanediol Dehydratases Reveals Distinct Mechanisms for B12-Dependent and Glycyl Radical Enzymes.

Propanediol dehydratase (PD), a recently characterized member of the glycyl radical enzyme (GRE) family, uses protein-based radicals to catalyze the chemically challenging dehydration of ( S)-1,2-propanediol. This transformation is also performed by the well-studied enzyme B12-dependent propanediol dehydratase (B12-PD) using an adenosylcobalamin cofactor. Despite the prominence of PD in anaerobic microorganisms, it remains unclear if the mechanism of this enzyme is similar to that of B12-PD. Here we report 18O labeling experiments that suggest PD and B12-PD employ distinct mechanisms. Unlike B12-PD, PD appears to catalyze the direct elimination of a hydroxyl group from an initially formed substrate-based radical, avoiding the generation of a 1,1- gem diol intermediate. Our studies provide further insights into how GREs perform elimination chemistry and highlight how nature has evolved diverse strategies for catalyzing challenging reactions.

Switch I-dependent allosteric signaling in a G-protein chaperone-B12 enzyme complex.

G-proteins regulate various processes ranging from DNA replication and protein synthesis to cytoskeletal dynamics and cofactor assimilation and serve as models for uncovering strategies deployed for allosteric signal transduction. MeaB is a multifunctional G-protein chaperone, which gates loading of the active 5'-deoxyadenosylcobalamin cofactor onto methylmalonyl-CoA mutase (MCM) and precludes loading of inactive cofactor forms. MeaB also safeguards MCM, which uses radical chemistry, against inactivation and rescues MCM inactivated during catalytic turnover by using the GTP-binding energy to offload inactive cofactor. The conserved switch I and II signaling motifs used by G-proteins are predicted to mediate allosteric regulation in response to nucleotide binding and hydrolysis in MeaB. Herein, we targeted conserved residues in the MeaB switch I motif to interrogate the function of this loop. Unexpectedly, the switch I mutations had only modest effects on GTP binding and on GTPase activity and did not perturb stability of the MCM-MeaB complex. However, these mutations disrupted multiple MeaB chaperone functions, including cofactor editing, loading, and offloading. Hence, although residues in the switch I motif are not essential for catalysis, they are important for allosteric regulation. Furthermore, single-particle EM analysis revealed, for the first time, the overall architecture of the MCM-MeaB complex, which exhibits a 2:1 stoichiometry. These EM studies also demonstrate that the complex exhibits considerable conformational flexibility. In conclusion, the switch I element does not significantly stabilize the MCM-MeaB complex or influence the affinity of MeaB for GTP but is required for transducing signals between MeaB and MCM.

Coordination chemistry controls the thiol oxidase activity of the B12-trafficking protein CblC.

The cobalamin or B12 cofactor supports sulfur and one-carbon metabolism and the catabolism of odd-chain fatty acids, branched-chain amino acids, and cholesterol. CblC is a B12-processing enzyme involved in an early cytoplasmic step in the cofactor-trafficking pathway. It catalyzes the glutathione (GSH)-dependent dealkylation of alkylcobalamins and the reductive decyanation of cyanocobalamin. CblC from Caenorhabditis elegans (ceCblC) also exhibits a robust thiol oxidase activity, converting reduced GSH to oxidized GSSG with concomitant scrubbing of ambient dissolved O2 The mechanism of thiol oxidation catalyzed by ceCblC is not known. In this study, we demonstrate that novel coordination chemistry accessible to ceCblC-bound cobalamin supports its thiol oxidase activity via a glutathionyl-cobalamin intermediate. Deglutathionylation of glutathionyl-cobalamin by a second molecule of GSH yields GSSG. The crystal structure of ceCblC provides insights into how architectural differences at the α- and ß-faces of cobalamin promote the thiol oxidase activity of ceCblC but mute it in wild-type human CblC. The R161G and R161Q mutations in human CblC unmask its latent thiol oxidase activity and are correlated with increased cellular oxidative stress disease. In summary, we have uncovered key architectural features in the cobalamin-binding pocket that support unusual cob(II)alamin coordination chemistry and enable the thiol oxidase activity of ceCblC.

Glycerol Dehydratases: Biochemical Structures, Catalytic Mechanisms, and Industrial Applications in 1,3-Propanediol Production by Naturally Occurring and Genetically Engineered Bacterial Strains.

