Structural basis for the catalytic mechanism of ethylenediamine-N,N′-disuccinic acid lyase, a carbon-nitrogen bond-forming enzyme with broad substrate scope

The natural aminocarboxylic acid product ethylenediamine-N,N′-disuccinic acid [(S,S)-EDDS] is able to form a stable complex with metal ions, making it an attractive biodegradable alternative for the synthetic metal chelator ethylenediamine tetraacetic acid (EDTA), which is currently used at large scale in numerous applications. Previous studies have demonstrated that biodegradation of (S,S)-EDDS may be initiated by an EDDS lyase, converting (S,S)-EDDS via the intermediate N-(2-aminoethyl)aspartic acid (AEAA) into ethylenediamine and two molecules of fumarate. However, current knowledge of this enzyme is limited due to the absence of structural data. Here, we describe the identification and characterization of an EDDS lyase from Chelativorans sp. BNC1, which has a broad substrate scope, accepting various mono- and diamines for addition to fumarate. We report crystal structures of the enzyme in an unliganded state and in complex with formate, succinate, fumarate, AEAA and (S,S)-EDDS. The structures reveal a tertiary and quaternary fold that is characteristic of the aspartase/fumarase superfamily and support a mechanism that involves general base-catalyzed, sequential two-step deamination of (S,S)-EDDS. This work broadens our understanding of mechanistic diversity within the aspartase/fumarase superfamily and will aid in the optimization of EDDS lyase for asymmetric synthesis of valuable (metal-chelating) aminocarboxylic acids

Characterization of a thermostable methylaspartate ammonia lyase from Carboxydothermus hydrogenoformans

Methylaspartate ammonia lyase (MAL; EC 4.3.1.2) catalyzes the reversible addition of ammonia to mesaconate to give (2S,3S)-3-methylaspartate and (2S,3R)-3-methylaspartate as products. MAL is of considerable biocatalytic interest because of its potential use for the asymmetric synthesis of substituted aspartic acids, which are important building blocks for synthetic enzymes, peptides, chemicals, and pharmaceuticals. Here, we have cloned the gene encoding MAL from the thermophilic bacterium Carboxydothermus hydrogenoformans Z-2901. The enzyme (named Ch-MAL) was overproduced in Escherichia coli and purified to homogeneity by immobilized metal affinity chromatography. Ch-MAL is a dimer in solution, consisting of two identical subunits (∼49 kDa each), and requires Mg(2+) and K(+) ions for maximum activity. The optimum pH and temperature for the deamination of (2S,3S)-3-methylaspartic acid are 9.0 and 70°C (k(cat) = 78 s(−1) and K(m) = 16 mM). Heat inactivation assays showed that Ch-MAL is stable at 50°C for >4 h, which is the highest thermal stability observed among known MALs. Ch-MAL accepts fumarate, mesaconate, ethylfumarate, and propylfumarate as substrates in the ammonia addition reaction. The enzyme also processes methylamine, ethylamine, hydrazine, hydroxylamine, and methoxylamine as nucleophiles that can replace ammonia in the addition to mesaconate, resulting in the corresponding N-substituted methylaspartic acids with excellent diastereomeric excess (>98% de). This newly identified thermostable MAL appears to be a potentially attractive biocatalyst for the stereoselective synthesis of aspartic acid derivatives on large (industrial) scale. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00253-011-3615-6) contains supplementary material, which is available to authorized users

Characterization and biocatalytic applications of aspartate and methylaspartate ammonia lyases

