Aspartate Aminotransferase

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Yunpeng Hua - One of the best experts on this subject based on the ideXlab platform.

Cristina M Vega - One of the best experts on this subject based on the ideXlab platform.

  • structure and mechanism of a cysteine sulfinate desulfinase engineered on the Aspartate Aminotransferase scaffold
    Biochimica et Biophysica Acta, 2012
    Co-Authors: Francisco J Fernandez, Heinz Gehring, Philipp Christen, Dominique De Vries, Esther Penasoler, Miquel Coll, Cristina M Vega
    Abstract:

    The joint substitution of three active-site residues in Escherichia coli (L)-Aspartate Aminotransferase increases the ratio of l-cysteine sulfinate desulfinase to transaminase activity 10(5)-fold. This change in reaction specificity results from combining a tyrosine-shift double mutation (Y214Q/R280Y) with a non-conservative substitution of a substrate-binding residue (I33Q). Tyr214 hydrogen bonds with O3 of the cofactor and is close to Arg374 which binds the α-carboxylate group of the substrate; Arg280 interacts with the distal carboxylate group of the substrate; and Ile33 is part of the hydrophobic patch near the entrance to the active site, presumably participating in the domain closure essential for the transamination reaction. In the triple-mutant enzyme, k(cat)' for desulfination of l-cysteine sulfinate increased to 0.5s(-1) (from 0.05s(-1) in wild-type enzyme), whereas k(cat)' for transamination of the same substrate was reduced from 510s(-1) to 0.05s(-1). Similarly, k(cat)' for β-decarboxylation of l-Aspartate increased from<0.0001s(-1) to 0.07s(-1), whereas k(cat)' for transamination was reduced from 530s(-1) to 0.13s(-1). l-Aspartate Aminotransferase had thus been converted into an l-cysteine sulfinate desulfinase that catalyzes transamination and l-Aspartate β-decarboxylation as side reactions. The X-ray structures of the engineered l-cysteine sulfinate desulfinase in its pyridoxal-5'-phosphate and pyridoxamine-5'-phosphate form or liganded with a covalent coenzyme-substrate adduct identified the subtle structural changes that suffice for generating desulfinase activity and concomitantly abolishing transaminase activity toward dicarboxylic amino acids. Apparently, the triple mutation impairs the domain closure thus favoring reprotonation of alternative acceptor sites in coenzyme-substrate intermediates by bulk water.

  • structure and mechanism of a cysteine sulfinate desulfinase engineered on the Aspartate Aminotransferase scaffold proteins and proteomics
    Biochimica et Biophysica Acta, 2012
    Co-Authors: Francisco J Fernandez, Heinz Gehring, Philipp Christen, Dominique De Vries, Esther Penasoler, Miquel Coll, Cristina M Vega
    Abstract:

    The joint substitution of three active-site residues in Escherichia colil-Aspartate Aminotransferase increases the ratio of l-cysteine sulfinate desulfinase to transaminase activity 10⁵-fold. This change in reaction specificity results from combining a tyrosine-shift double mutation (Y214Q/R280Y) with a non-conservative substitution of a substrate-binding residue (I33Q). Tyr214 hydrogen bonds with O3 of the cofactor and is close to Arg374 which binds the α-carboxylate group of the substrate; Arg280 interacts with the distal carboxylate group of the substrate; and Ile33 is part of the hydrophobic patch near the entrance to the active site, presumably participating in the domain closure essential for the transamination reaction. In the triple-mutant enzyme, kcₐₜ′ for desulfination of l-cysteine sulfinate increased to 0.5s⁻¹ (from 0.05s⁻¹ in wild-type enzyme), whereas kcₐₜ′ for transamination of the same substrate was reduced from 510s⁻¹ to 0.05s⁻¹. Similarly, kcₐₜ′ for β-decarboxylation of l-Aspartate increased from<0.0001s⁻¹ to 0.07s⁻¹, whereas kcₐₜ′ for transamination was reduced from 530s⁻¹ to 0.13s⁻¹. l-Aspartate Aminotransferase had thus been converted into an l-cysteine sulfinate desulfinase that catalyzes transamination and l-Aspartate β-decarboxylation as side reactions. The X-ray structures of the engineered l-cysteine sulfinate desulfinase in its pyridoxal-5′-phosphate and pyridoxamine-5′-phosphate form or liganded with a covalent coenzyme–substrate adduct identified the subtle structural changes that suffice for generating desulfinase activity and concomitantly abolishing transaminase activity toward dicarboxylic amino acids. Apparently, the triple mutation impairs the domain closure thus favoring reprotonation of alternative acceptor sites in coenzyme–substrate intermediates by bulk water.