To date, two types of glycerol dehydratases have been reported: coenzyme B12-dependent and coenzyme B12-independent glycerol dehydratases. The three-dimensional structure of the former is a dimer of αßγ heterotrimer, while that of the latter is a homodimer. Their mechanisms of reaction are typically enzymatic radical catalysis. Functional radical in both the glycerol dehydratases is the adenosyl radical. However, the adenosyl radical in the former originates from coenzyme B12 by homolytic cleavage, and that in the latter from S-adenosyl-methionine. Until some years ago, Clostridium butyricum VPI 1718 was the only microorganism known to possess B12-independent glycerol dehydratase, but since then, several other bacteria with this unique capability have been identified. This article focuses on the glycerol dehydratases and on 1,3-propanediol production from glycerol by naturally occurring and genetically engineered bacterial strains containing glycerol dehydratase.

High resolution melting analysis of the MMAB gene in cblB patients and in those with undiagnosed methylmalonic aciduria.

Isolated methylmalonic aciduria (MMA) results either from a defect in the mitochondrial enzyme methylmalonylCoA mutase (MCM), or in the intracellular conversion of vitamin B12 (cobalamin) into its active coenzyme adenosylcobalamin (AdoCbl). Mutations in the MMAB gene affect the function of the enzyme ATP:cob(I)alamin adenosyltransferase (ATR) and the production of AdoCbl. Measurement of MCM function in cultured patient fibroblasts, followed by somatic cell complementation analysis in cases where MCM function is decreased, has classically been used to diagnose the cblB cobalamin disorder. A patient with persistent MMA, who could not be diagnosed using traditional somatic cell studies, was subsequently shown by sequencing in a clinical laboratory to contain two variants in the MMAB gene. This observation brings into question whether somatic cell studies have failed to diagnose other cblB patients with mild cellular phenotypes. A high resolution melting analysis (HRMA) assay was developed for the MMAB gene. It was used to scan 96 reference samples and two cohorts of patients: 42 patients diagnosed with cblB by complementation studies; and 181 patients with undiagnosed MMA. MMAB mutations, including one novel nonsense mutation (c.12 C>A [p.C4X]), were identified in all members of the cblB cohort. Four patients with undiagnosed MMA, including the index case described above, were found to contain variants in the MMAB gene: c.185C>T (p.T62M), c.394T>C (p.C132R), c.398C>T (p.S133F), c.521C>T (p.S174L), c.572G>A (p.R191Q). Only the index case was found to have two variants, suggesting that somatic cell studies diagnose almost all cblB patients.

Loss of allostery and coenzyme B12 delivery by a pathogenic mutation in adenosyltransferase.

ATP-dependent cob(I)alamin adenosyltransferase (ATR) is a bifunctional protein: an enzyme that catalyzes the adenosylation of cob(I)alamin and an escort that delivers the product, adenosylcobalamin (AdoCbl or coenzyme B(12)), to methylmalonyl-CoA mutase (MCM), resulting in holoenzyme formation. Failure to assemble holo-MCM leads to methylmalonic aciduria. We have previously demonstrated that only 2 equiv of AdoCbl bind per homotrimer of ATR and that binding of ATP to the vacant active site triggers ejection of 1 equiv of AdoCbl from an adjacent site. In this study, we have mimicked in the Methylobacterium extorquens ATR, a C-terminal truncation mutation, D180X, described in a patient with methylmalonic aciduria, and characterized the associated biochemical penalties. We demonstrate that while k(cat) and K(M)(Cob(I)) for D180X ATR are only modestly decreased (by 3- and 2-fold, respectively), affinity for the product, AdoCbl, is significantly diminished (400-fold), and the negative cooperativity associated with its binding is lost. We also demonstrate that the D180X mutation corrupts ATP-dependent cofactor ejection, which leads to transfer of AdoCbl from wild-type ATR to MCM. These results suggest that the pathogenicity of the corresponding human truncation mutant results from its inability to sequester AdoCbl for direct transfer to MCM. Instead, cofactor release into solution is predicted to reduce the capacity for holo-MCM formation, leading to disease.

Structures of the human GTPase MMAA and vitamin B12-dependent methylmalonyl-CoA mutase and insight into their complex formation.