In zijn promotieonderzoek heeft Vinod Puthan Veetil het katalytische mechanisme van aspartaat ammonia lyase, dat lange tijd onbekend was, opgehelderd. Ook heeft hij het biokatalytische potentieel van een variant van methylaspartaat ammonia lyase benut voor de asymmetrische synthese van N-gesubstitueerde-L-aspartaatverbindingen. Aspartaat ammonia lyases (aspartases) spelen een belangrijke rol in het microbiële stikstofmetabolisme door het katalyseren van de reversibele deaminering van L-aspartaat, resulterend in fumaraat en ammonia. Aspartases behoren tot de meest specifieke enzymen die bekend zijn en de hoge specificiteit en katalytische activiteit van deze enzymen zijn gedurende decennia benut voor de commerciële productie van L-aspartaat. Hoewel aspartases voor het eerst gerapporteerd werden in 1929 en de eerste gedetailleerde studies dateren van de jaren 50 van de 20e eeuw, was het exacte katalytische mechanisme nog onbekend. In zijn promotieonderzoek is Vinod Puthan Veetil er uiteindelijk in geslaagd het katalytische mechanisme van aspartase op te helderen. Hij heeft zich hierbij gericht op het aspartase (AspB) uit de thermofiele bacterie Bacillus species YM55-1 en heeft door middel van gerichte mutagenese, kinetiek- en inhibitiestudies de rol van de ‘active site’ residuen in substraatbinding en katalyse bestudeerd. Bovendien werd de kristalstructuur van AspB in complex met het natuurlijke substraat (L-aspartaat) opgelost. De structuurstudies onthulden dat substraatbinding een grote conformationele verandering induceert in een geconserveerde lus, waardoor belangrijke katalytische residuen in de nabijheid van het substraat worden gebracht. Er is een katalytisch mechanisme voorgesteld voor AspB en de resultaten versterken de veronderstelling dat AspB en andere leden van de aspartase/fumarase superfamilie eenzelfde wijze van substraatbinding gebruiken en een gemeenschappelijk katalytisch mechanisme hebben. De bevindingen vormen het uitgangspunt voor ‘mechanism-based engineering’ van aspartase met als doel het gebruik van aspartase in de biokatalytische synthese van aspartaatderivaten. In zijn proefschrift rapporteert Vinod Puthan Veetil ook de biokatalytische synthese van een grote reeks N-gesubstitueerde aspartaatverbindingen, waarbij gebruik wordt gemaakt van een variant van methylaspartaat ammonia lyase (MAL). N-gesubstitueerde aspartaatverbindingen zijn belangrijke bouwstenen voor synthetische peptides, voedingssupplementen en medicijnen; voor zulke toepassingen is de optische zuiverheid van de verbinding van het grootste belang. De MAL-variant katalyseert de additie van structureel verschillende amines aan fumaraat met hoge enantioselectiviteit, resulterend in alleen de L-enantiomeren van de aminozuurproducten. n his PhD work, Vinod Puthan Veetil unraveled the catalytic mechanism of aspartate ammonia lyase, which had for long eluded enzymologists, and exploited the biocatalytic potential of an engineered variant of methylaspartate ammonia lyase for the asymmetric synthesis of N-substituted-L-aspartic acids Aspartate ammonia lyases (aspartases) play an important role in microbial nitrogen metabolism by catalyzing the reversible deamination of L-aspartate to yield fumarate and ammonia. Aspartases are among the most specific enzymes known and, for decades, the high specificity and catalytic activity of these enzymes have been exploited for the commercial production of L-aspartic acid. Although aspartases were first reported as early as 1929, and the first detailed studies were reported in the 1950’s, their exact catalytic mechanism was still unknown. In his PhD work, Vinod Puthan Veetil has finally unraveled the catalytic mechanism of aspartase. By focusing on the aspartase (AspB) from the thermophilic bacterium Bacillus species YM55-1, he applied site-directed mutagenesis, kinetic, and inhibition studies to evaluate the role of active-site residues in substrate binding and catalysis. Additionally, the crystal structure of AspB in complex with the natural substrate (L-aspartate) was solved. The structural data revealed that substrate binding induces a large conformational change in a conserved loop which brings key catalytic residues in close proximity to the substrate. A catalytic mechanism for AspB has been proposed and the results have provided credence for the notion that AspB and other members of the aspartase/fumarase superfamily use a common substrate binding mode and catalytic mechanism. Furthermore, the findings set the stage for mechanism-based engineering of aspartase for synthesis of aspartic acid derivatives. In his thesis, Vinod Puthan Veetil also reports the biocatalytic synthesis of a large variety of N-substituted aspartic acids using an engineered variant of the enzyme methylaspartate ammonia lyase. N-substituted aspartic acids are important building blocks for synthetic peptides, nutraceuticals and pharmaceuticals, and for such applications optical purity is paramount. The engineered enzyme indeed catalyzes additions of various structurally diverse amines to fumaric acid with very high enantioselectivity, yielding only the desired L-enantiomers of the amino acid products.

Structural Basis for the Catalytic Mechanism of Aspartate Ammonia Lyase

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 Å 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 G317SSIMPGKVN326) 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.

Catalytic Mechanisms and Biocatalytic Applications of Aspartate and Methylaspartate Ammonia Lyases

Ammonia lyases catalyze the formation of alpha-beta-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 a-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.