Abstract
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.
Highlights
The enzyme methylaspartate ammonia-lyase (MAL; EC 4.3.1.2) catalyzes the reversible addition of ammonia to mesaconate (1) to yield l-threo-(2S,3S)-3-methylaspartate (2) and l-erythro-(2S,3R)-3methylaspartate (3) as products (Fig. 1) [1,2]
On the basis of these structural studies [15,16,17], combined with kinetic isotope measurements [18,19] and mutagenesis experiments [20], a minimal mechanism has emerged for the MAL-catalyzed reaction
On the basis of previous kinetic isotope measurements [18,19], structural studies [15,16], and mutagenesis experiments [20], a minimal mechanism has emerged for the MAL-catalyzed reaction
Summary
The enzyme methylaspartate ammonia-lyase (MAL; EC 4.3.1.2) catalyzes the reversible addition of ammonia to mesaconate (1) to yield l-threo-(2S,3S)-3-methylaspartate (2) and l-erythro-(2S,3R)-3methylaspartate (3) as products (Fig. 1) [1,2]. On the basis of these structural studies [15,16,17], combined with kinetic isotope measurements [18,19] and mutagenesis experiments [20], a minimal mechanism has emerged for the MAL-catalyzed reaction. In this proposed mechanism, an (S)-specific (Lys-331) or (R)-specific (His-194) catalytic base abstracts the C3 proton of the respective stereoisomer of 3-methylaspartate to generate an enolate anion intermediate that is stabilized by a hydrogen bond interaction with Gln-329 and coordination to the essential active site Mg2 + ion [20]. This mechanism, is far from complete and the importance of other residues for the catalytic activity of MAL has not been investigated yet
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