Hans Raj

Engineering methylaspartate ammonia lyase for the asymmetric synthesis of unnatural amino acids

The redesign of enzymes to produce catalysts for a predefined transformation remains a major challenge in protein engineering. Here, we describe the structure-based engineering of methylaspartate ammonia lyase (which in nature catalyses the conversion of 3-methylaspartate to ammonia and 2-methylfumarate) to accept a variety of substituted amines and fumarates and catalyse the asymmetric synthesis of aspartic acid derivatives. We obtained two single-active-site mutants, one exhibiting a wide nucleophile scope including structurally diverse linear and cyclic alkylamines and one with broad electrophile scope including fumarate derivatives with alkyl, aryl, alkoxy, aryloxy, alkylthio and arylthio substituents at the C2 position. Both mutants have an enlarged active site that accommodates the new substrates while retaining the high stereo- and regioselectivity of the wild-type enzyme. As an example, we demonstrate a highly enantio- and diastereoselective synthesis of threo-3-benzyloxyaspartate (an important inhibitor of neuronal excitatory glutamate transporters in the brain). Substituted aspartic acids are highly valuable as tools for biological research and as chiral building blocks for pharmaceuticals. Here, engineering of the enzyme methylaspartate ammonia lyase to accept a large variety of substituted amines and fumarates and catalyse the asymmetric synthesis of aspartic acid derivatives is described.

Catalytic mechanisms and biocatalytic applications of aspartate and methylaspartate ammonia lyases.

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.

Aspartase/Fumarase Superfamily: A Common Catalytic Strategy Involving General Base-Catalyzed Formation of a Highly Stabilized aci-Carboxylate Intermediate

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.

Alteration of the Diastereoselectivity of 3-Methylaspartate Ammonia Lyase by Using Structure-Based Mutagenesis

3‐Methylaspartate ammonia‐lyase (MAL) catalyzes the reversible amination of mesaconate to give both (2S,3S)‐3‐methylaspartic acid and (2S,3R)‐3‐methylaspartic acid as products. The deamination mechanism of MAL is likely to involve general base catalysis, in which a catalytic base abstracts the C3 proton of the respective stereoisomer to generate an enolate anion intermediate that is stabilized by coordination to the essential active‐site MgII ion. The crystal structure of MAL in complex with (2S,3S)‐3‐methylaspartic acid suggests that Lys331 is the only candidate in the vicinity that can function as a general base catalyst. The structure of the complex further suggests that two other residues, His194 and Gln329, are responsible for binding the C4 carboxylate group of (2S,3S)‐3‐methylaspartic acid, and hence are likely candidates to assist the MgII ion in stabilizing the enolate anion intermediate. In this study, the importance of Lys331, His194, and Gln329 for the activity and stereoselectivity of MAL was investigated by site‐directed mutagenesis. His194 and Gln329 were replaced with either an alanine or arginine, whereas Lys331 was mutated to a glycine, alanine, glutamine, arginine, or histidine. The properties of the mutant proteins were investigated by circular dichroism (CD) spectroscopy, kinetic analysis, and 1H NMR spectroscopy. The CD spectra of all mutants were comparable to that of wild‐type MAL, and this indicates that these mutations did not result in any major conformational changes. Kinetic studies demonstrated that the mutations have a profound effect on the values of kcat and kcat/KM; this implicates Lys331, His194 and Gln329 as mechanistically important. The 1H NMR spectra of the amination and deamination reactions catalyzed by the mutant enzymes K331A, H194A, and Q329A showed that these mutants have strongly enhanced diastereoselectivities. In the amination direction, they catalyze the conversion of mesaconate to yield only (2S,3S)‐3‐methylaspartic acid, with no detectable formation of (2S,3R)‐3‐methylaspartic acid. The results are discussed in terms of a mechanism in which Lys331, His194, and Gln329 are involved in positioning the substrate and in formation and stabilization of the enolate anion intermediate.

Site‐directed mutagenesis, kinetic and inhibition studies of aspartate ammonia lyase from Bacillus sp. YM55‐1

