Propanediol utilization genes (pdu) of Salmonella typhimurium: three genes for the propanediol dehydratase

The propanediol utilization (pdu) operon of Salmonella typhimurium encodes proteins required for the catabolism of propanediol, including a coenzyme B12-dependent propanediol dehydratase. A clone that expresses propanediol dehydratase activity was isolated from a Salmonella genomic library. DNA sequence analysis showed that the clone included part of the pduF gene, the pduABCDE genes, and a long partial open reading frame (ORF1). The clone included 3.9 kbp of pdu DNA which had not been previously sequenced. Complementation and expression studies with subclones constructed via PCR showed that three genes (pduCDE) are necessary and sufficient for propanediol dehydratase activity. The function of ORF1 was not determined. Analyses showed that the S. typhimurium propanediol dehydratase was related to coenzyme B12-dependent glycerol dehydratases from Citrobacter freundii and Klebsiella pneumoniae. Unexpectedly, the S. typhimurium propanediol dehydratase was found to be 98% identical in amino acid sequence to the Klebsiella oxytoca propanediol dehydratase; this is a much higher identity than expected, given the relationship between these organisms. DNA sequence analyses also supported previous studies indicating that the pdu operon was inherited along with the adjacent cobalamin biosynthesis operon by a single horizontal gene transfer.     

Molecular cloning, sequencing and characterization of the genes for adenosylcobalamin-dependent diol dehydratase of Klebsiella pneumoniae

Klebsiella pneumoniae and some of the other Enterobacteriaceae form both diol dehydratase and glycerol dehydratase in response to growth substrates. To compare these enzymes produced by the same bacterium, the pdd genes of K. pneumoniae encoding adenosylcobalamin-dependent diol dehydratase were cloned and sequenced. The sequential three open reading frames (pddA, pddB, and pddC genes) encoded polypeptides of 554, 228, and 174 amino acid residues with predicted molecular weights of 60,379(alpha), 24,401(beta), and 19,489(gamma), respectively. The deduced amino acid sequences of the subunits were 84-100% and 54-71% identical with those reported for diol dehydratases and glycerol dehydratases, respectively.     

Cloning, sequencing, and overexpression of the genes encoding coenzyme B12-dependent glycerol dehydratase of Citrobacter freundii

The genes encoding coenzyme B12-dependent glycerol dehydratase of Citrobacter freundii were cloned and overexpressed in Escherichia coli. The B12-free enzyme was purified to homogeneity. It consists of three types of subunits whose N-terminal sequences are in accordance with those deduced from the open reading frames dhaB, dhaC, and dhaE, coding for subunits of 60,433 (alpha), 21,487 (beta), and 16,121 (gamma) Da, respectively. The enzyme complex has the composition alpha2beta2gamma2. Amino acid alignments with the subunits of the recently sequenced diol dehydratase of Klebsiella oxytoca (T. Tobimatsu, T. Hara, M. Sakaguchi, Y. Kishimoto, Y. Wada, M. Isoda, T. Sakai, and T. Toraya, J. Biol. Chem. 270:7142-7148, 1995) revealed identities between 51.8 and 70.9%.     

Purification and properties of an iron-sulfur and FAD-containing 4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA delta 3-delta 2-isomerase from Clostridium aminobutyricum