Baogang Peng - One of the best experts on this subject based on the ideXlab platform.

Philipp Christen - One of the best experts on this subject based on the ideXlab platform.

  • structure and mechanism of a cysteine sulfinate desulfinase engineered on the Aspartate Aminotransferase scaffold
    Biochimica et Biophysica Acta, 2012
    Co-Authors: Francisco J Fernandez, Heinz Gehring, Philipp Christen, Dominique De Vries, Esther Penasoler, Miquel Coll, Cristina M Vega
    Abstract:

    The joint substitution of three active-site residues in Escherichia coli (L)-Aspartate Aminotransferase increases the ratio of l-cysteine sulfinate desulfinase to transaminase activity 10(5)-fold. This change in reaction specificity results from combining a tyrosine-shift double mutation (Y214Q/R280Y) with a non-conservative substitution of a substrate-binding residue (I33Q). Tyr214 hydrogen bonds with O3 of the cofactor and is close to Arg374 which binds the α-carboxylate group of the substrate; Arg280 interacts with the distal carboxylate group of the substrate; and Ile33 is part of the hydrophobic patch near the entrance to the active site, presumably participating in the domain closure essential for the transamination reaction. In the triple-mutant enzyme, k(cat)' for desulfination of l-cysteine sulfinate increased to 0.5s(-1) (from 0.05s(-1) in wild-type enzyme), whereas k(cat)' for transamination of the same substrate was reduced from 510s(-1) to 0.05s(-1). Similarly, k(cat)' for β-decarboxylation of l-Aspartate increased from<0.0001s(-1) to 0.07s(-1), whereas k(cat)' for transamination was reduced from 530s(-1) to 0.13s(-1). l-Aspartate Aminotransferase had thus been converted into an l-cysteine sulfinate desulfinase that catalyzes transamination and l-Aspartate β-decarboxylation as side reactions. The X-ray structures of the engineered l-cysteine sulfinate desulfinase in its pyridoxal-5'-phosphate and pyridoxamine-5'-phosphate form or liganded with a covalent coenzyme-substrate adduct identified the subtle structural changes that suffice for generating desulfinase activity and concomitantly abolishing transaminase activity toward dicarboxylic amino acids. Apparently, the triple mutation impairs the domain closure thus favoring reprotonation of alternative acceptor sites in coenzyme-substrate intermediates by bulk water.

  • structure and mechanism of a cysteine sulfinate desulfinase engineered on the Aspartate Aminotransferase scaffold proteins and proteomics
    Biochimica et Biophysica Acta, 2012
    Co-Authors: Francisco J Fernandez, Heinz Gehring, Philipp Christen, Dominique De Vries, Esther Penasoler, Miquel Coll, Cristina M Vega
    Abstract:

