TL;DR: Its broad nucleophile specificity and high catalytic activity make AspB an attractive enzyme for the enantioselective synthesis of N-substituted aspartic acids, which are interesting building blocks for peptide and pharmaceutical synthesis as well as for peptidomimetics.
Abstract: The gene encoding aspartate ammonia lyase (aspB) from Bacillus sp. YM55-1 has been cloned and overexpressed, and the recombinant enzyme containing a C-terminal His(6) tag has been purified to homogeneity and subjected to kinetic characterization. Kinetic studies have shown that the His(6) tag does not affect AspB activity. The enzyme processes L-aspartic acid, but not D-aspartic acid, with a K(m) of approximately 15 mM and a k(cat) of approximately 40 s(-1). By using this recombinant enzyme in the reverse reaction, a set of four N-substituted aspartic acids were prepared by the Michael addition of hydroxylamine, hydrazine, methoxylamine, and methylamine to fumarate. Both hydroxylamine and hydrazine were found to be excellent substrates for AspB. The k(cat) values are comparable to those observed for the AspB-catalyzed addition of ammonia to fumarate ( approximately 90 s(-1)), whereas the K(m) values are only slightly higher. The products of the enzyme-catalyzed addition of hydrazine, methoxylamine, and methylamine to fumarate were isolated and characterized by NMR spectroscopy and HPLC analysis, which revealed that AspB catalyzes all the additions with excellent enantioselectivity (>97 % ee). Its broad nucleophile specificity and high catalytic activity make AspB an attractive enzyme for the enantioselective synthesis of N-substituted aspartic acids, which are interesting building blocks for peptide and pharmaceutical synthesis as well as for peptidomimetics.
TL;DR: The mechanism leading to the formation of D-phenylalanine derivatives was explored through mutagenesis of key active site residues and isotopic labeling studies and the results obtained demonstrate that D-amino acid formation occurs via a previously unobserved competing MIO-independent pathway which proceeds in a non-stereoselective manner.
Abstract: The research presented in this thesis describes the application of phenylalanine ammonia lyase from the bacteria Anabaena variabilis (AvPAL), as a biocatalyst for the asymmetric hydroamination of cinnamic acid derivatives. PALs from eukaryotic sources such as the plant Petroselinum crispum (PcPAL) and yeast Rhodotorula glutinis (RgPAL) have been widely used as biocatalysts for the synthesis of non-natural amino acids. For example the PAL catalyzed hydroamination of 2?-chlorocinnamic acid has been implemented by DSM Pharma Chemicals on a tonne scale. However, there are very few examples of prokaryotic PALs and to our knowledge their activity towards unnatural substrates has not been investigated. Herein we explore the activity of AvPAL towards a panel of cinnamic acid analogues. For comparison, the activity of the commonly studied eukaryotic PcPAL and RgPAL towards the same substrate panel was also investigated. Although the difference in substrate conversions between the three PALs was fairly unremarkable, a significant reduction in product e.e was observed following prolonged reaction times with all three PALs towards substrates bearing electron deficient aromatic rings. A time dependence on e.e. has not been previously reported for ammonia lyases and all previously described biotransformations have been reported to proceed with excellent e.e. in favour of the L-enantiomer. The mechanism leading to the formation of D-phenylalanine derivatives was explored through mutagenesis of key active site residues and isotopic labeling studies. The results obtained demonstrate that D-amino acid formation occurs via a previously unobserved competing MIO-independent pathway which proceeds in a non-stereoselective manner. In addition, the observations are consistent with amino acid deamination occurring via a stepwise E1cB elimination mechanism. In order to develop a more general biocatalytic method for asymmetric hydroamination reactions, the activity of PAL towards substrates lacking the carboxylic acid functionality was investigated. The synthesis of a panel of substrates and subsequent screening with AvPAL and RgPAL is described. Unfortunately, the wild-type enzymes demonstrated no activity towards any of the substrates screened. These enzymes were also screened for their promiscuity towards the nucleophilic amine partner and although deamination activity towards N-methyl-L-phenylalanine was observed, no hydroamination activity was detected using primary amines as nucleophiles. In order to broaden the substrate specificity of PAL enzymes, a number of screening methods have been developed. Herein we present both liquid phase and colony based colorimetric screens for the detection of PAL catalyzed hydroamination activity. Furthermore these screens have been used to screen libraries of variants for increased D-selectivity and hydroamination activity towards ?-methylstyrene and cinnamyl alcohol derivatives.
