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Journal ArticleDOI

Biocatalytic Enantioselective Synthesis of N-Substituted Aspartic Acids by Aspartate Ammonia Lyase

10 Nov 2008-Chemistry: A European Journal (WILEY-V C H VERLAG GMBH)-Vol. 14, Iss: 32, pp 10094-10100
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.

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  • A hydrogen balloon was placed on top of the Schlenck flask and the solution stirred vigorously over night.
  • The mixture was filtered over celite and the filtrate was evaporated.

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University of Groningen
Biocatalytic Enantioselective Synthesis of N-Substituted Aspartic Acids by Aspartate
Ammonia Lyase
Weiner, Barbara; Poelarends, Gerrit J.; Janssen, Dick B.; Feringa, Ben L.
Published in:
Chemistry
DOI:
10.1002/chem.200801407
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below.
Document Version
Final author's version (accepted by publisher, after peer review)
Publication date:
2008
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Weiner, B., Poelarends, G. J., Janssen, D. B., & Feringa, B. L. (2008). Biocatalytic Enantioselective
Synthesis of N-Substituted Aspartic Acids by Aspartate Ammonia Lyase.
Chemistry
,
14
(32), 10094-10100.
https://doi.org/10.1002/chem.200801407
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Supporting Information
© Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2008

Biocatalytic enantioselective synthesis of N-substituted aspartic acids by
aspartate ammonia lyase
Barbara Weiner
[a]
, Gerrit J. Poelarends*
[b]
, Dick B. Janssen*
[c]
and Ben L. Feringa*
[a]
[a] Department of Organic and Molecular Inorganic Chemistry, Stratingh Institute of Chemistry,
University of Groningen, Nijenborgh 4, 9747AG Groningen, The Netherlands.
[b] Department of Pharmaceutical Biology, Institute of Pharmacy, University of Groningen, Antonius
Deusinglaan 1, 9713 AV Groningen, The Netherlands.
[c] Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747AG Groningen, The
Netherlands.

General methods
Reagents were purchased from Aldrich, Acros, Merck or Fluka and were used as
provided, unless stated otherwise. All solvents were reagent grade. Enzymes used for the
molecular biology procedures, DNA ladders, protein molecular weight standards,
deoxynucleotide triphosphates (dNTPs), the high pure plasmid isolation kit, the high pure
PCR product purification kit, and multipurpose agarose were purchased from F.
Hoffmann-La Roche, Ltd. Oligonucleotides for DNA amplification and sequencing were
synthesized by Sigma-Aldrich. All biocatalytic reactions were performed in 50 mL
Greiner tubes, which were shaken at ~100 rpm in a waterbath at 37°C. Buffer and stock
solutions of fumarate were prepared in distilled water and stored at 4°C. The pH of the
solutions was adjusted with a Professional Meter PP-15 pH-meter from Sartorius. All
moisture sensitive reactions were performed in round bottomed or modified Schlenk
flasks, previously heated with a heatgun under oilpump vacuum, which were fitted with
rubber septa under a positive pressure of nitrogen. Air- and moisture-sensitive liquids and
solutions were transferred via syringe. Organic solutions were concentrated by rotary
evaporation at 4060°C. Lyophilization was performed with a ALPHA 2-4 LD plus
freeze dryer from Christ. Flash column chromatography was performed as described by
Still et al.
[1]
As stationary phase, Silia-P flash silica gel from Silicycle, size 40-63 µm,
was used. For TLC analysis silica gel 60 from Merck (0.25 mm) impregnated with a
fluorescent indicator (254 nm) was used. TLC plates were visualized by exposure to
ninhydrin or phosphomolybdic acid (PMA) stain followed by brief heating with a
heatgun. Ion exchange chromatography was performed with either Dowex 50 (H
+
form)
activated with 1N HCl and rinsed with distilled water until a neutral pH was obtained (as
assessed with pH indicator paper), or Amberlite IRA 140 (Cl
form) activated with 1N
NaOH until chloride free and washed with distilled water until a neutral pH was obtained.
SPE SCX columns were purchased from IST. Optical rotations were recorded with a
Polartronic MH8 polarimeter from Schmidt + Haensch. The concentrations are given in
g/100 mL.
1
H and
13
C NMR spectra were recorded on a Varian VXR-300 (300 MHz) or a
Varian Mercury Plus (400 MHz) spectrometer. Chemical shifts for protons are reported
in parts per million scale (δ scale) downfield from tetramethylsilane and are referenced to
residual protium in the NMR solvents (CHCl
3
: δ = 7.25, H
2
O: δ = 4.67). Chemical shifts
for carbon are calibrated to the middle signal of the
13
C-triplet of the solvent CDCl
3
(δ =
77.0). HPLC spectra were obtained using a Shimadzu LC-20AD equipped with a
Chiralpak OD-H column. Reversed phase HPLC was performed on a Shimadzu LC-
10AD VP using either a C6 Crownpack column or an Astec CLC-L column. Kinetic data
were obtained on a Jasko V-550, V-560, or V-570 UV-spectrophotometer. Protein was
analyzed by polyacrylamide gel electrophoresis (PAGE) under either denaturing
conditions using sodium dodecyl sulfate (SDS) or native conditions on gels containing
12% polyacrylamide. The gels were stained with Coomassie brilliant blue. Protein
concentrations were measured using the Waddell method.
[2]
DNA sequencing was
performed by GATC Biotech.
[1] W. C. Still, M. Kahn, A. Mitra, J. Org. Chem. 1978, 43, 2923-2925.
[2] W. J. Waddell, J. Lab. Clin. Med. 1956, 48, 311-314.


