Structural Basis for the Catalytic Activity of Aspartate Aminotransferase K258H Lacking the Pyridoxal 5'-Phosphate-Binding Lysine Residue

Malashkevich, V.N.; Jäger, Joachim; Ziak, Martin; Sauder, Ursula; Gehring, Heinz; Christen, Philipp; Jansonius, Johan N.

doi:10.1021/bi00002a004

Cited by 27 publications

(34 citation statements)

References 32 publications

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“…However, PLP is clearly bound to the active site. This is similar to what was described for a K258H mutant of chicken mitochondria AAT and E. coli AAT (Malashkevich, Jä ger et al, 1995). The density for PLP is strong, and the occupancy of PLP was refined to 0.88 and 0.89 in the two chains, with B factors that were very similar to those for the environment (Fig.…”

Section: Resultssupporting

confidence: 78%

Structures of aspartate aminotransferases fromTrypanosoma brucei,Leishmania majorandGiardia lamblia

et al. 2015

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The structures of three aspartate aminotransferases (AATs) from eukaryotic pathogens were solved within the Seattle Structural Genomics Center for Infectious Disease (SSGCID). Both the open and closed conformations of AAT were observed. Pyridoxal phosphate was bound to the active site via a Schiff base to a conserved lysine. An active-site mutant showed that Trypanosoma brucei AAT still binds pyridoxal phosphate even in the absence of the tethering lysine. The structures highlight the challenges for the structure-based design of inhibitors targeting the active site, while showing options for inhibitor design targeting the N-terminal arm.

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Section: Resultssupporting

confidence: 78%

Structures of aspartate aminotransferases fromTrypanosoma brucei,Leishmania majorandGiardia lamblia

et al. 2015

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“…However, in all of these enzymes water molecules are believed to play no catalytic role; rather, they seem to contribute to the binding of the substrate. On the other hand, for the K258H variant of aspartate aminotransferase (18) it was suggested that the 1,3-prototropic shift catalyzed by this mutant protein is mediated by a water molecule. Water molecules have also been suggested as representing excellent candidates for the protonation of external aldimine of threonine aldolase with glycine (19).…”

Section: Discussionmentioning

confidence: 99%

Site-directed Mutagenesis Provides Insight into Racemization and Transamination of Alanine Catalyzed by Treponema denticola Cystalysin

Cellini

Bertoldi

Paiardini³

et al. 2004

Journal of Biological Chemistry

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In addition to ␣, ␤-elimination of L-cysteine, Treponema denticola cystalysin catalyzes the racemization of both enantiomers of alanine accompanied by an overall transamination. Lys-238 and Tyr-123 or a water molecule located on the si and re face of the cofactor, respectively, have been proposed to act as the acid/base catalysts in the proton abstraction/donation at C␣/C4 of the external aldimine. In this investigation, two site-directed mutants, K238A and Y123F, have been characterized. The Lys 3 Ala mutation results in the complete loss of either lyase activity or racemase activity in both directions or transaminase activity toward L-alanine. However, the K238A mutant is able to catalyze the overall transamination of D-alanine, and only D-alanine is the product of the reverse transamination. For Y123F the k cat /K m is reduced 3.5-fold for ␣,␤-elimination, whereas it is reduced 300 -400-fold for racemization. Y123F has ϳ18% of wild type transaminase activity with L-alanine and an extremely low transaminase activity with D-alanine. Moreover, the catalytic properties of the Y124F and Y123F/Y124F mutants rule out the possibility that the residual racemase and transaminase activities displayed by Y123F are due to Tyr-124. All these data, together with computational results, indicate a two-base racemization mechanism for cystalysin in which Lys-238 has been unequivocally identified as the catalyst acting on the si face of the cofactor. Moreover, this study highlights the importance of the interaction of Tyr-123 with water molecules for efficient proton abstraction/donation function on the re face.Treponema denticola cystalysin catalyzes the pyridoxal 5Ј-phosphate (PLP) 1 -dependent ␣,␤-elimination of L-cysteine to generate pyruvate, ammonia, and H 2 S (1). It has been shown that several sulfur-and non-sulfur-containing amino acids as well as disulfidic amino acids also serve as substrates for this reaction (2). The protein is composed of two identical subunits, each of which comprises 399 amino acid residues. The threedimensional structure of the enzyme has been solved to 1.9 Å (3). The PLP cofactor in the active site forms a Schiff base with the ⑀-amino group of Lys-238 of cystalysin (3). Through sitedirected mutagenesis, spectroscopic, and kinetic studies, we have shown recently that Lys-238 is an essential residue for ␣,␤-elimination catalyzed by the enzyme cystalysin. In addition to strengthening the coenzyme binding and facilitating transimination, this residue seems to participate as a general base abstracting the C␣ proton from the substrate and possibly as a general acid, protonating the ␤-leaving group (4).In a recent paper we have shown that, in addition to the lyase activity, the enzyme exhibits an alanine racemase activity accompanied by an overall transaminase activity toward both enantiomers of alanine (5). Considering that racemization is a side reaction of cystalysin, it occurs with a significant k cat (ϳ1 s Ϫ1 ), even if it is ϳ1000-fold lower than that of alanine racemase (6). The structure of cy...