Vitamin B(12) (cobalamin, Cbl) is essential to the function of two human enzymes, methionine synthase (MS) and methylmalonyl-CoA mutase (MUT). The conversion of dietary Cbl to its cofactor forms, methyl-Cbl (MeCbl) for MS and adenosyl-Cbl (AdoCbl) for MUT, located in the cytosol and mitochondria, respectively, requires a complex pathway of intracellular processing and trafficking. One of the processing proteins, MMAA (methylmalonic aciduria type A), is implicated in the mitochondrial assembly of AdoCbl into MUT and is defective in children from the cblA complementation group of cobalamin disorders. To characterize the functional interplay between MMAA and MUT, we have crystallized human MMAA in the GDP-bound form and human MUT in the apo, holo, and substrate-bound ternary forms. Structures of both proteins reveal highly conserved domain architecture and catalytic machinery for ligand binding, yet they show substantially different dimeric assembly and interaction, compared with their bacterial counterparts. We show that MMAA exhibits GTPase activity that is modulated by MUT and that the two proteins interact in vitro and in vivo. Formation of a stable MMAA-MUT complex is nucleotide-selective for MMAA (GMPPNP over GDP) and apoenzyme-dependent for MUT. The physiological importance of this interaction is highlighted by a recently identified homoallelic patient mutation of MMAA, G188R, which, we show, retains basal GTPase activity but has abrogated interaction. Together, our data point to a gatekeeping role for MMAA by favoring complex formation with MUT apoenzyme for AdoCbl assembly and releasing the AdoCbl-loaded holoenzyme from the complex, in a GTP-dependent manner.

Functional analysis of the nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase (CobT) enzyme, involved in the late steps of coenzyme B12 biosynthesis in Salmonella enterica.

In Salmonella enterica, the CobT enzyme activates the lower ligand base during the assembly of the nucleotide loop of adenosylcobalamin (AdoCbl) and other cobamides. Previously, mutational analysis identified a class of alleles (class M) that failed to restore AdoCbl biosynthesis during intragenic complementation studies. To learn why class M cobT mutations were deleterious, we determined the nature of three class M cobT alleles and performed in vivo and in vitro functional analyses guided by available structural data on the wild-type CobT (CobT(WT)) enzyme. We analyzed the effects of the variants CobT(G257D), CobT(G171D), CobT(G320D), and CobT(C160A). The latter was not a class M variant but was of interest because of the potential role of a disulfide bond between residues C160 and C256 in CobT activity. Substitutions G171D, G257D, and G320D had profound negative effects on the catalytic efficiency of the enzyme. The C160A substitution rendered the enzyme fivefold less efficient than CobT(WT). The CobT(G320D) protein was unstable, and results of structure-guided site-directed mutagenesis suggest that either variants CobT(G257D) and CobT(G171D) have less affinity for 5,6-dimethylbenzimidazole (DMB) or access of DMB to the active site is restricted in these variant proteins. The reported lack of intragenic complementation among class M cobT alleles is caused in some cases by unstable proteins, and in others it may be caused by the formation of dimers between two mutant CobT proteins with residual activity that is so low that the resulting CobT dimer cannot synthesize sufficient product to keep up with even the lowest demand for AdoCbl.

Demonstration that CobG, the monooxygenase associated with the ring contraction process of the aerobic cobalamin (vitamin B12) biosynthetic pathway, contains an Fe-S center and a mononuclear non-heme iron center.

The ring contraction process that occurs during cobalamin (vitamin B(12)) biosynthesis is mediated via the action of two enzymes, CobG and CobJ. The first of these generates a tertiary alcohol at the C-20 position of precorrin-3A by functioning as a monooxygenase, a reaction that also forms a gamma lactone with the acetic acid side chain on ring A. The product, precorrin-3B, is then acted upon by CobJ, which methylates at the C-17 position and promotes ring contraction of the macrocycle by catalyzing a masked pinacol rearrangement. Here, we report the characterization of CobG enzymes from Pseudomonas denitrificans and Brucella melitensis. We show that both contain a [4Fe-4S] center as well as a mononuclear non-heme iron. Although both enzymes are active in vivo, the P. denitrificans enzyme was found to be inactive in vitro. Further analysis of this enzyme revealed that the mononuclear non-heme iron was not reducible, and it was concluded that it is rapidly inactivated once it is released from the bacterial cell. In contrast, the B. melitensis enzyme was found to be fully active in vitro and the mononuclear non-heme iron was reducible by dithionite. The reduced mononuclear non-heme was able to react with the oxygen analogue NO, but only in the presence of the substrate precorrin-3A. The cysteine residues responsible for binding the Fe-S center were identified by site-directed mutagenesis. A mechanism for CobG is presented.