Citations
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TL;DR: The authors show that fosfazinomycin and kinamycin share a common pathway for N-N bond formation that is different from pathways found for other natural products.
Abstract: Fosfazinomycin and kinamycin are natural products that contain nitrogen-nitrogen (N-N) bonds but that are otherwise structurally unrelated Despite their considerable structural differences, their biosynthetic gene clusters share a set of genes predicted to facilitate N-N bond formation In this study, we show that for both compounds, one of the nitrogen atoms in the N-N bond originates from nitrous acid Furthermore, we show that for both compounds, an acetylhydrazine biosynthetic synthon is generated first and then funneled via a glutamyl carrier into the respective biosynthetic pathways Therefore, unlike other pathways to N-N bond-containing natural products wherein the N-N bond is formed directly on a biosynthetic intermediate, during the biosyntheses of fosfazinomycin, kinamycin, and related compounds, the N-N bond is made in an independent pathway that forms a branch of a convergent route to structurally complex natural products

51 citations

Journal ArticleDOI
TL;DR: The biosynthetic origin of a unique hydrazide moiety in the phosphonate natural product fosfazinomycin is investigated.
Abstract: The biosynthetic origin of a unique hydrazide moiety in the phosphonate natural product fosfazinomycin is unknown. This study presents the activities of five proteins encoded in its gene cluster. The flavin-dependent oxygenase FzmM catalyses the oxidation of L-Asp to N-hydroxy-Asp. When FzmL is added, fumarate is produced in addition to nitrous acid. The adenylosuccinate lyase homolog FzmR eliminates acetylhydrazine from N-acetyl-hydrazinosuccinate, which in turn is the product of FzmQ-catalysed acetylation of hydrazinosuccinate. Collectively, these findings suggest a path to N-acetylhydrazine from L-Asp. The incorporation of nitrogen from L-Asp into fosfazinomycin was confirmed by isotope labelling studies. Installation of the N-terminal Val of fosfazinomycin is catalysed by FzmI in a Val-tRNA dependent process.