4-Hydroxybutyryl-CoA dehydratase, the key enzyme in the metabolism of gamma-aminobutyrate in Clostridium aminobutyricum, represents approximately 15-25% of the soluble protein. The enzyme was purified to homogeneity under anaerobic conditions to a specific activity of 209 nkat mg-1. The dehydratase catalyses the reversible conversion of 4-hydroxybutyryl-CoA (Km = 50 microM) to crotonyl-CoA and possesses a probably intrinsic vinylacetyl-CoA delta 3-delta 2-isomerase with a specific activity of 223 nkat mg-1. The equilibrium of the reversible dehydration was determined from both sides as K = [crotonyl-CoA]/[4-hydroxybutyryl-CoA] = 4.2 +/- 0.3. Cyclopropylcarboxyl-CoA was not converted to crotonyl-CoA. The native enzyme has an apparent molecular mass of 232 kDa and is composed of four apparently identical subunits (molecular mass = 56 kDa), indicating a homotetrameric structure. Under anaerobic conditions the active enzyme revealed a brown colour and contained 2 +/- 0.2 mol FAD (64 +/- 5% oxidized), 16 +/- 0.8 mol Fe and 14.4 +/- 1.2 mol inorganic sulfur, which probably form iron-sulfur clusters. Exposure to air resulted initially in a slight activation followed by irreversible inactivation. Concomitantly the vinylacetyl-CoA delta-isomerase activity was lost and the colour of the enzyme changed to yellow. Reduction by sodium dithionite yielded inactive enzyme which could be completely reactivated by oxidation with potassium hexacyanoferrate(III). The data indicate that the active enzyme contains oxidized FAD despite its sensitivity towards oxygen. During the dehydration a non activated C-H bond at C-3 of 4-hydroxybutyryl-CoA has to be cleaved. A putative mechanism for 4-hydroxybutyryl-CoA dehydratase is proposed in which this cleavage is achieved by a FAD-dependent oxidation of 4-hydroxybutyryl-CoA to 4-hydroxycrotonyl-CoA. In a second step the hydroxyl group is substituted by a hydride derived from the now reduced FAD in an SN2' reaction leading to vinylacetyl-CoA. Finally isomerisation yields crotonyl-CoA. 4-Hydroxybutyryl-CoA dehydratase is quite distinct from 3-hydroxyacyl-CoA dehydratase (crotonase) and 2-hydroxyacyl-CoA dehydratases. Contrary to the latter enzyme [e.g. (R)-lactyl-CoA dehydratase and (R)-2-hydroxyglutaryl-CoA dehydratase] which are composed of three different subunits and similarly catalyse the cleavage of a non activated C-H bond at C-3, 4-hydroxybutyryl-CoA dehydratase does not require ATP, MgCl2 and Ti(III)citrate for activity. Furthermore 4-hydroxybutyryl-CoA dehydratase is not inactivated by oxidants such as 5 mM 4-nitrophenol, 5 mM chloramphenicol and 5 mM hydroxylamine.     

A reactivating factor for coenzyme B12-dependent diol dehydratase

Adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca undergoes suicide inactivation by glycerol, a physiological substrate. The coenzyme is modified through irreversible cleavage of its cobalt-carbon bond, resulting in inactivation of the enzyme by tight binding of the modified coenzyme to the active site. Recombinant DdrA and DdrB proteins of K. oxytoca were co-purified to homogeneity from cell-free extracts of Escherichia coli overexpressing the ddrAB genes. They existed as a tight complex, i.e. a putative reactivating factor, with an apparent molecular weight of 150,000. The factor consists of equimolar amounts of the two subunits with Mr of 64,000 (A) and 14,000 (B), encoded by the ddrA and ddrB genes, respectively. Therefore, its subunit structure is most likely A2B2. The factor not only reactivated glycerol-inactivated and O2-inactivated holoenzymes but also activated enzyme-cyanocobalamin complex in the presence of free adenosylcobalamin, ATP, and Mg2+. The reactivating factor mediated ATP-dependent exchange of the enzyme-bound cyanocobalamin for free 5-adeninylpentylcobalamin in the presence of ATP and Mg2+, but the reverse was not the case. Thus, it can be concluded that the inactivated holoenzyme becomes reactivated by exchange of the enzyme-bound, adenine-lacking cobalamins for free adenosylcobalamin, an adenine-containing cobalamin.     

Characterization of the coenzyme-B12-dependent glutamate mutase from Clostridium cochlearium produced in Escherichia coli

