-3-methylaspartic Acid Research Articles

Isolation and mechanism analysis of a catalytic efficiency improved L-aspartate β-decarboxylase toward 3-methylaspartic acid

L-Aspartate β-decarboxylase from Acinetobacter radioresistens (ArASD) has been modified to convert 3-methylaspartic acid into 2-aminobutyric acid, which activated a novel process for biosynthesis of 2-aminobutyric acid. However, the process is limited by the low activity of the ArASD. Here, the activity of ArASD was significantly improved by modification based on sequence alignment and structural analysis. The 38th residue of ArASD is speculated to be the key residue for regulating the conformation of the internal aldimine, and site-directed mutagenesis on R38 residue was carried out. A variant, K18A/R38K/V287I, with 2.2 times higher specific activity was isolated. Molecular dynamics simulation indicated that the torsion angle of the imine bond of the variant decreased, which was beneficial to the protonation of the internal aldimine and the increase in the initial energy of the enzyme. Therefore, the energy barrier of the transition state was reduced, resulting in improved catalytic activity toward 3-methylaspartic acid. These results provide a reference and a new point of view for enzyme modification by increasing the energy of the initial state.

Structure-function investigation of 3-methylaspartate ammonia lyase reveals substrate molecular determinants for the deamination reaction.

The enzymatic reactions leading to the deamination of β-lysine, lysine, or 2-aminoadipic acid are of great interest for the metabolic conversion of lysine to adipic acid. Enzymes able to carry out these reactions are not known, however ammonia lyases (EC 4.3.1.-) perform deamination on a wide range of substrates. We have studied 3-methylaspartate ammonia lyase (MAL, EC 4.3.1.2) as a potential candidate for protein engineering to enable deamination towards β-lysine, that we have shown to be a competitive inhibitor of MAL. We have characterized MAL activity, binding and inhibition properties on six different compounds that would allow to define the molecular determinants necessary for MAL to deaminate our substrate of interest. Docking calculations showed that β-lysine as well as the other compounds investigated could fit spatially into MAL catalytic pocket, although they probably are weak or very transient binders and we identified molecular determinants involved in the binding of the substrate. The hydrophobic interactions formed by the methyl group of 3-methylaspartic acid, together with the presence of the amino group on carbon 2, play an essential role in the appropriate binding of the substrate. The results showed that β-lysine is able to fit and bind in MAL catalytic pocket and can be potentially converted from inhibitor to substrate of MAL upon enzyme engineering. The characterization of the binding and inhibition properties of the substrates tested here provide the foundation for future and more extensive studies on engineering MAL that could lead to a MAL variant able to catalyse this challenging deamination reaction.

Dolyemycins A and B, two novel cyclopeptides isolated from Streptomyces griseus subsp. griseus HYS31.

Two novel cyclopeptides with special skeleton, namely, dolyemycins A (1) and B (2) were isolated from Streptomyces griseus subsp. griseus HYS31 by bio-guided isolation. Their structures were elucidated by detailed analysis of spectroscopic data. These two compounds were cyclopeptides containing eleven amino acids including five unusual amino acids (hydroxyglycine, 3-hydroxyleucine, 3-phenylserine, β-hydroxy-O-methyltyrosine, 2,3-diaminobutyric acid) in both of them and an extra nonprotein amino acids (3-methylaspartic acid) in Dolyemycin B only. Dolyemycins A and B performed antiproliferative activity against human lung cancer A549 cells with IC50 values of 1.0 and 1.2 µM, respectively.

Stereoselective alkylation of chiral pyrrolidin-2-ones leading to novel conformationally restricted analogues of 3-methylaspartic acid: a computational investigation

The stereoselectivity of the alkylation of chiral pyrrolidin-2-ones leading to conformationally restricted analogues of 3-methylaspartic acid was investigated using density functional theory calculations. The overall stereocontrol is regulated by the relative stability of the lithium enolate intermediates which are also involved in the rate-determining step leading to the methylaspartic acid derivative. The computed results explain the observed data and the different diastereoselectivity can be ascribed to the nature of the alkylating group.

A novel conformationally restricted analogue of 3-methylaspartic acid via stereoselective methylation of chiral pyrrolidin-2-ones

Conformationally restricted analogues of β-methylaspartic acid were easily prepared starting from chiral N-protected trans-3-amino-4-methoxycarbonyl pyrrolidin-2-ones. The key step of the synthesis was the methylation reaction at C-4, proceeding with high diastereoselection syn to the protected amino group lying at C-3 of the pyrrolidin-2-one ring.