Aspartate ammonia lyases (also referred to as aspartases) catalyze the reversible deamination of l‐aspartate to yield fumarate and ammonia. In the proposed mechanism for these enzymes, an active site base abstracts a proton from C3 of l‐aspartate to form an enzyme‐stabilized enediolate intermediate. Ketonization of this intermediate eliminates ammonia and yields the product, fumarate. Although two crystal structures of aspartases have been determined, details of the catalytic mechanism have not yet been elucidated. In the present study, eight active site residues (Thr101, Ser140, Thr141, Asn142, Thr187, His188, Lys324 and Asn326) were mutated in the structurally characterized aspartase (AspB) from Bacillus sp. YM55‐1. On the basis of a model of the complex in which l‐aspartate was docked manually into the active site of AspB, the residues responsible for binding the amino group of l‐aspartate were predicted to be Thr101, Asn142 and His188. This postulate is supported by the mutagenesis studies: mutations at these positions resulted in mutant enzymes with reduced activity and significant increases in the Km for l‐aspartate. Studies of the pH dependence of the kinetic parameters of AspB revealed that a basic group with a pKa of approximately 7 and an acidic group with a pKa of approximately 10 are essential for catalysis. His188 does not play the typical role of active site base or acid because the H188A mutant retained significant activity and displayed an unchanged pH‐rate profile compared to that of wild‐type AspB. Mutation of Ser140 and Thr141 and kinetic analysis of the mutant enzymes revealed that these residues are most likely involved in substrate binding and in stabilizing the enediolate intermediate. Mutagenesis studies corroborate the essential role of Lys324 because all mutations at this position resulted in mutant enzymes that were completely inactive. The substrate‐binding model and kinetic analysis of mutant enzymes suggest that Thr187 and Asn326 assist Lys324 in binding the C1 carboxylate group of the substrate. A catalytic mechanism for AspB is presented that accounts for the observed properties of the mutant enzymes. Several features of the mechanism that are also found in related enzymes are discussed in detail and may help to define a common substrate binding mode for the lyases in the aspartase/fumarase superfamily.

PvdP is a tyrosinase that drives maturation of the pyoverdine chromophore in Pseudomonas aeruginosa

The iron binding siderophore pyoverdine constitutes a major adaptive factor contributing to both virulence and survival in fluorescent pseudomonads. For decades, pyoverdine production has allowed the identification and classification of fluorescent and nonfluorescent pseudomonads. Here, we demonstrate that PvdP, a periplasmic enzyme of previously unknown function, is a tyrosinase required for the maturation of the pyoverdine chromophore in Pseudomonas aeruginosa. PvdP converts the nonfluorescent ferribactin, containing two iron binding groups, into a fluorescent pyoverdine, forming a strong hexadentate complex with ferrous iron, by three consecutive oxidation steps. PvdP represents the first characterized member of a small family of tyrosinases present in fluorescent pseudomonads that are required for siderophore maturation and are capable of acting on large peptidic substrates.

Enantioselective Synthesis of N-Substituted Aspartic Acids Using an Engineered Variant of Methylaspartate Ammonia Lyase

N-Substituted aspartic acids are important building blocks for pharmaceuticals, artificial sweeteners, synthetic enzymes, and peptidomimetics. In such applications, the optical purities of the compounds are paramount. The most common synthetic route to N-substituted aspartic acids is through the direct Michael-type addition of amines to maleic acid, maleic anhydride, or the ester or amide derivatives of maleic acid. Recently, a novel one-step method for the preparation of N-substituted aspartic acids from maleic anhydride and amines via the monolithium salt of maleic acid was reported. However, the products of all these reactions were racemic mixtures, and further separation steps were needed to obtain an optically pure amino acid. A general method for the synthesis of enantiomerically pure N-substituted aspartic acids is the reductive amination of carbonyl compounds with enantiopure aspartic acid. 11] However, this method is limited by the availability of the corresponding carbonyl compounds and the formation of multiple undesired side products. In addition to these chemical synthesis routes, two classes of ammonia lyases, aspartate ammonia lyases (EC 4.3.1.1) and 3-methylaspartate ammonia lyases (EC 4.3.1.2), have recently been explored for the enzymatic synthesis of enantiopure N-substituted aspartic acids (Scheme 1). 14] Aspartate ammonia lyases play a key role in microbial nitrogen metabolism by catalyzing the reversible amination of fumaric acid to produce l-aspartic acid. 15, 16] These enzymes are highly enantioselective and have been used in industry for the production of l-aspartic acid, which in turn is an ingredient in the synthesis of the artificial sweetener aspartame. 18] The aspartate ammonia lyase from Bacillus species YM55-1 (AspB) is highly active, thermostable, and lacks allosteric regulation by substrate or metal ions. AspB processes different amines in the addition reaction and has been used to synthesize Nsubstituted l-aspartic acids with high enantiomeric excess (> 97 % ee). However, the enzyme only accepts a few small amines such as hydroxylamine, hydrazine, methylamine, and methoxylamine in the addition reaction, which yields a limited number of aspartic acid derivatives (Scheme 1). To overcome this limitation, we have attempted to broaden the amine scope of AspB by rational design and by structure-guided saturation mutagenesis. However, we were unsuccessful in these engineering attempts, which may have to do with complex loop movement upon substrate binding, 23] and this makes protein engineering of AspB (and other aspartate ammonia lyases) a formidable challenge. 3-Methylaspartate ammonia lyases catalyze the reversible addition of ammonia to mesaconic acid (2-methylfumaric acid) to give threo-(2S,3S)-3-methylaspartic acid and erythro-(2S,3R)3-methylaspartic acid as products. 24–28] These enzymes are part of glutamate catabolic pathways in anaerobic bacteria and require the presence of Mg + and K ions for full activity. The methylaspartate ammonia lyase from Clostridium tetanomorphum (MAL) accepts fumaric acid as well as a range of 2substituted fumaric acids (ethyl, propyl, isopropyl, and halide derivatives) in the ammonia addition reaction. 30] MAL also accepts hydrazine, hydroxylamine, methylamine, methoxylamine, and ethylamine in the addition reaction. The MAL-catalyzed additions of these amines to fumaric acid were highly enantioselective and yielded the corresponding N-substituted l-aspartic acids as the only products (Scheme 1). Unfortunately, as with AspB, MAL has a very narrow amine scope and only accepts these small amines as substrates. Interestingly, previous protein engineering work on MAL, which involved structure-guided saturation mutagenesis and activity screening, resulted in the MAL variant Q73A, which accepts many structurally distinct amines in addition reactions to mesaconic acid. In the present study, we exploited the synthetic potential of this engineered MAL variant for the preparation of a large variety of N-substituted aspartic acids by the addition Scheme 1. Biocatalytic synthesis of a few N-substituted l-aspartic acids.