The glutamate mutase dependent on adenosylcobalamin (coenzyme B12) catalyzes the carbon skeleton rearrangement of (S)-glutamate to (2S,3S)-3-methylaspartate, the first step of the glutamate fermentation pathway of the anaerobic bacterium Clostridium cochlearium. The enzyme consists of two protein components, E, a dimer epsilon 2 (epsilon, 53.5 kDa) and S, a monomer (sigma, 14.8 kDa). The corresponding genes (glmE and glmS) were cloned, sequenced and over-expressed in Escherichia coli. The genes glmS and glmE are separated by glmL encoding a protein of unknown function. The deduced amino acid sequence of GlmL contains an ATP-binding motif which is common to chaperones of the HSP70-type, actin and procaryotic cell-cycle proteins. Both components of glutamate mutase were purified with excellent yields from cell-free extracts of E. coli carrying the corresponding genes. In contrast to component E, component S was shown to bind coenzyme B12. This observation strongly supports the idea that significant similarities of the amino acid sequences of component S and several other cobamide-dependent enzymes represent a common binding motif. Incubation of pure components E and S with coenzyme B12 resulted in the formation of a fully active glutamate mutase heterotetramer (epsilon 2 sigma 2) containing one molecule of coenzyme B12. EPR spectra of recombinant glutamate mutase, now available in sufficiency large amounts, were recorded after incubation of the enzyme with coenzyme B12 and (S)-glutamate. The EPR signals (gx,y approximately 2.1, gz = 1.985) were of much better resolution than observed earlier with the clostridial enzyme. Their typical hyperfine splitting is clearly derived from Co(II), which is involved in the formation of the paramagnetic species but is different from cob(II)alamin (gx,y = 2.25). The spin concentration was 34-50% of the concentration of the enzyme (epsilon 2 sigma 2) coenzyme complex. The competitive inhibitors (2S, 4S)-4-fluoroglutamate and 2-methyleneglutarate induced similar but not identical signals with spin concentrations of 134-148% of the enzyme concentration. Even (S)-[2,3,3,4,4-2H5]glutamate induced a signal significantly different to that of (S)-glutamate with an intensity of only 7%. These data suggest an involvement of the Co(II)-containing paramagnetic species in catalysis, the concentration of which reflects a steady state between its formation and decomposition. The large difference in the spin concentrations observed with (S)-glutamate as compared to the predeuterated glutamate is probably due to a kinetic isotope effect and indicates a cleavage of a C-H bond during formation of the paramagnetic species.(ABSTRACT TRUNCATED AT 400 WORDS)     

The metabolism of 6-deoxyhexoses in bacterial and animal cells

L-fucose and L-rhamnose are two 6-deoxyhexoses naturally occurring in several complex carbohydrates. In prokaryotes both of them are found in polysaccharides of the cell wall, while in animals only L-fucose has been described, which mainly participates to the structure of glycoconjugates, either in the cell membrane or secreted in biological fluids, such as ABH blood groups and Lewis system antigens. L-fucose and L-rhamnose are synthesized by two de novo biosynthetic pathways starting from GDP-D-mannose and dTDP-D-glucose, respectively, which share several common features. The first step for both pathways is a dehydration reaction catalyzed by specific nucleotide-sugar dehydratases. This leads to the formation of unstable 4-keto-6-deoxy intermediates, which undergo a subsequent epimerization reaction responsible for the change from D- to L-conformation, and then a NADPH-dependent reduction of the 4-keto group, with the consequent formation of either GDP-L-fucose or dTDP-L-rhamnose. These compounds are then the substrates of specific glycosyltransferases which are responsible for insertion of either L-fucose or L-rhamnose in the corresponding glycoconjugates. The enzyme involved in the first step of GDP-L-fucose biosynthesis in E. coli, i.e., GDP-D-mannose 4,6 dehydratase, has been recently expressed as recombinant protein and characterized in our laboratory. We have also cloned and fully characterized a human protein, formerly named FX, and an E. coli protein, WcaG, which display both the epimerase and the reductase activities, thus indicating that only two enzymes are required for GDP-L-fucose production. Fucosylated complex glycoconjugates at the cell surface can then be recognized by specific counter-receptors in interacting cells, these mechanisms initiating important processes including inflammation and metastasis. The second pathway starting from dTDP-D-glucose leads to the synthesis of antibiotic glycosides or, alternatively, to the production of dTDP-L-rhamnose. While several sets of data are available on the first enzyme of the pathway, i.e., dTDP-D-glucose dehydratase, the enzymes involved in the following steps still need to be identified and characterized.     