    The joint substitution of three active-site residues in Escherichia colil-Aspartate Aminotransferase increases the ratio of l-cysteine sulfinate desulfinase to transaminase activity 10⁵-fold. This change in reaction specificity results from combining a tyrosine-shift double mutation (Y214Q/R280Y) with a non-conservative substitution of a substrate-binding residue (I33Q). Tyr214 hydrogen bonds with O3 of the cofactor and is close to Arg374 which binds the α-carboxylate group of the substrate; Arg280 interacts with the distal carboxylate group of the substrate; and Ile33 is part of the hydrophobic patch near the entrance to the active site, presumably participating in the domain closure essential for the transamination reaction. In the triple-mutant enzyme, kcₐₜ′ for desulfination of l-cysteine sulfinate increased to 0.5s⁻¹ (from 0.05s⁻¹ in wild-type enzyme), whereas kcₐₜ′ for transamination of the same substrate was reduced from 510s⁻¹ to 0.05s⁻¹. Similarly, kcₐₜ′ for β-decarboxylation of l-Aspartate increased from<0.0001s⁻¹ to 0.07s⁻¹, whereas kcₐₜ′ for transamination was reduced from 530s⁻¹ to 0.13s⁻¹. l-Aspartate Aminotransferase had thus been converted into an l-cysteine sulfinate desulfinase that catalyzes transamination and l-Aspartate β-decarboxylation as side reactions. The X-ray structures of the engineered l-cysteine sulfinate desulfinase in its pyridoxal-5′-phosphate and pyridoxamine-5′-phosphate form or liganded with a covalent coenzyme–substrate adduct identified the subtle structural changes that suffice for generating desulfinase activity and concomitantly abolishing transaminase activity toward dicarboxylic amino acids. Apparently, the triple mutation impairs the domain closure thus favoring reprotonation of alternative acceptor sites in coenzyme–substrate intermediates by bulk water.

  • mutant Aspartate Aminotransferase k258h without pyridoxal 5 phosphate binding lysine residue structural and catalytic properties
    FEBS Journal, 1993
    Co-Authors: Martin Ziak, Rolf Jaussi, Heinz Gehring, Johan N Jansonius, Joachim Jager, Vladimir N Malashkevich, Philipp Christen
    Abstract:

    If the pyridoxal-phosphate-binding lysine residue 258 of Aspartate Aminotransferase is exchanged for a histidine residue, the enzyme retains partial catalytic competence [Ziak, M., Jaussi, R., Gehring, H. and Christen, P. (1990) Eur. J. Biochem. 187, 329-333]. The three-dimensional structures of the mutant enzymes of both chicken mitochondria and Escherichia coli were determined at high resolution. The folding patterns of the polypeptide chains proved to be identical to those of the wild-type enzymes, small conformational differences being restricted to parts of the active site. If Aspartate or glutamate was added to the pyridoxal form of the mutant enzyme [lambda max 392 nm and 330 nm (weak); negative CD at 420 nm, positive CD at 370 nm and 330 nm], the external aldimine (lambda max = 430 nm; negative CD at 360 nm and 430 nm) transiently accumulated. Upon addition of 2-oxoglutarate to the pyridoxamine form (lambda max 330 nm, positive CD), a putative ketamine intermediate could be detected; however, with oxalacetate, an equilibrium between external aldimine and the pyridoxal form, which was strongly in favour of the former, was established within seconds. The transamination cycle with glutamate and oxalacetate proceeds only three orders of magnitude more slowly than the overall reaction of the wild-type enzyme. The specific activity of the mutant enzyme is 0.1 U/mg at 25 degrees C and constant from pH 6.0 to 8.5. Reconstitution of the mutant apoenzyme with [4'-3H]pyridoxamine 5'-phosphate resulted in rapid release of 3H with a first-order rate constant kappa' = 5 x 10(-4) s-1 similar to that of the wild-type enzyme. Apparently, in Aspartate Aminotransferase, histidine can to some extent substitute for the active-site lysine residue. The imidazole ring of H258, however, seems too distant from C alpha and C4' to act efficiently as proton donor/acceptor in the aldimine-ketamine tautomerization, suggesting that the prototropic shift might be mediated by an intervening water molecule. Transmination of the internal to the external aldimine apparently can be replaced by de novo formation of the latter, and by its hydrolysis in the reverse direction.

Jack F Kirsch - One of the best experts on this subject based on the ideXlab platform.