51 citations

Journal ArticleDOI
TL;DR: Recent progress in the engineering and application of these enzymes to prepare enantiopure l-aspartic acid derivatives, which are highly valuable as tools for biological research and as chiral building blocks for pharmaceuticals and food additives, is discussed.
Abstract: Ammonia lyases catalyze the formation of α,β-unsaturated bonds by the elimination of ammonia from their substrates. This conceptually straightforward reaction has been the emphasis of many studies, with the main focus on the catalytic mechanism of these enzymes and/or the use of these enzymes as catalysts for the synthesis of enantiomerically pure α-amino acids. In this Review aspartate ammonia lyase and 3-methylaspartate ammonia lyase, which represent two different enzyme superfamilies, are discussed in detail. In the past few years, the three-dimensional structures of these lyases in complex with their natural substrates have revealed the details of two elegant catalytic strategies. These strategies exploit similar deamination mechanisms that involve general-base catalyzed formation of an enzyme-stabilized enolate anion (aci-carboxylate) intermediate. Recent progress in the engineering and application of these enzymes to prepare enantiopure l-aspartic acid derivatives, which are highly valuable as tools for biological research and as chiral building blocks for pharmaceuticals and food additives, is also discussed.

49 citations

Journal ArticleDOI
TL;DR: In this paper, crystal structures of AspB, the aspartase from Bacillus sp. YM55-1, in an unliganded state and in complex with L-aspartate at 2.4 and 2.6 A resolution were reported.
Abstract: Aspartate ammonia lyases (or aspartases) catalyze the reversible deamination of L-aspartate into fumarate and ammonia. The lack of crystal structures of complexes with substrate, product, or substrate analogues so far precluded determination of their precise mechanism of catalysis. Here, we report crystal structures of AspB, the aspartase from Bacillus sp. YM55-1, in an unliganded state and in complex with L-aspartate at 2.4 and 2.6 A resolution, respectively. AspB forces the bound substrate to adopt a high-energy, enediolate-like conformation that is stabilized, in part, by an extensive network of hydrogen bonds between residues Thr101, Ser140, Thr141, and Ser319 and the substrate's β-carboxylate group. Furthermore, substrate binding induces a large conformational change in the SS loop (residues G(317)SSIMPGKVN(326)) from an open conformation to one that closes over the active site. In the closed conformation, the strictly conserved SS loop residue Ser318 is at a suitable position to act as a catalytic base, abstracting the Cβ proton of the substrate in the first step of the reaction mechanism. The catalytic importance of Ser318 was confirmed by site-directed mutagenesis. Site-directed mutagenesis of SS loop residues, combined with structural and kinetic analysis of a stable proteolytic AspB fragment, further suggests an important role for the small C-terminal domain of AspB in controlling the conformation of the SS loop and, hence, in regulating catalytic activity. Our results provide evidence supporting the notion that members of the aspartase/fumarase superfamily use a common catalytic mechanism involving general base-catalyzed formation of a stabilized enediolate intermediate.

47 citations

Journal ArticleDOI
TL;DR: Structural work, combined with earlier mechanistic studies, revealed that members of the aspartase/fumarase superfamily use a common catalytic strategy, which involves general base-catalyzed formation of a stabilized aci-carboxylate intermediate and the participation of a highly flexible loop, containing the signature sequence GSSxxPxKxN (named the SS loop), in substrate binding and catalysis.
Abstract: Members of the aspartase/fumarase superfamily share a common tertiary and quaternary fold, as well as a similar active site architecture; the superfamily includes aspartase, fumarase, argininosuccinate lyase, adenylosuccinate lyase, δ-crystallin, and 3-carboxy-cis,cis-muconate lactonizing enzyme (CMLE). These enzymes all process succinyl-containing substrates, leading to the formation of fumarate as the common product (except for the CMLE-catalyzed reaction, which results in the formation of a lactone). In the past few years, X-ray crystallographic analysis of several superfamily members in complex with substrate, product, or substrate analogues has provided detailed insights into their substrate binding modes and catalytic mechanisms. This structural work, combined with earlier mechanistic studies, revealed that members of the aspartase/fumarase superfamily use a common catalytic strategy, which involves general base-catalyzed formation of a stabilized aci-carboxylate (or enediolate) intermediate and the participation of a highly flexible loop, containing the signature sequence GSSxxPxKxN (named the SS loop), in substrate binding and catalysis.

47 citations

References
More filters
Book
01 Jan 1995
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.

273 citations

Journal ArticleDOI
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].

100 citations