L-serine and L-threonine dehydratase from Clostridium propionicum. Two enzymes with different prosthetic groups

L-Serine dehydratase from the Gram-positive bacterium Peptostreptococcus asaccharolyticus is novel in the group of enzymes deaminating 2-hydroxyamino acids in that it is an iron-sulfur protein and lacks pyridoxal phosphate [Grabowski, R. and Buckel, W. (1991) Eur. J. Biochem. 199, 89-94]. It was proposed that this type of L-serine dehydratase is widespread among bacteria but has escaped intensive characterization due to its oxygen lability. Here, we present evidence that another Gram-positive bacterium, Clostridium propionicum, contains both an iron-sulfur-dependent L-serine dehydratase and a pyridoxal-phosphate-dependent L-threonine dehydratase. These findings support the notion that two independent mechanisms exist for the deamination of 2-hydroxyamino acids. L-Threonine dehydratase was purified 400-fold to apparent homogeneity and revealed as being a tetramer of identical subunits (m = 39 kDa). The purified enzyme exhibited a specific activity of 5 mu kat/mg protein and a Km for L-threonine of 7.7 mM. L-Serine (Km = 380 mM) was also deaminated, the V/Km ratio, however, being 118-fold lower than the one for L-threonine. L-Threonine dehydratase was inactivated by borohydride, hydroxylamine and phenylhydrazine, all known inactivators of pyridoxal-phosphate-containing enzymes. Incubation with NaB3H4 specifically labelled the enzyme. Activity of the phenylhydrazine-inactivated enzyme could be restored by pyridoxal phosphate. L-Serine dehydratase was also purified 400-fold, but its extreme instability did not permit purification to homogeneity. The enzyme was specific for L-serine (Km = 5 mM) and was inhibited by L-cysteine (Ki = 0.5 mM) and D-serine (Ki = 8 mM). Activity was insensitive towards borohydride, hydroxylamine and phenylhydrazine but was rapidly lost upon exposure to air. Fe2+ specifically reactivated the enzyme. L-Serine dehydratase was composed of two different subunits (alpha, m = 30 kDa; beta, m = 26 kDa), their apparent molecular masses being similar to the ones of the two subunits of the iron-sulfur-dependent enzyme from P. asaccharolyticus. Moreover, the N-terminal sequences of the small subunits from these two organisms were found to be 47% identical. In addition, 38% identity with the N-terminus of one of the two L-serine dehydratases of Escherichia coli was detected.     

Involvement of Fas/Fas ligand system-mediated apoptosis in the development of concanavalin A-induced hepatitis

A series of 2H- and 13C-labeled glutamates were used as substrates for coenzyme B12-dependent glutamate mutase, which equilibrates (S)-glutamate with (2S,3S)-3-methylaspartate. These compounds contained the isotopes at C-2, C-3, or C-4 of the carbon chain: [2-2H], [3,3-2H2], [4,4-2H2], [2,3,3,4,4-2H5], [2-13C], [3-13C], and [4-13C]glutamate. Each reaction was monitored by electron paramagnetic resonance (EPR) spectroscopy and revealed a similar signal characterized by g'xy = 2.1, g'z = 1.985, and A' = 5.0 mT. The interpretation of the spectral data was aided by simulations which gave close agreement with experiment. This approach underpinned the idea of the formation of a radical pair, consisting of cob(II)alamin interacting with an organic radical at a distance of 6.6 +/- 0.9 A. Comparison of the hyperfine couplings observed with unlabeled glutamate with those from the labeled glutamates enabled a principal contributor to the radical pair to be identified as the 4-glutamyl radical. These findings support the currently accepted mechanism for the glutamate mutase reaction, i.e., the process is initiated through hydrogen atom abstraction from C-4 of glutamate by the 5'-deoxyadenosyl radical, which is derived by homolysis of the Co-C sigma-bond of coenzyme B12.     

Genetic characterization of the pdu operon: use of 1,2-propanediol in Salmonella typhimurium

Salmonella typhimurium is able to catabolize 1,2-propanediol for use as the sole carbon and energy source; the first enzyme of this pathway requires the cofactor adenosyl cobalamin (Ado-B12). Surprisingly, Salmonella can use propanediol as the sole carbon source only in the presence of oxygen but can synthesize Ado-B12 only anaerobically. To understand this situation, we have studied the pdu operon, which encodes proteins for propanediol degradation. A set of pdu mutants defective in aerobic degradation of propanediol (with exogenous vitamin B12) defines four distinct complementation groups. Mutations in two of these groups (pduC and pduD) eliminate propanediol dehydratase activity. Based on mutant phenotypes, a third complementation group (pduG) appears to encode a cobalamin adenosyl transferase activity. No function has been assigned to the pduJ complementation group. Propionaldehyde dehydrogenase activity is eliminated by mutations in any of the four identified complementation groups, suggesting that this activity may require a complex of proteins encoded by the operon. None of the mutations analyzed affects either of the first two genes of the operon (pduA and pduB), which were identified by DNA sequence analysis. Available data suggest that the pdu operon includes enough DNA for about 15 genes and that the four genetically identified genes are the only ones required for aerobic use of propanediol.     