Natural and Artificial Functionalized Biopolyesters I. Chemoenzymatic Mediated Synthesis of Chiral Precursors

Design of optically active polymeric materials for temporary therapeutic and environmental applications requires the working-out of functionalized polymers with structures and properties adjusted to the considered applications. Biocompatibility of selected polymers is a required property in regard to the interactions between living organisms and macromolecular systems. It is therefore important to prepare highly optically active monomers and their corresponding hydrolyzable and biocompatible polymers. Bacterium Clostridium tetanomorphum is an useful source of enzymes for bioconversion and particularly in the chemoenzyrnatic route to optically active alkylmalolactonic acid esters and their optically pure stereoisomers. 3-Methylaspartase, involved in the glutamate fermentation pathway, is a very interesting enzyme, which can provide chiral precursors with high optically purity. This chemoenzymatic strategy can be used to prepare the four stereoisomers of 3-methylmalolaconic acid esters, by enzymatic resolution of natural and artificial stereoisomers of 3-methylaspartic acid. A series of 3-alkymalolactonic acid esters with alkyl equal to ethyl or isopropyl, have been also synthesized and transformed into corresponding optically active polyesters.

Synthesis of 3-methylaspartic acids by ring-contraction of a nickelacycle derived from glutamic anhydride

The synthesis of protected methylaspartic acids from glutamic acid has been achieved by means of ring contraction of the derived nickelacycle followed by insertion of isocyanides.

Formation of N-carbamoylaspartic acid and its cyclisation to orotic acid

The stereochemistries of the reactions between cyanate and aspartic acid and 3-methylaspartic acid have been examined. The product of the first reaction, N-carbamoylaspartic acid 3. can cyclise to give either dihydroorotic acid 5 or 5-carboxymethylhydantoin 4. Acid catalysed cyclisation gives the latter while catalysis by the enzyme dihydroorotase gives the former. Introduction of a double bond into 3 changes the course of non-enzymatic cyclisation and a six-membered ring compound orotic acid 1 is the product. It is proposed that the double bond prevents intramolecular hydrogen bonding.

Synthesis of protected 3-methylaspartic acids from glutamic anhydride via nickelacycles

The synthesis of fully protected ß-methyl aspartic acids from (±)-glutamic anhydride has been achieved by means of ring contraction of the derived nickelacycle followed by insertion of an isocyanide. The intermediate five-membered ring nickelacycles were isolated and characterized spectroscopically.

Syntheses of (2S,3R)- and (2S,3R)[3- 2h]- 3-methylaspartic acid: slow substrates for a syn-elimination reaction catalysed by methylaspartase.

Methylaspartase catalyses the slow syn-elimination of ammonia from the (2S,3R)-[L- erythro]-diastereomer of the natural substrate, (2S,3S)-3-methylaspartic acid, to give mesaconic acid. To provide material of sufficient stereochemical purity to probe the mechanism of the reaction, two synthetic routes to (2S,3R)- and (2S,3R)[3- 2H]- 3-methylaspartic acid were devised. The use of these (2S,3R)-3-methylaspartic adds revealed that the enzymic reaction does not involve C-3 epimerisation followed by normal anti-elimination, ruling-out the possibility of a carbanion intermediate. Conversely, the substrate displayed very large primary deuterium isotope effects indicating rate-limiting CH bond cleavage.

Primary deuterium isotope effects for the 3-methylaspartase-catalyzed deamination of (2S)-aspartic acid, (2S,3S)-3-methylaspartic acid, and (2S,3S)-3-ethylaspartic acid.