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“…These two enzymes do not show any mutual sequence similarity and have different folds and different enantiomeric selectivites. mAspAT is one of the most thoroughly characterized aminotransferases; in particular, the catalytic activity of its K258H variant lacking the PLP binding lysine residue has been explained on the basis of its crystal structure (27). DaAT has the same enantiomeric selectivity as 15A9.…”

Section: Resultsmentioning

confidence: 99%

Structural Basis for D-Amino Acid Transamination by the Pyridoxal 5′-Phosphate-dependent Catalytic Antibody 15A9

Golinelli‐Pimpaneau

Lüthi

Christen

2006

Journal of Biological Chemistry

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Antibody 15A9, raised with 5-phosphopyridoxyl (PPL)-N⑀ -acetyl-L-lysine as hapten, catalyzes the reversible transamination of hydrophobic D-amino acids with pyridoxal 5-phosphate (PLP). The crystal structures of the complexes of Fab 15A9 with PPL-L-alanine, PPL-D-alanine, and the hapten were determined at 2.3, 2.3, and 2.5 Å resolution, respectively, and served for modeling the complexes with the corresponding planar imine adducts. The conformation of the PLP-amino acid adduct and its interactions with 15A9 are similar to those occurring in PLPdependent enzymes, except that the amino acid substrate is only weakly bound, and, due to the immunization and selection strategy, the lysine residue that covalently binds PLP in these enzymes is missing. However, the N-acetyl-L-lysine moiety of the hapten appears to have selected for aromatic residues in hypervariable loop H3 (Trp-H100e and Tyr-H100b), which, together with Lys-H96, create an anion-binding environment in the active site. The structural situation and mutagenesis experiments indicate that two catalytic residues facilitate the transamination reaction of the PLP-D-alanine aldimine. The space vacated by the absent L-lysine side chain of the hapten can be filled, in both PLP-alanine aldimine complexes, by mobile Tyr-H100b. This group can stabilize a hydroxide ion, which, however, abstracts the C␣ proton only from D-alanine. Together with the absence of any residue capable of deprotonating C␣ of L-alanine, Tyr-H100b thus underlies the enantiomeric selectivity of 15A9. The reprotonation of C4 of PLP, the rate-limiting step of 15A9-catalyzed transamination, is most likely performed by a water molecule that, assisted by Lys-H96, produces a hydroxide ion stabilized by the anion-binding environment.The most often used approach for generating catalytic antibodies is to elicit antibodies against a haptenic group that mimics the transition state of the target reaction (1). The crystal structures of several antibodies obtained in this way validate this procedure and show the catalytic antibodies to bind and stabilize the transition state through van der Waals, hydrogen bonding, and electrostatic interactions (2, 3). Only by chance, these antibodies possess residues at the combining site that directly participate in the covalence changes of the reaction. The lack of catalytic residues is one of the reasons why the rate enhancement brought about by catalytic antibodies only occasionally approaches that of enzymes. The "bait and switch" strategy, which uses a charged hapten to elicit a programmed chemically reactive charged residue (4, 5), and the related procedure of "reactive immunization" (6) have been developed for obtaining catalytic antibodies endowed with reactive residues. Another possibility is to expand the catalytic scope of antibodies by incorporating a nonproteinaceous cofactor (7) that contributes to the catalytic efficacy while the antibody ensures substrate specificity and possibly reaction specificity. As yet, the structure of only one catalytic antibody tha...

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Structural Basis for the Catalytic Activity of Aspartate Aminotransferase K258H Lacking the Pyridoxal 5'-Phosphate-Binding Lysine Residue

Cited by 27 publications

References 32 publications

Structures of aspartate aminotransferases fromTrypanosoma brucei,Leishmania majorandGiardia lamblia

Structures of aspartate aminotransferases fromTrypanosoma brucei,Leishmania majorandGiardia lamblia

Site-directed Mutagenesis Provides Insight into Racemization and Transamination of Alanine Catalyzed by Treponema denticola Cystalysin

Structural Basis for D-Amino Acid Transamination by the Pyridoxal 5′-Phosphate-dependent Catalytic Antibody 15A9

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