Failure to detect beta-leucine in human blood or leucine 2,3-aminomutase in rat liver using capillary gas chromatography-mass spectrometry

It has been reported (Poston, J. (1976) J. Biol. Chem. 251, 1859-1863; (1982) 255, 10067-10072; (1984) 259, 2059-2061) that mammalian tissues contain an adenosylcobalamin-dependent enzyme, leucine 2,3-aminomutase, which catalyzes the interconversion of beta-leucine and leucine. It was also reported that beta-leucine is detectable in normal human serum (mean = 4.8 mumol/liter, n = 37) and is elevated in serum from patients with cobalamin deficiency (mean = 24.7 mumol/liter, n = 17). Serum levels of leucine were claimed to be decreased in the cobalamin deficient patients (mean = 52 mumol/liter) as compared with the normal subjects (mean = 81 mumol/liter). It was also reported that rat liver supernatant catalyzed the formation of beta-leucine, leucine, or both amino acids from iso-fatty acids, and that the generation of leucine from iso-fatty acids was stimulated by adenosylcobalamin and inhibited by unsaturated cobalamin-binding protein. We have synthesized t-butyldimethylsilyl derivatives of beta-leucine and leucine and have used capillary gas chromatography-mass spectrometry for their analysis. Using forms of beta-leucine and leucine that contain several deuterium atoms in place of several hydrogen atoms as internal standards, techniques have been developed which make it possible to detect and quantitate as little as 0.1 mumol/liter of beta-leucine or leucine in human serum and in incubations containing rat liver supernatant. beta-Leucine was not detectable, i.e. less than 0.1 mumol/liter, in any sera from 50 normal human subjects or in any sera from 50 cobalamin-deficient patients. The mean level of leucine in the 50 cobalamin-deficient sera was 219 mumol/liter, which was not decreased with respect to that in the 50 control sera (167 mumol/liter). Experiments in which beta-leucine, leucine, isostearic acid, or isocaproic acid were incubated with rat liver supernatant in the presence or absence of adenosylcobalamin or cobalamin-binding protein failed to demonstrate the formation of leucine or beta-leucine or their interconversion under any of the conditions studied. We conclude that beta-leucine is not present in human blood and that the existence of leucine 2,3-aminomutase in mammalian tissues remains to be established.     

Separation by high-performance liquid chromatography of alpha- and beta-amino acids: application to assays of lysine 2,3-aminomutase and leucine 2,3-aminomutase

Several pairs of alpha- and beta-amino acids were well separated as the ortho-phthalaldehyde derivatives by reversed phase HPLC. These included alpha- and beta-lysine, leucine, tyrosine, serine, aminoisobutyric acid, and beta-hydroxyvaline and its positional isomer. The separation was applicable to assays of lysine 2,3-aminomutase and leucine 2,3-aminomutase. However, for unknown reasons, no leucine 2,3-aminomutase activity could be found in rat liver homogenate.     

The S subunit of D-ornithine aminomutase from Clostridium sticklandii is responsible for the allosteric regulation in D-alpha-lysine aminomutase

Ornithine and lysine are degraded in quite a similar way in Clostridium sticklandii. Both pathways involve adenosylcobalamin-dependent enzymes, d-ornithine 4,5-aminomutase and lysine 5,6-aminomutase. According to previous reports, lysine 5,6-aminomutase is an ATP-dependent allosteric enzyme with many different activators and inhibitors. However, recent studies indicate that ATP does not have a regulatory effect on the recombinant enzyme. To monitor the activity of lysine aminomutase, a novel capillary electrophoresis-based assay method was developed. The present results demonstrate that the S subunit of d-ornithine aminomutase, OraS, is capable of forming a complex with KamDE of lysine 5,6-aminomutase and restores the enzyme's ATP-dependent allosteric regulation. Not only does ATP lower the K(m) of the KamDE-OraS complex for adenosylcobalamin and pyridoxal phosphate, but also OraS protein alone lowers the K(m) of KamDE for adenosylcobalamin and pyridoxal phosphate. The activity of reconstituted enzyme can also be activated by ammonium ion as reported by Morley and Stadtman.     