3-Methylaspartate ammonia-lyase catalyzes the deamination of (2S)-aspartic acid 137 times more slowly than the deamination of (2S,3S)-3-methylaspartic acid but catalyzes the amination of fumaric acid 1.8 times faster than the amination of mesaconic acid [Botting, N.P., Akhtar, M., Cohen, M. A., & Gani, D. (1988) Biochemistry (preceding paper in this issue)]. In order to understand the mechanistic basis for these observations, the deamination reaction was examined kinetically with (2S)-aspartic acid, (2S,3S)-3-methylaspartic acid, (2S,3S)-3-ethylaspartic acid, and the corresponding C-3-deuteriated isotopomers. Comparison of the double-reciprocal plots of the initial reaction velocities for each of the three pairs of substrates revealed that the magnitude of the primary isotope effect on both Vmax and V/K varied with the substituent at C-3 of the substrate. 3-Methylaspartic acid showed the largest isotope effect (1.7 on Vmax and V/K), 3-ethylaspartic acid showed a smaller isotope effect (1.2 on Vmax and V/K), and aspartic acid showed no primary isotope effect at all. These results, which are inconsistent with earlier reports that there is no primary isotope effect for 3-methylaspartic acid [Bright, H. J. (1964) J. Biol. Chem. 239, 2307], suggest that for both 3-methylaspartic acid and 3-ethylaspartic acid elimination occurs via a predominantly concerted mechanism whereas for aspartic acid an E1cb mechanism prevails.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of the enzymic elimination of ammonia from 3-substituted aspartic acids by 3-methylaspartase

Kinetic experiments with 3-methylaspartase, using aspartic, 3-methylaspartic, and 3-ethylaspartic acid and the appropriate C-3 deuteriated isotopomers as substrates, reveal that C(3)–H bond cleavage is partially rate-limiting for 3-methylaspartic acid, much less rate-limiting for 3-ethylaspartic acid, and not rate-limiting at all for aspartic acid.

PROPIONIC ACID METABOLISM: MECHANISM OF THE METHYLMALONYL ISOMERASE REACTION AND THE REDUCTION OF ACRYLYL COENZYME A TO PROPIONYL COENZYME A IN PROPIONIBACTERIA

step is the transfer of a carboxyl group from methylmalonyl CoAl to pyruvate with the formation of oxalacetate and propionyl CoA.2 3 The reaction was found to involve biotin;2 the purified enzyme, methylmalonyl-oxalacetic transcarboxylase, has now been shown to contain this factor.4 Methylmalonyl CoA arises from succinyl CoA by an isomerization the mechanism of which remains obscure. The original proposal that it occurred via a transcarboxylation' was questioned on theoretical grounds as well as by indirect experimental evidenlce.d The discovery that the reaction was dependent on the presence of the various cobamide coenzymes6 that the rearrangement was analogous to the isomerization of glutamic acid to 3-methylaspartic acid observed in Clostridium tetanomorphum.7 In the latter case the rearrangement can be considered to be a cleavage of a propionate moiety with subsequent reattachment of the glycine residue to the a position of propionate. Using an extract of propionibacteria, Eggerer et al.8 showed that the isomerization of methylmalonyl CoA involved a similar migrationl of the formyl thioester group from one methylene position of the propionate moiety to the other. Similar results were obtained in our laboratory with methylmalonyl isomerase isolated from sheep kidneys, as well as from propionibacteria. Eggerer et al.8 that the rearrangement of methylmalonyl CoA involved a free radical intermediate. In accord with this hypothesis, Phares et al.9 recently reported an inhibition of the reaction by chlorpromazine, an efficient electron donor. It has also been suggested that the reaction might involve a dehydrogenation which would weaken the bond of the formyl CoA group, permitting its translocation to the other side of the double bond. Since there has been no successful demon-

Failure of [formula omitted] to form thymine from 3-methylaspartic acid

Cinnamamides with aminoalkyl groups on the amide nitrogen were conceived as structural analogs of serotonin which would act as antimetabolites of it. Several such amides were synthesized and tested for antiserotonin activity on the isolated rat uterus and in living animal. All such amides that were tested acted as antagonists of the hormone. When the structural analogy to serotonin was increased by introduction of a meta-methoxyl group in the. benzene ring, antiserotonin potency was increased considerably. A further increase in potency was achieved by introduction of a benzyl group on the amide nitrogen. The most active cinnamamide proved to be more active as a serotonin antagonist in the rat uterus assay than the previously known and highly potent 1-benzyl-2-methyl-5-methoxytryptamine (BAS), and was much easier to synthesize. It was, however, less active than BAS in the in-vivo assay that was used. The compounds were not antagonistic toward acetylcholine or bradykinin on rat uterus. It was suggested that some previously known cinnamamides may owe some of their pharmacological activity (e.g. local anesthetic) to their ability to act as antimetabolites of serotonin.

3-Methylaspartic Acid as a Potent Antimetabolite of Aspartic Acid in Pyrimidine Biosynthesis

3-Methylaspartic Acid as a Potent Antimetabolite of Aspartic Acid in Pyrimidine Biosynthesis