Inactivation of dehydratase [4Fe-4S] clusters and disruption of iron homeostasis upon cell exposure to peroxynitrite

Phagocytes produce both nitric oxide and superoxide as components of the oxidative defense against pathogens. Neither molecule is likely at physiological concentrations to kill cells. However, two of their reaction products, hydrogen peroxide and peroxynitrite, are strong oxidants, cell-permeant, and toxic. Hydrogen peroxide generates oxidative DNA damage, while the primary mechanism of toxicity of peroxynitrite has not yet been determined. Recent in vitro studies indicated that peroxynitrite is capable of oxidizing the [4Fe-4S] clusters of a family of dehydratases (Hausladen, A., and Fridovich, I. (1994) J. Biol. Chem. 269, 29405-29408; Castro, L., Rodriguez, M., and Radi, R. (1994) J. Biol. Chem. 269, 29409-29415). We demonstrate here that peroxynitrite at 1% of its lethal dose almost fully inactivated the labile dehydratases in Escherichia coli. The rate at which peroxynitrite inactivated the clusters substantially exceeded the rate at which it oxidized thiols or spontaneously decomposed. These results suggest that these dehydratases may be primary targets of peroxynitrite in vivo. Another consequence of the cluster damage was the release of 100 microM iron into the cytosol. During phagocytosis, this intracellular free iron could increase lethal DNA damage by hydrogen peroxide or protein modification by additional peroxynitrite. In response to peroxynitrite challenges, E. coli rapidly sequestered the intracellular free iron using an undefined scavenging system. The iron-sulfur clusters were more gradually repaired by a process that drew iron from its iron-storage proteins. These are likely to be critical events in the struggle between phagocyte and pathogen.     

Crystal structure of methyl-coenzyme M reductase: the key enzyme of biological methane formation

Methyl-coenzyme M reductase (MCR), the enzyme responsible for the microbial formation of methane, is a 300-kilodalton protein organized as a hexamer in an alpha2beta2gamma2 arrangement. The crystal structure of the enzyme from Methanobacterium thermoautotrophicum, determined at 1.45 angstrom resolution for the inactive enzyme state MCRox1-silent, reveals that two molecules of the nickel porphinoid coenzyme F430 are embedded between the subunits alpha, alpha', beta, and gamma and alpha', alpha, beta', and gamma', forming two identical active sites. Each site is accessible for the substrate methyl-coenzyme M through a narrow channel locked after binding of the second substrate coenzyme B. Together with a second structurally characterized enzyme state (MCRsilent) containing the heterodisulfide of coenzymes M and B, a reaction mechanism is proposed that uses a radical intermediate and a nickel organic compound.     

Elastic modulus of equine hoof horn, tested in wall samples, sole samples and frog samples at varying levels of moisture

In mammals and yeast, 5-aminolaevulinic acid dehydratase is a zinc-dependent enzyme that catalyses the synthesis of porphobilinogen-the pyrrole building block that is incorporated into all modified tetrapyrroles, including haem, chlorophyll and vitamin B12. The X-ray structure of this enzyme reveals how substitution of the catalytically important zinc ion by lead inactivates the enzyme and causes a form of pseudo-porphyria.     

Amino acid sequence of piguamerin, an antistasin-type protease inhibitor from the blood sucking leech Hirudo nipponia

Dimethylamine:5-hydroxybenzimidazolylcobamide methyltransferase (DMA-MT) was purified from cells of Methanosarcina barkeri Fusaro grown on trimethylamine. In the presence of methylcobalamine:coenzyme M methyltransferase isoenzyme II [MT2(II)] the enzyme quite specifically catalyzed the stoichiometric conversion of dimethylamine (apparent Km = 0.45 mM) and 2-mercaptoethane-sulfonate (coenzyme M) to monomethylamine and methyl-coenzyme M. Monomethylamine was a competitive inhibitor of the reaction (Ki = 4.5 mM). The apparent molecular mass of DMA-MT was 100 kDa and the enzyme was found to be a dimer, composed of identical 50-kDa subunits. A corrinoid content of 0.9 +/- 0.1 mol B12/mol holoenzyme was calculated from HPLC analysis. The as-isolated methyltransferase was inactive, but it could be reductively reactivated. Activation required the presence of methyltransferase-activating protein, ATP and dimethylamine. Incubation with these compounds resulted in the methylation of the corrinoid prosthetic group.     