Lysine 2,3-aminomutase. Support for a mechanism of hydrogen transfer involving S-adenosylmethionine

The conversion of L-lysine to L-beta-lysine is catalyzed by lysine 2,3-aminomutase. The reaction involves the interchange of the 2-amino group of lysine with a hydrogen at carbon 3. As such the reaction is formally analogous to adenosylcobalamin-dependent rearrangements. However, the enzyme does not contain and is not activated by this coenzyme. Instead it contains iron and pyridoxal phosphate and is activated by S-adenosylmethionine. Earlier experiments implicated adenosyl-C-5' of S-adenosylmethionine in the hydrogen transfer mechanism, apparently in a role similar or analogous to that of adenosyl moiety of adenosylcobalamin in the B12-dependent rearrangements. The question of whether both hydrogens or only one hydrogen at adenosyl-C-5' participate in the hydrogen-transfer process has been addressed by carrying out the lysine 2,3-aminomutase reaction with S-[5'-3H] adenosylmethionine in the presence of 10 times its molar concentration of enzyme. Under these conditions all of the tritium appeared in lysine and beta-lysine, showing that C-5'-hydrogens participate. To determine whether hydrogen transfer is compulsorily intermolecular and intramolecular, various molar ratios of [3,3-2H2]lysine and unlabeled lysine were submitted to the action of lysine 2,3-aminomutase under conditions in which 10-15% conversion to beta-lysine occurred. Mass spectral analysis of the beta-lysine for monodeutero and dideutero species showed conclusively that hydrogen transfer is both intramolecular and intermolecular. The results quantitatively support our postulate that activation of the enzyme involves a transformation of S-adenosylmethionine into a form that promotes the generation of an adenosyl-5' free radical, which abstracts hydrogen from lysine to form 5'-deoxyadenosine as an intermediate.     

Leucine 2,3-aminomutase, an enzyme of leucine catabolism.

The initial step in the fermentation of leucine to acetate, isobutyrate, and ammonia by Clostridium sporogenes is the B12 coenzyme-dependent conversion of alpha-leucine to beta-leucine (3-amino-4-methylpentanoate). The amino group migration reaction, catalyzed by leucine 2,3-aminomutase, is reversible and is inhibited by intrinsic factor. The enzyme activity has been found in several clostridia, in rat, sheep, rhesus, and African green monkey livers, and in human leukocytes.     

Pyridoxal-5'-phosphate as the catalyst for radical isomerization in reactions of PLP-dependent aminomutases

PLP catalyzes the 1,2 shifts of amino groups in free radical-intermediates at the active sites of amino acid aminomutases. Free radical forms of the substrates are created upon H atom abstractions carried out by the 5'-deoxyadenosyl radical. In most of these enzymes, the 5'-deoxyadenosyl radical is generated by an iron-sulfur cluster-mediated reductive cleavage of S-adenosyl-(S)-methionine. However, in lysine 5,6-aminomutase and ornithine 4,5-aminomutase, the radical is generated by homolytic cleavage of the cobalt-carbon bond of adenosylcobalamin. The imine linkages in the initial radical forms of the external aldimines undergo radical addition to form azacyclopropylcarbinyl radicals as central intermediates in the catalytic cycles. In the case of lysine 2,3-aminomutase, the multistep catalytic mechanism is corroborated by direct spectroscopic observation and characterization of a product radical trapped during steady-state turnover. Analogues of the substrate-related radical having substituents adjacent to the radical center to stabilize the unpaired electron are also observed and characterized spectroscopically. A functional allylic analogue of the 5'-deoxyadenosyl radical is observed spectroscopically. A high-resolution crystal structure fully supports the mechanistic proposals. Evidence for a similar free radical mediated amino group transfer in the adenosylcobalamin-dependent lysine 5,6-aminomutase is provided by spectroscopic detection and characterization of radicals generated from the 4-thia analogues of the lysine substrates. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.     

An organic radical in the lysine 2,3-aminomutase reaction.

Lysine 2,3-aminomutase from Clostridium SB4 has been studied by electron paramagnetic resonance (EPR) spectroscopy at 77 K. Although the reaction catalyzed by this enzyme is similar to rearrangements catalyzed by enzymes requiring adenosylcobalamin, lysine 2,3-aminomutase does not utilize this cofactor. The enzyme instead contains iron-sulfur clusters, cobalt, and pyridoxal phosphate and is activated by S-adenosylmethionine. Subsequent to a reductive incubation procedure that is required to activate the enzyme, EPR studies reveal the appearance of an organic radical signal (g = 2.001) upon addition of both L-lysine and S-adenosylmethionine. The radical signal is complex, having multiple hyperfine transitions. The total radical concentration is proportional to enzyme activity and decreases in parallel with the approach to chemical equilibrium between alpha-lysine and beta-lysine. The signal changes over the time course of the reaction in a way that suggests the presence of more than one radical species, with different relative proportions of species in the steady state and equilibrium state. Isotopic substitution experiments show that unpaired spin density resides on the molecular framework of lysine and that solvent-exchangeable protons do not participate in strong hyperfine coupling to the radical. The results indicate that lysine radicals participate in the rearrangement mechanism.     