Roles of adenine anchoring and ion pairing at the coenzyme B12-binding site in diol dehydratase catalysis.

The X-ray structure of the diol dehydratase-adeninylpentylcobalamin complex revealed that the adenine moiety of adenosylcobalamin is anchored in the adenine-binding pocket of the enzyme by hydrogen bonding of N3 with the side chain OH group of Seralpha224, and of 6-NH(2), N1 and N7 with main chain amide groups of other residues. A salt bridge is formed between the epsilon-NH(2) group of Lysbeta135 and the phosphate group of cobalamin. To assess the importance of adenine anchoring and ion pairing, Seralpha224 and Lysbeta135 mutants of diol dehydratase were prepared, and their catalytic properties investigated. The Salpha224A, Salpha224N and Kbeta135E mutants were 19-2% as active as the wild-type enzyme, whereas the Kbeta135A, Kbeta135Q and Kbeta135R mutants retained 58-76% of the wild-type activity. The presence of a positive charge at the beta135 residue increased the affinity for cobalamins but was not essential for catalysis, and the introduction of a negative charge there prevented the enzyme-cobalamin interaction. The Salpha224A and Salpha224N mutants showed a k(cat)/k(inact) value that was less than 2% that of the wild-type, whereas for Lysbeta135 mutants this value was in the range 25-75%, except for the Kbeta135E mutant (7%). Unlike the wild-type holoenzyme, the Salpha224N and Salpha224A holoenzymes showed very low susceptibility to oxygen in the absence of substrate. These findings suggest that Seralpha224 is important for cobalt-carbon bond activation and for preventing the enzyme from being inactivated. Upon inactivation of the Salpha224A holoenzyme during catalysis, cob(II)alamin accumulated, and a trace of doublet signal due to an organic radical disappeared in EPR. 5'-Deoxyadenosine was formed from the adenosyl group, and the apoenzyme itself was not damaged. This inactivation was thus considered to be a mechanism-based one.

Construction and characterization of hybrid dehydratases between adenosylcobalamin-dependent diol and glycerol dehydratases.

Adenosylcobalamin-dependent diol dehydratase and glycerol dehydratase are isofunctional enzymes that catalyze the dehydration of 1,2-diols to the corresponding aldehydes. Although they bear different metabolic roles, both enzymes consist of three different subunits and possess a common (alphabetagamma)2 structure. To elucidate the roles of each subunit, we constructed expression plasmids for the hybrid dehydratases between diol dehydratase of Klebsiella oxytoca and glycerol dehydratase of Klebsiella pneumoniae in all the combinations of subunits by gene engineering techniques. All of the hybrid enzymes were produced in Escherichia coli at high levels, but only two hybrid enzymes consisting of the alpha subunit from glycerol dehydratase and the beta subunits from diol dehydratase showed high activity. The substrate specificity, the susceptibility to inactivation by glycerol, and the monovalent cation specificity of the wild type and hybrid enzymes were primarily determined by the origin of their alpha subunits.

Minimal functions and physiological conditions required for growth of salmonella enterica on ethanolamine in the absence of the metabolosome.

During growth on ethanolamine, Salmonella enterica synthesizes a multimolecular structure that mimics the carboxysome used by some photosynthetic bacteria to fix CO(2). In S. enterica, this carboxysome-like structure (hereafter referred to as the ethanolamine metabolosome) is thought to contain the enzymatic machinery needed to metabolize ethanolamine into acetyl coenzyme A (acetyl-CoA). Analysis of the growth behavior of mutant strains of S. enterica lacking specific functions encoded by the 17-gene ethanolamine utilization (eut) operon established the minimal biochemical functions needed by this bacterium to use ethanolamine as a source of carbon and energy. The data obtained support the conclusion that the ethanolamine ammnonia-lyase (EAL) enzyme (encoded by the eutBC genes) and coenzyme B(12) are necessary and sufficient to grow on ethanolamine. We propose that the EutD phosphotransacetylase and EutG alcohol dehydrogenase are important to maintain metabolic balance. Glutathione (GSH) had a strong positive effect that compensated for the lack of the EAL reactivase EutA protein under aerobic growth on ethanolamine. Neither GSH nor EutA was needed during growth on ethanolamine under reduced-oxygen conditions. GSH also stimulated growth of a strain lacking the acetaldehyde dehydrogenase (EutE) enzyme. The role of GSH in ethanolamine catabolism is complex and requires further investigation. Our data show that the ethanolamine metabolosome is not involved in the biochemistry of ethanolamine catabolism. We propose the metabolosome is needed to concentrate low levels of ethanolamine catabolic enzymes, to keep the level of toxic acetaldehyde low, to generate enough acetyl-CoA to support cell growth, and to maintain a pool of free CoA.