  • cofactor directed reversible denaturation pathways the cofactor stabilized escherichia coli Aspartate Aminotransferase homodimer unfolds through a pathway that differs from that of the apoenzyme
    Biochemistry, 2007
    Co-Authors: Edgar Deu, Jack F Kirsch
    Abstract:

    While the urea-mediated unfolding pathway of the Escherichia coli Aspartate Aminotransferase (eAATase) homodimer proceeds through a reversible three-state process with a partially folded dimeric intermediate, D ⇄ D* ⇄ 2U (E. Deu and J. F. Kirsch, accompanying paper), that of a cofactor-stabilized form differs. Pyridoxal phosphate, which binds at the intersubunit active sites, stabilizes the native form by 6 kcal mol-1 and dissociates during the D ⇄ D* transition. Reductive trapping of the cofactor to a nondissociable derivative (PPL-eAATase) precludes the formation of D*. A novel monomeric intermediate (M‘-PPL) with 70% of the native secondary structure (circular dichroism) was identified in the unfolding pathway of PPL-eAATase:  D-PPL2 ⇄ 2M‘-PPL ⇄ 2U-PPL. The combined results define two structural regions with distinct stabilities:  the active site region (ASR) and the generally more stable, dimerization region (DMR). The DMR includes the key intersubunit contacts. It is responsible for the multimeric na...

  • redesign of the substrate specificity of escherichia coli Aspartate Aminotransferase to that of escherichia coli tyrosine Aminotransferase by homology modeling and site directed mutagenesis
    Protein Science, 1995
    Co-Authors: James J Onuffer, Jack F Kirsch
    Abstract:

    Although several high-resolution X-ray crystallographic structures have been determined for Escherichia coli Aspartate Aminotransferase (eAATase), efforts to crystallize E. coli tyrosine Aminotransferase (eTATase) have been unsuccessful. Sequence alignment analyses of eTATase and eAATase show 43% sequence identity and 72% sequence similarity, allowing for conservative substitutions. The high similarity of the two sequences indicates that both enzymes must have similar secondary and tertiary structures. Six active site residues of eAATase were targeted by homology modeling as being important for aromatic amino acid reactivity with eTATase. Two of these positions (Thr 109 and Asn 297) are invariant in all known Aspartate Aminotransferase enzymes, but differ in eTATase (Ser 109 and Ser 297). The other four positions (Val 39, Lys 41, Thr 47, and Asn 69) line the active site pocket of eAATase and are replaced by amino acids with more hydrophobic side chains in eTATase (Leu 39, Tyr 41, Ile 47, and Leu 69). These six positions in eAATase were mutated by site-directed mutagenesis to the corresponding amino acids found in eTATase in an attempt to redesign the substrate specificity of eAATase to that of eTATase. Five combinations of the individual mutations were obtained from mutagenesis reactions. The redesigned eAATase mutant containing all six mutations (Hex) displays second-order rate constants for the transamination of Aspartate and phenylalanine that are within an order of magnitude of those observed for eTATase. Thus, the reactivity of eAATase with phenylalanine was increased by over three orders of magnitude without sacrificing the high transamination activity with Aspartate observed for both enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

  • accumulation of the quinonoid intermediate in the reaction catalyzed by Aspartate Aminotransferase with cysteine sulfinic acid
    Archives of Biochemistry and Biophysics, 1995
    Co-Authors: N C Furumo, Jack F Kirsch
    Abstract:

    Abstract The pyridoxal phosphate form of Aspartate Aminotransferase from Escherichia coli catalyzes the irreversible conversion of L-cysteine sulfinate to the pyridoxamine phosphate form of the enzyme, bisulfite, and pyruvate. The addition of L-cysteine sulfinate to a solution containing a high concentration of enzyme (≍10 μM) yields a rapidly appearing red color (λ max = 520 nm) which decays with a rate constant which is only about 1% of k cat (2-3 s −1 versus 250 s −1 at 15°C, pH 7). The red color can be assigned to the quinonoid form of the enzyme substrate complex, which accumulates under these single turnover conditions. The rate of decay of this species is dependent on that for the decomposition of β-sulfinylpyruvate (β-SP), the initial product of the reaction between Aspartate Aminotransferase and L-cysteine sulfinate. Trapping β-SP with morpholine or malate dehydrogenase plus NADH abolishes the transient red color; therefore, the intermediate accumulates by virtue of the reverse reaction of β-SP with the pyridoxamine phosphate form of the enzyme. The association and dissociation rate constants of β-SP with the pyridoxamine-5′-phosphate form of the enzyme are 2 × 10 7 M −1 s −1 and 400 s −1 , respectively, at 15°C. No red transient species is observed under these conditions when Aspartate is substituted for L-cysteine sulfinate.

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