Distribution of leucine 2,3-aminomutase activity in various organs of the rat and in subcellular organelles of rat liver

Leucine 2,3-aminomutase, the cobalamine-dependent enzyme involved in the conversion of β-leucine to leucine, has been shown to be widely distributed in rat organs. The activity is highest in skeletal and cardiac muscle, lower in brain, liver, and kidney, and very low in small intestine. In rat liver, the activity is nearly all in the cytosolic fraction.     

Interference by methylcobalamin analogues with synthesis of cobalamin coenzymes in human lymphocytes in vitro.

1. 72 h uptake of cyano[57Co]cobalamin and formation of 57Co-labelled methylcobalamin, adenosylcobalamin and hydroxocobalamin has been estimated with and without the addition of methylcobalamin analogues in phytohaemagglutinin-stimulated lymphocytes from healthy human subjects. 2. Difluorochloromethylcobalamin reduced cell uptake of cyanocobalamin and caused a disproportionate reduction in synthesis of adenosylcobalamin. 3. Methylcobalamin-palladium trichloride reduced cell uptake of cyanobalamin more effectively than did difluorochloromethylcobalamin and reduced the formation of methylcobalamin, adenosylcobalamin and hydroxocobalamin in proportion. 4. The results suggest that in addition to inhibiting uptake of cyanocobalamin, one or both compounds may have interfered directly with the mechanism of synthesis of the cobalamin coenzymes.     

Cloning, sequencing, heterologous expression, purification, and characterization of adenosylcobalamin-dependent D-lysine 5, 6-aminomutase from Clostridium sticklandii

D-Lysine 5,6-aminomutase from Clostridium sticklandii catalyzes the 1,2-shift of the epsilon-amino group of D-lysine and reverse migration of C5(H). The two genes encoding 5,6-aminomutase have been cloned, sequenced, and expressed in Escherchia coli. They are adjacent on the Clostridial chromosome and encode polypeptides of 57. 3 and 29.2 kilodaltons. The predicted amino acid sequence includes a conserved base-off 5'-deoxyadenosylcobalamin binding motif and a 3-cysteine cluster in the small subunit, as well as a P-loop sequence in the large subunit. Activity of the recombinant enzyme exceeds that of the 5,6-aminomutase purified from C. sticklandii by 6-fold, presumably due to the absence of bound, inactive corrinoids in the recombinant enzyme. The K(m) values for adenosylcobalamin and pyridoxal 5'-phosphate are 6.6 and 1.0 microM, respectively. ATP does not have a regulatory effect on the recombinant protein. The rapid turnover associated inactivation reported for the enzyme purified from Clostridium is also seen with the recombinant form. Aminomutase activity does not depend on structural or catalytic metal ions. Electron paramagnetic resonance experiments with [(15)N-dimethylbenz-imidazole]adenosylcobalamin demonstrate base-off binding, consistent with other B(12)-dependent enzymes that break unactivated C-H bonds.     

Glutamate 2,3-aminomutase: a new member of the radical SAM superfamily of enzymes

A gene eam in Clostridium difficile encodes a protein that is homologous to lysine 2,3-aminomutase (LAM) in many other species but does not have the lysyl-binding residues Asp293 and Asp330 in LAM from Clostridium subterminale SB4. The C. difficile protein has Lys and Asn, respectively, in the sequence positions of the essential Asp residues in LAM. The C. difficile gene has been cloned into an E. coli expression vector, expressed in E. coli, and the protein purified and characterized. The recombinant protein displays excellent activity as a glutamate 2,3-aminomutase and no activity toward l-lysine. The PLP-, iron-, and sulfide-content and ultraviolet/visible spectrum are similar to LAM, and the enzyme requires SAM and dithionite as activators, as does LAM. Freeze-quench EPR experiments in the presence of l-glutamate reveal a glutamate-based free radical in the steady state of the reaction. A number of other bacterial genomes include genes encoding proteins homologous to the glutamate 2,3-aminomutase from C. difficile, and four of these proteins display the activity of glutamate 2,3-aminomutase when produced in E. coli. All of the homologous proteins have the cysteine motif CSMYCRHC corresponding to the motif CxxxCxxC characteristic of radical SAM enzymes. It is concluded that glutamate 2,3-aminomutase from C. difficile is a representative of a family found in a number of bacteria. It is likely that the beta-glutamate found in a few bacterial and archeal species as an osmolyte arises from the action of glutamate 2,3-aminomutase.     