Complementation studies in the cblA class of inborn error of cobalamin metabolism: evidence for interallelic complementation and for a new complementation class (cblH).

AIM: To investigate genetic heterogeneity within the cblA class of inborn error of cobalamin metabolism. CONTEXT: The cblA disorder is characterised by vitamin B12 (cobalamin) responsive methylmalonic aciduria and deficient synthesis of adenosylcobalamin, required for activity of the mitochondrial enzyme methylmalonyl CoA mutase. The cblA gene has not been identified or cloned. We have previously described a patient with the clinical and biochemical phenotype of the cblA disorder whose fibroblasts complemented cells from patients with all known types of inborn error of adenosylcobalamin synthesis, including cblA. METHODS: We have performed somatic cell complementation analysis of the cblA variant fibroblast line with a panel of 28 cblA lines. We have also performed detailed complementation analysis on a panel of 10 cblA fibroblast lines, not including the cblA variant line. RESULTS: The cblA variant line complemented all 28 cell lines of the panel. There was evidence for interallelic complementation among the 10 cblA lines used for detailed complementation analysis; no cell line in this panel complemented all other members. CONCLUSIONS: These results strongly suggest that the cblA variant represents a novel complementation class, which we have designated cblH and which represents a mutation at a distinct gene. They also suggest that the cblA gene encodes a protein that functions as a multimer, allowing for extensive interallelic complementation.

[Neural tube defects (NTD)--assessment from the perspective of 25 years of studies]. / Wady cewy nerwowej (WCN)--ocena z perspektywy 25 lat badan.

The present paper illustrates the authors 25-year experience in step by step approach to the definition of environmental and genetic background of neural tube defects. Based on the birth defects registry, a complete ascertainment of all deliveries was performed in Southern Poland during two period: 1970-1972, and 1979-1981. The birth prevalence of neural tube defects (NTD), as well as other CNS malformations was determined. The empiric recurrence risk was calculated as 3.2% +/- 1.6. Based on this figure, the relative risk (RR = 37.6 p < 0.001) and heritability (h2 = 74.7 +/- 6.7) were estimated. Our own modification of Morton's complex segregation analysis was applied. Three Mendelian (dominant, additive and recessive) and one multifactorial model were tested. The results did not provide a clear cut discrimination between different models; however the lowest 2 value was obtained for additive inheritance with 61% of penetrance and the frequency of sporadic cases equaled 55%. A search for genetic markers did not support the hypothesis that HLA-A,B,C loci are equivalents of T/t like locus in mice. The results of the study on transcobalamine levels in amniotic fluid may suggests that different transcobalamine metabolism reflects phenotypic expression of genetic susceptibility to NTD development. Current research and future perspectives on genetic and environmental background of NTD are also presented.

Repression of the cob operon of Salmonella typhimurium by adenosylcobalamin is influenced by mutations in the pdu operon.

The cob operon encodes functions needed for the biosynthesis of adenosylcobalamin (Ado-B12). Propanediol induces transcription of the cob operon and the neighboring pdu operon, which encodes proteins for the B12-dependent degradation of propanediol. Expression of the cob (but not the pdu) operon is repressed by exogenous cyanocobalamin. Evidence is provided that cob operon repression is signaled by internally generated Ado-B12, which can be formed either by the CobA adenosyltransferase or by an alternative adenosyltransferase (AdoT) that we infer is encoded within the pdu operon. Repression is also affected by mutations (AdoB) in the pdu operon that map upstream of the inferred pdu adenosyltransferase gene. Such mutations allow cobalamin to mediate repression at concentrations 100-fold lower than those needed in the wild type. It is proposed that these mutations eliminate a component of the propanediol dehydratase enzyme complex (PduCDE) and that this complex competes with the cob regulatory mechanism for a limited supply of Ado-B12.