Novel keto acid formate-lyase and propionate kinase enzymes are components of an anaerobic pathway in Escherichia coli that degrades L-threonine to propionate

An immunological analysis of an Escherichia coli strain unable to synthesize the main pyruvate formate-lyase enzyme Pfl revealed the existence of a weak, cross-reacting 85 kDa polypeptide that exhibited the characteristic oxygen-dependent fragmentation typical of a glycyl radical enzyme. Polypeptide fragmentation of this cross-reacting species was shown to be dependent on Pfl activase. Cloning and sequence analysis of the gene encoding this protein revealed that it coded for a new enzyme, termed TdcE, which has 82% identity with Pfl. On the basis of RNA analyses, the tdcE gene was shown to be part of a large operon that included the tdcABC genes, encoding an anaerobic threonine dehydratase, tdcD, coding for a propionate kinase, tdcF, the function of which is unknown, and the tdcG gene, which encodes a L-serine dehydratase. Expression of the tdcABCDEFG operon was strongly catabolite repressed. Enzyme studies showed that TdcE has both pyruvate formate-lyase and 2-ketobutyrate formate-lyase activity, whereas the TdcD protein is a new propionate/acetate kinase. By monitoring culture supernatants from various mutants using 1H nuclear magnetic resonance (NMR), we followed the anaerobic conversion of L-threonine to propionate. These studies confirmed that 2-ketobutyrate, the product of threonine deamination, is converted in vivo by TdcE to propionyl-CoA. These studies also revealed that Pfl and an as yet unidentified thiamine pyrophosphate-dependent enzyme(s) can perform this reaction. Double null mutants deficient in phosphotransacetylase (Pta) and acetate kinase (AckA) or AckA and TdcD were unable to metabolize threonine to propionate, indicating that propionyl-CoA and propionyl-phosphate are intermediates in the pathway and that ATP is generated during the conversion of propionyl-P to propionate by AckA or TdcD.     

How a protein prepares for B12 binding: structure and dynamics of the B12-binding subunit of glutamate mutase from Clostridium tetanomorphum

BACKGROUND: Glutamate mutase is an adenosylcobamide (coenzyme B12) dependent enzyme that catalyzes the reversible rearrangement of (2S)-glutamate to (2S,3S)-3-methylaspartate. The enzyme from Clostridium tetanomorphum comprises two subunits (of 53.7 and 14.8 kDa) and in its active form appears to be an alpha 2 beta 2 tetramer. The smaller subunit, termed MutS, has been characterized as the B12-binding component. Knowledge on the structure of a B12-binding apoenzyme does not exist. RESULTS: The solution structure and important dynamical aspects of MutS have been determined from a heteronuclear NMR study. The global fold of MutS in solution resembles that determined by X-ray crystallography for the B12-binding domains of Escherichia coli methionine synthase and Propionibacterium shermanii methylmalonyl CoA mutase. In these two proteins a histidine residue displaces the endogenous cobalt-coordinating ligand of the B12 cofactor. In MutS, however, the segment of the protein containing the conserved histidine residue forms part of an unstructured and mobile extended loop. CONCLUSIONS: A comparison of the crystal structures of two B12-binding domains, with bound B12 cofactor, and the solution structure of the apoprotein MutS has helped to clarify the mechanism of B12 binding. The major part of MutS is preorganized for B12 binding, but the B12-binding site itself is only partially formed. Upon binding B12, important elements of the binding site appear to become structured, including an alpha helix that forms one side of the cleft accommodating the nucleotide 'tail' of the cofactor.     