Coenzyme B12-dependent enzymes in potatoes: Leucine 2,3-aminomutase and methylmalonyl-coa mutase

Extracts of potato tubers contain endogenous activities of two coenzyme B₁₂-dependent enzymes, leucine 2,3-aminomutase and methylmalonyl-CoA mutase. These activities are stimulated by the addition of coenzyme B₁₂ and are inhibited by intrinsic factor. The inhibition is overcome by coenzyme B₁₂.     

Enzymatic activation of lysine 2,3-aminomutase from Porphyromonas gingivalis

The development of lysine 2,3-aminomutase as a robust biocatalyst hinges on the development of an in vivo activation system to trigger catalysis. This is the first report to show that, in the absence of chemical reductants, lysine 2,3-aminomutase activity is dependent upon the presence of flavodoxin, ferredoxin, or flavodoxin-NADP(+) reductase.     

The cblD defect causes either isolated or combined deficiency of methylcobalamin and adenosylcobalamin synthesis

Intracellular cobalamin is converted to adenosylcobalamin, coenzyme for methylmalonyl-CoA mutase and to methylcobalamin, coenzyme for methionine synthase, in an incompletely understood sequence of reactions. Genetic defects of these steps are defined as cbl complementation groups of which cblC, cblD (described in only two siblings), and cblF are associated with combined homocystinuria and methylmalonic aciduria. Here we describe three unrelated patients belonging to the cblD complementation group but with distinct biochemical phenotypes different from that described in the original cblD siblings. Two patients presented with isolated homocystinuria and reduced formation of methionine and methylcobalamin in cultured fibroblasts, defined as cblD-variant 1, and one patient with isolated methylmalonic aciduria and deficient adenosylcobalamin synthesis in fibroblasts, defined as cblD-variant 2. Cell lines from the cblD-variant 1 patients clearly complemented reference lines with the same biochemical phenotype, i.e. cblE and cblG, and the cblD-variant 2 cell line complemented cells from the mutant classes with isolated deficiency of adenosylcobalamin synthesis, i.e. cblA and cblB. Also, no pathogenic sequence changes in the coding regions of genes associated with the respective biochemical phenotypes were found. These findings indicate heterogeneity within the previously defined cblD mutant class and point to further complexity of intracellular cobalamin metabolism.     

Enzymatic Activation of Lysine 2,3-Aminomutase from Porphyromonas gingivalis

The development of lysine 2,3-aminomutase as a robust biocatalyst hinges on the development of an in vivo activation system to trigger catalysis. This is the first report to show that, in the absence of chemical reductants, lysine 2,3-aminomutase activity is dependent upon the presence of flavodoxin, ferredoxin, or flavodoxin-NADP⁺ reductase.     

Basis for the equilibrium constant in the interconversion of l-lysine and l-beta-lysine by lysine 2,3-aminomutase

l-beta-lysine and beta-glutamate are produced by the actions of lysine 2,3-aminomutase and glutamate 2,3-aminomutase, respectively. The pK(a) values have been titrimetrically measured and are for l-beta-lysine: pK(1)=3.25 (carboxyl), pK(2)=9.30 (beta-aminium), and pK(3)=10.5 (epsilon-aminium). For beta-glutamate the values are pK(1)=3.13 (carboxyl), pK(2)=3.73 (carboxyl), and pK(3)=10.1 (beta-aminium). The equilibrium constants for reactions of 2,3-aminomutases favor the beta-isomers. The pH and temperature dependencies of K(eq) have been measured for the reaction of lysine 2,3-aminomutase to determine the basis for preferential formation of beta-lysine. The value of K(eq) (8.5 at 37 degrees C) is independent of pH between pH 6 and pH 11; ruling out differences in pK-values as the basis for the equilibrium constant. The K(eq)-value is temperature-dependent and ranges from 10.9 at 4 degrees C to 6.8 at 65 degrees C. The linear van't Hoff plot shows the reaction to be enthalpy-driven, with DeltaH degrees =-1.4 kcal mol(-1) and DeltaS degrees =-0.25 cal deg(-1) mol(-1). Exothermicity is attributed to the greater strength of the bond C(beta)-N(beta) in l-beta-lysine than C(alpha)-N(alpha) in l-lysine, and this should hold for other amino acids.     