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Cites background or methods from "Biocatalytic Enantioselective Synth..."
...4.6 Nucleophile Promiscuity
The aspartate ammonia lyase (AAL) catalyzed hydroamination of fumerate with small primary amines has been successfully achieved by Feringa et al.[123] However AALs belong to the fumerase/ enolase superfamily and react via an alternative catalytic mechanism which does not involve an MIO-cofactor....
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...6 Nucleophile Promiscuity The aspartate ammonia lyase (AAL) catalyzed hydroamination of fumerate with small primary amines has been successfully achieved by Feringa et al.[123] However AALs belong to the fumerase/ enolase superfamily and react via an alternative catalytic mechanism which does not involve an MIO-cofactor....
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...A variety of nucleophiles including methylamine, hydrazine, methoxyamine and hydroxylamine have been previously used in the aspartate ammonia lyase (AAL) catalyzed hydroamination of fumarate 25.[123] Furthermore, Rétey et al....
TL;DR: The diastereoselective conjugate addition of secondary homoallylamines, obtained in the enantioenriched form via allylmetallation of imines, to α,β-unsaturated esters is reported.
Abstract: The diastereoselective conjugate addition of secondary homoallylamines, obtained in the enantioenriched form via allylmetallation of imines, to α,β-unsaturated esters is reported. This method allows access to valuable building blocks as well as heterocyclic skeletons, providing tertiary amines bearing two chains integrating a stereogenic center adjacent to the nitrogen atom.
TL;DR: Carbonelloxal-5'-phosphate-dependent enzymes as discussed by the authors catalyze the elimination reactions of C-S bond in various sulfur-containing amino acids to yield corresponding sulfurcontaining molecules, α-keto acid, and ammonia.
Abstract: Lyases are enzymes that catalyze the bond generation and the bond breaking with the addition or removal of groups from their substrates. Enzyme-catalyzed stereoselective reactions forming new carbon–carbon bonds are of utmost important in synthetic organic chemistry. Ammonia lyases are a class of enzymes capable of catalyzing the reversible cleavage of carbon–nitrogen bonds, typically α-amino acids, to release ammonia and corresponding α,β-unsaturated or cyclic derivatives without employing hydrolysis or oxidation mechanism. Enzymes that catalyze the cleavage of a carbon–sulfur bond by means other than hydrolysis or oxidation are carbon–sulfur lyases (C–S lyases). Specifically, carbon–sulfur bond lyases are pyridoxal-5'-phosphate-dependent enzyme, which catalyze the elimination reactions of C–S bond in various sulfur-containing amino acids to yield corresponding sulfur-containing molecules, α-keto acid, and ammonia.
TL;DR: A tabular survey of commercially available enzymes can be found in this paper, with a focus on enzymes that are used in organic synthesis, such as: production and isolation of enzymes, immobilization of enzymes reaction techniques, use of growing or resting cells, applications of enqumes in organically synthesized organic synthesis hydrolysis and formation of C-O bonds, C-N bonds, P-O bond formation, reduction reactions oxidation reactions isomerizations introduction and removal of protecting groups extremophiles catalytic antibodies enqumatic analysis and biosensors prtoein engineering
Abstract: Production and isolation of enzymes immobilization of enzymes reaction techniques use of growing or resting cells applications of enqumes in organic synthesis hydrolysis and formation of C-O bonds, C-N bonds, P-O bonds formation of C-C bonds reduction reactions oxidation reactions isomerizations introduction and removal of protecting groups extremophiles catalytic antibodies enqumatic analysis and biosensors prtoein engineering tabular survey of commercially available enzymes.
TL;DR: The structure of the apoenzyme has made it possible to identify some of the residues that are involved in binding the substrate, and their putative roles have been assigned.
Abstract: The X-ray crystal structure of l-aspartate ammonia-lyase has been determined to 2.8 A resolution. The enzyme contains three domains, and each domain is composed almost completely of α helices. The central domain is composed of five long helices. In the tetramer, these five helices form a 20-helix cluster. Such clusters have also been seen in δ-crystallin and in fumarase. The active site of aspartase has been located in a region that contains side chains from three different subunits. The structure of the apoenzyme has made it possible to identify some of the residues that are involved in binding the substrate. These residues have been examined by site-directed mutagenesis, and their putative roles have been assigned [Jayasekera, M. M. K., Shi, W., Farber, G. K., & Viola, R. E. (1997) Biochemistry 36, 9145−9150].