Human pterin-4 alpha-carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor-1 alpha. Characterization and kinetic analysis of wild-type and mutant enzymes

Pterin-4a-carbinolamine dehydratase/dimerization cofactor for hepatocyte nuclear factor-1 alpha is a protein with two different functions. We have overexpressed and purified the human wild-type protein, and its Cys81Ser and Cys81Arg mutants. The Cys81Arg mutant has been proposed to be causative in a hyperphenylalaninaemic patient [Citron, B. A., Kaufman, S., Milstien, S., Naylor, E. W., Greene, C. L. & Davis, M. D. (1993) Am. J. Hum. Genet. 53, 768-774]. The dehydratase behaves as a tetramer on gel filtration, while cross-linking experiments showed mono-, di-, tri-, and tetrameric forms, irrespective of the presence of the single Cys81. Sulfhydryl-modifying reagents did not affect the activity, but rather showed that Cys81 is exposed. Various pterins bind and quench the tryptophan fluorescence suggesting the presence of a specific binding site. The fluorescence is destroyed upon light irradiation. Wild-type and the Cys81Ser protein enhance the rate of the phenylalanine hydroxylase assay approximately 10-fold, a value similar to that of native dehydratase from rat liver; the Cys81Arg mutant, in contrast, has significantly lower activity. This is compatible with the hypothesis that the dehydratase is a rate-limiting factor for the in vivo phenylalanine hydroxylase reaction. The three proteins enhance the spontaneous dehydration of the synthetic substrate 6,6-dimethyl-7,8-dihydropterin-4a-carbinolamine approximately 50-70-fold at 4 degrees C and pH 8.5. The results are discussed in view of the recently solved three-dimensional structure of the enzyme [Ficner, R., Sauer, U. W., Stier, G. & Suck, D. (1995) EMBO J. 14, 2032-2042].     

Aspartate dehydrogenase in vitamin B12-producing Klebsiella pneumoniae IFO 13541

We found a new reaction of aspartic acid dehydrogenation, catalyzed by NADP(+)-dependent aspartate dehydrogenase, in vitamin B12-producing Klebsiella pneumoniae IFO 13541. The enzyme, which was purified from a crude extract of K.pneumoniae IFO 13541, catalyzes the oxidative deamination of aspartic acid to form oxaloacetic acid. This enzyme had a molecular mass of about 124 kDa consisting of two identical subunits. L-Aspartic acid was a substrate, although D-aspartic acid and L-glutamic acid were inactive. The enzyme showed maximal activity at about pH 7.0-8.0 for the oxidative deamination of L-aspartic acid, and it required NADP+ as a coenzyme, while NAD+ was inactive.     

Thiol:fumarate reductase (Tfr) from Methanobacterium thermoautotrophicum--identification of the catalytic sites for fumarate reduction and thiol oxidation

Most methanogenic Archaea contain an unusual cytoplasmic fumarate reductase which catalyzes the reduction of fumarate with coenzyme M (CoM-S-H) and coenzyme B (CoB-S-H) as electron donors forming succinate and CoM-S-S-CoB as products. We report here on the purification and characterization of this thiol:fumarate reductase (Tfr) from Methanobacterium thermoautotrophicum (strain Marburg). The purified enzyme, which was composed of two different subunits with apparent molecular masses of 58 kDa (TfrA) and 50 kDa (TfrB), was found to catalyze the following reactions: (a) the reduction of fumarate with CoM-S-H and CoB-S-H (150 U/mg); (b) the reduction of fumarate with reduced benzyl viologen (620 U/mg); (c) the oxidation of CoM-S-H and CoB-S-H to CoM-S-S-CoB with methylene blue (95 U/mg); and (d) the reduction of CoM-S-S-CoB with reduced benzyl viologen (250 U/mg). The flavoprotein contained 12 mol non-heme iron and approximately the same amount of acid-labile sulfur/mol heterodimer. The genes encoding TfrA and TfrB were cloned and sequenced. Sequence comparisons with fumarate reductases and succinate dehydrogenases from Bacteria and Eucarya and with heterodisulfide reductases from M. thermoautotrophicum and Methanosarcina barkeri revealed that TfrA harbors FAD-binding motifs and the catalytic site for fumarate reduction and that TfrB harbors one [2Fe-2S] cluster and two [4Fe-4S] clusters and the catalytic site for CoM-S-H and CoB-S-H oxidation.