S-adenosylmethionine-dependent enzyme activation

S-Adenosylmethionine (SAM)-dependent activations of pyruvate formate-lyase, lysine 2,3-aminomutase and cobalamin-dependent methionine synthase are discussed. In each case, cleavage of SAM is accompanied by the formation of a catalytically active enzyme. The chemistry of activation of these three enzymes falls into three distinct classes: generation of an essential enzyme radical (pyruvate formate-lyase), formation of a catalytically active 5'-deoxyadenosyl radical (lysine 2,3-aminomutase) and reductive methylation to form a required methylcobalamin complex (methionine synthase).     

Adenosylcobalamin and cob(II)alamin as prosthetic groups of 2-methyleneglutarate mutase from Clostridium barkeri.

The ultraviolet/visible spectrum of the pure pink-orange 2-methyleneglutarate mutase from Clostridium barkeri between 300-600 nm showed the presence of cobalamins; notably the peaks at 470 and 528 nm were indicative of oxygen-stable cob(II)alamin and adenosylcobalamin (coenzyme B12), respectively. Using the absorption coefficients of the isosbestic points at 340, 393 and 489 nm, the total cobalamin content was estimated as 3.7 +/- 0.3 mol/mol tetrameric enzyme (m = 300 kDa). Denaturation with 8 M urea in the presence of 2 mM dithiothreitol followed by gel chromatography and renaturation afforded an inactive enzyme which contained 40-50% of the initially bound cobalamin. This preparation could be reactivated to 95-100% by addition of adenosylcobalamin. The cobalamins were removed to 85% from the mutase by denaturation with 8 M urea in the presence of 1 M cyanide (pH 12) with irreversible loss of activity. 2-Methyleneglutarate mutase was inactivated by incubation with aquo-, cyano- or methylcobalamin; up to 50% of the activity was recovered by addition of adenosylcobalamin. Upon incubation of the mutase with [5'-3H]adenosylcobalamin about 30% of the total cobalamin was exchanged by the tritium-labelled cofactor without loss of activity. During aerobic catalysis the enzyme became sensitive towards oxygen which was accompanied by loss of activity and formation of aquocobalamin from adenosylcobalamin. EPR spectroscopy demonstrated the presence of 0.8 mol base-on cob(II)alamin/mol enzyme. Upon addition of 2-methyleneglutarate a second EPR signal of about equal intensity at g = 2.13 arose. The question of whether the oxygen-stable cob(II)alamin participates in catalysis or its complex with the enzyme represents an inactive form is currently under investigation.     

High-pressure liquid chromatography of cobalamins and cobalamin analogs

High-pressure liquid chromatography has been used to separate, identify, and quantitate 37 different cyanocobalamin analogs, including the most commonly occurring analogs that result from bacterial synthesis. This technique has also been used to simultaneously separate, identify, and quantitate five naturally occurring cobalamins that differ in their upper axial ligands: methylcobalamin, adenosylcobalamin, hydroxocobalamin, cyanocobalamin, and sulfitocobalamin. This method permits rapid quantitative detection and identification of cobalamins and naturally occurring and synthetic cobalamin analogs in complex mixtures.     

Computational study on the difference between the Co–C bond dissociation energy in methylcobalamin and adenosylcobalamin

The bond dissociation energies of the Co–C bonds in the cobalamin cofactors methylcobalamin and adenosylcobalamin were calculated using the hybrid quantum mechanics/molecular mechanics method IMOMM (integrated molecular orbital and molecular mechanics). Calculations were performed on models of differing complexities as well as on the full systems. We investigated the origin of the different experimental values for the Co–C bond dissociation energies in methylcobalamin and adenosylcobalamin, and have provided an explanation for the difficulties encountered when we attempt to reproduce this difference in quantum chemistry. Additional calculations have been performed using the Miertus–Scrocco–Tomasi method in order to estimate the influence of solvent effects on the homolytic Co–C bond cleavage. Introduction of these solvation effects is shown to be necessary for the correct reproduction of experimental trends in bond dissociation energies in solution, which consequently have no direct correlation with dissociation processes in the enzyme.