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

Raj H, Poelarends GJ - FEBS Open Bio (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.

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 Mg(2+) 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.

No MeSH data available.


Crystal structure of MAL in complex with the natural substrate l-threo-(2S,3S)-3-methylaspartate (PDB: 1KKR) [16]. (a) Close-up of the active site showing the hydrogen-bond interactions between the substrate's carboxylate groups (C1 and C4) and the side chains of His-194, Gln-329, Gln-172 and Thr-360, or the main chain NH of Cys-361. The carbon atoms of the active site residues are shown in green, and those of the substrate in cyan. Hydrogen bonds are represented as dashed lines. The distance (in Å) between the C3 atom of the substrate and side chain of Lys-331 is shown (atoms connected by a solid line). The magnesium ion is shown as a magenta sphere. (b) Close-up of the active site showing the hydrogen-bond interactions between the substrate's amino group and the side chains of Gln-73 (via a water molecule) and Gln-172, as well as the observed distances (in Å, atoms connected by solid lines) between the substrate's methyl group and the side chains of Leu-384, Phe-170 and Tyr-356. The magnesium ion and water molecule are shown as magenta and yellow spheres, respectively. Colour scheme as in (a). The figures were prepared with Pymol (http://www.pymol.org) [30]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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fig0002: Crystal structure of MAL in complex with the natural substrate l-threo-(2S,3S)-3-methylaspartate (PDB: 1KKR) [16]. (a) Close-up of the active site showing the hydrogen-bond interactions between the substrate's carboxylate groups (C1 and C4) and the side chains of His-194, Gln-329, Gln-172 and Thr-360, or the main chain NH of Cys-361. The carbon atoms of the active site residues are shown in green, and those of the substrate in cyan. Hydrogen bonds are represented as dashed lines. The distance (in Å) between the C3 atom of the substrate and side chain of Lys-331 is shown (atoms connected by a solid line). The magnesium ion is shown as a magenta sphere. (b) Close-up of the active site showing the hydrogen-bond interactions between the substrate's amino group and the side chains of Gln-73 (via a water molecule) and Gln-172, as well as the observed distances (in Å, atoms connected by solid lines) between the substrate's methyl group and the side chains of Leu-384, Phe-170 and Tyr-356. The magnesium ion and water molecule are shown as magenta and yellow spheres, respectively. Colour scheme as in (a). The figures were prepared with Pymol (http://www.pymol.org) [30]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Mentions: The crystal structure of MAL complexed with its natural substrate 2 shows that the surface of the enzyme lining the substrate binding pocket is provided, in part, by residues Gln-73, Phe-170, Gln-172, Tyr-356, Thr-360, Cys-361 and Leu-384 (Fig. 2) [16]. These residues are highly conserved in known MALs [16], but not in other members of the enolase superfamily [15]. In the present study, we performed site-directed mutagenesis experiments on all these residues to provide insight into their roles in the catalytic mechanism for the MAL-catalyzed reaction. 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 recent enzyme engineering work, we present a detailed catalytic mechanism for the MAL-catalyzed reaction, with important roles for Gln-73, Gln-172, Tyr-356, Thr-360, and Leu-384 in substrate binding and activation.


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

Raj H, Poelarends GJ - FEBS Open Bio (2013)

Crystal structure of MAL in complex with the natural substrate l-threo-(2S,3S)-3-methylaspartate (PDB: 1KKR) [16]. (a) Close-up of the active site showing the hydrogen-bond interactions between the substrate's carboxylate groups (C1 and C4) and the side chains of His-194, Gln-329, Gln-172 and Thr-360, or the main chain NH of Cys-361. The carbon atoms of the active site residues are shown in green, and those of the substrate in cyan. Hydrogen bonds are represented as dashed lines. The distance (in Å) between the C3 atom of the substrate and side chain of Lys-331 is shown (atoms connected by a solid line). The magnesium ion is shown as a magenta sphere. (b) Close-up of the active site showing the hydrogen-bond interactions between the substrate's amino group and the side chains of Gln-73 (via a water molecule) and Gln-172, as well as the observed distances (in Å, atoms connected by solid lines) between the substrate's methyl group and the side chains of Leu-384, Phe-170 and Tyr-356. The magnesium ion and water molecule are shown as magenta and yellow spheres, respectively. Colour scheme as in (a). The figures were prepared with Pymol (http://www.pymol.org) [30]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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fig0002: Crystal structure of MAL in complex with the natural substrate l-threo-(2S,3S)-3-methylaspartate (PDB: 1KKR) [16]. (a) Close-up of the active site showing the hydrogen-bond interactions between the substrate's carboxylate groups (C1 and C4) and the side chains of His-194, Gln-329, Gln-172 and Thr-360, or the main chain NH of Cys-361. The carbon atoms of the active site residues are shown in green, and those of the substrate in cyan. Hydrogen bonds are represented as dashed lines. The distance (in Å) between the C3 atom of the substrate and side chain of Lys-331 is shown (atoms connected by a solid line). The magnesium ion is shown as a magenta sphere. (b) Close-up of the active site showing the hydrogen-bond interactions between the substrate's amino group and the side chains of Gln-73 (via a water molecule) and Gln-172, as well as the observed distances (in Å, atoms connected by solid lines) between the substrate's methyl group and the side chains of Leu-384, Phe-170 and Tyr-356. The magnesium ion and water molecule are shown as magenta and yellow spheres, respectively. Colour scheme as in (a). The figures were prepared with Pymol (http://www.pymol.org) [30]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Mentions: The crystal structure of MAL complexed with its natural substrate 2 shows that the surface of the enzyme lining the substrate binding pocket is provided, in part, by residues Gln-73, Phe-170, Gln-172, Tyr-356, Thr-360, Cys-361 and Leu-384 (Fig. 2) [16]. These residues are highly conserved in known MALs [16], but not in other members of the enolase superfamily [15]. In the present study, we performed site-directed mutagenesis experiments on all these residues to provide insight into their roles in the catalytic mechanism for the MAL-catalyzed reaction. 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 recent enzyme engineering work, we present a detailed catalytic mechanism for the MAL-catalyzed reaction, with important roles for Gln-73, Gln-172, Tyr-356, Thr-360, and Leu-384 in substrate binding and activation.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.

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 Mg(2+) 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.

No MeSH data available.