Kinetic Resolution and Stereoselective Synthesis of 3-Substituted Aspartic Acids by Using Engineered Methylaspartate Ammonia Lyases

Enzymatic amino acid synthesis: Kinetic resolution and asymmetric synthesis of various valuable 3-substituted aspartic acids, which were obtained in fair to good yields with diastereomeric ratio values of up to >98:2 and enantiomeric excess values of up to >99 %, by using engineered methylaspartate ammonia lyases are described. These biocatalytic methodologies for the selective preparation of aspartic acid derivatives appear to be attractive alternatives for existing chemical methods.

Chemoenzymatic Synthesis of ortho-, meta-, and para-Substituted Derivatives of L-threo-3-Benzyloxyaspartate, An Important Glutamate Transporter Blocker

A simple, three‐step chemoenzymatic synthesis of L‐threo‐3‐benzyloxyaspartate (L‐TBOA), as well as L‐TBOA derivatives with F, CF3, and CH3 substituents at the aromatic ring, starting from dimethyl acetylenedicarboxylate was investigated. These chiral amino acids, which are extremely difficult to prepare by chemical synthesis, form an important class of inhibitors of excitatory amino acid transporters involved in the regulation of glutamatergic neurotransmission. In addition, a new chemical procedure for the synthesis of racemic mixtures of TBOA and its derivatives was explored. These chemically prepared racemates are valuable reference compounds in chiral‐phase HPLC to establish the enantiopurities of the corresponding chemoenzymatically prepared amino acids.

The roles of active site residues in the catalytic mechanism of methylaspartate ammonia-lyase

Methylaspartate ammonia‐lyase (MAL; EC 4.3.1.2) catalyzes the reversible addition of ammonia to mesaconate to yield l‐threo‐(2S,3S)‐3‐methylaspartate and l‐erythro‐(2S,3R)‐3‐methylaspartate as products. In the proposed minimal mechanism for MAL of Clostridium tetanomorphum, Lys‐331 acts as the (S)‐specific base catalyst and abstracts the 3S‐proton from l‐threo‐3‐methylaspartate, resulting in an enolate anion intermediate. This enolic intermediate is stabilized by coordination to the essential active site Mg2+ ion and hydrogen bonding to the Gln‐329 residue. Collapse of this intermediate results in the release of ammonia and the formation of mesaconate. His‐194 likely acts as the (R)‐specific base catalyst and abstracts the 3R‐proton from the l‐erythro isomer of 3‐methylaspartate, yielding the enolic intermediate. In the present study, we have investigated the importance of the residues Gln‐73, Phe‐170, Gln‐172, Tyr‐356, Thr‐360, Cys‐361 and Leu‐384 for the catalytic activity of C. tetanomorphum MAL. These residues, which are part of the enzyme surface lining the substrate binding pocket, were subjected to site‐directed mutagenesis and the mutant enzymes were characterized for their structural integrity, ability to catalyze the amination of mesaconate, and regio‐ and diastereoselectivity. Based on the observed properties of the mutant enzymes, combined with previous structural studies and protein engineering work, we propose a detailed catalytic mechanism for the MAL‐catalyzed reaction, in which the side chains of Gln‐73, Gln‐172, Tyr‐356, Thr‐360, and Leu‐384 provide favorable interactions with the substrate, which are important for substrate binding and activation. This detailed knowledge of the catalytic mechanism of MAL can serve as a guide for future protein engineering experiments.