Cocrystallization of a mutant aspartate aminotransferase with a C5-dicarboxylic substrate analog: structural comparison with the enzyme-C4-dicarboxylic analog complex

A mutant Escherichia coil aspartate aminotransferase with 17 amino acid substitutions (ATB17), previously created by directed evolution, shows increased activity for beta-branched amino acids and decreased activity for the native substrates, aspartate and glutamate. A new mutant (ATBSN) was generated by changing two of the 17 mutated residues back to the original ones. ATBSN recovered the activities for aspartate and glutamate to the level of the wild-type enzyme while maintaining the enhanced activity of ATB17 for the other amino acid substrates. The absorption spectrum of the bound coenzyme, pyridoxal 5'-phosphate, also returned to the original state. ATBSN shows significantly increased affinity for substrate analogs including succinate and glutarate, analogs of aspartate and glutamate, respectively. Hence, we could cocrystallize ATBSN with succinate or glutarate, and the structures show how the enzyme can bind two kinds of dicarboxylic substrates with different chain lengths. The present results may also provide an insight into the long-standing controversies regarding the mode of binding of glutamate to the wild-type enzyme.     

Reaction of aspartate aminotransferase with C5-dicarboxylic acids: comparison with the reaction with C4-dicarboxylic acids

The reaction of Escherichia coli aspartate aminotransferase (AspAT) with glutamate and other C5-dicarboxylates was analyzed in order to compare its mechanism of action toward C5 substrates with that toward C4 substrates, which had been extensively characterized. The association of the amino-group protonated and unprotonated forms of glutamate (SH(+) and S, respectively) with the Schiff-base protonated and unprotonated forms of the enzyme (E(L)H(+) and E(L), respectively) yields at least three forms of the Michaelis complex, whereas in the case of aspartate, only two species of this complex exist, E(L).SH(+) and E(L)H(+).S. The reaction of AspAT with 2-methylglutamate can be explained only when we consider all the protonation states of the Michaelis complex. Based on the previous crystallographic studies [Miyahara et al. (1994) J. Biochem. 116, 1001-1012], we consider that glutamate binds to the open form of AspAT and takes an extended conformation in the Michaelis complex, with the alpha-amino group of glutamate oriented in the opposite direction to the Schiff base. This is in contrast to the Michaelis complex of aspartate, in which a strong interaction of the alpha-amino group of aspartate and the Schiff base excludes the presence of the species E(L)H(+).SH(+). It is concluded that AspAT recognizes the two types of dicarboxylates with different chain lengths by changing the gross conformation of the enzyme protein.     

Structural basis for differences in substrate specificity of aspartate aminotransferase isoenzymes

X-Ray structural data concerning the substrate binding site of cytosolic chicken aspartate aminotransferase (AspAT) are reported. The structure of the complex of AspAT with the substrate-like inhibitor maleate has been refined at 2.2 A resolution. The lengths of hydrogen bonds between a bound molecule of maleate and side chains of amino acid residues in the active site are presented as well as other interatomic distances in the substrate binding site. The data obtained for the cytosolic AspAT have been compared with those for the mitochondrial chicken AspAT. It has been inferred that differences in substrate specificity of the AspAT isoenzymes are determined by interactions involving amino acid residues which are situated in the immediate vicinity of the active site and influence ionization or orientation of functional groups interacting with substrate. An explanation is suggested for different rates of transamination of aromatic amino acids in the active sites of the cytosolic and mitochondrial isoenzymes.     

Conformational change in aspartate aminotransferase on substrate binding induces strain in the catalytic group and enhances catalysis

Aspartate aminotransferase has been known to undergo a significant conformational change, in which the small domain approaches the large domain, and the residues at the entrance of the active site pack together, on binding of substrates. Accompanying this conformational change is a two-unit increase in the pK(a) of the pyridoxal 5'-phosphate-Lys(258) aldimine, which has been proposed to enhance catalysis. To elucidate how the conformational change is coupled to the shift in the aldimine pK(a) and how these changes are involved in catalysis, we analyzed structurally and kinetically an enzyme in which Val(39) located at both the domain interface and the entrance of the active site was replaced with a bulkier residue, Phe. The V39F mutant enzyme showed a more open conformation, and the aldimine pK(a) was lowered by 0.7 unit compared with the wild-type enzyme. When Asn(194) had been replaced by Ala in advance, the V39F mutation did not decrease the aldimine pK(a), showing that the domain rotation controls the aldimine pK(a) via the Arg(386)-Asn(194)-pyridoxal 5'-phosphate linkage system. The maleate-bound V39F enzyme showed the aldimine pK(a) 0.9 unit lower than that of the maleate-bound wild-type enzyme. However, the positions of maleate, Asn(194), and Arg(386) were superimposable between the mutant and the wild-type enzymes; therefore, the domain rotation was not the cause of the lowered aldimine pK(a) value. The maleate-bound V39F enzyme showed an altered side-chain packing pattern in the 37-39 region, and the lack of repulsion between Gly(38) carbonyl O and Tyr(225) Oeta seemed to be the cause of the reduced pK(a) value. Kinetic analysis suggested that the repulsion increases the free energy level of the Michaelis complex and promotes the catalytic reaction.     

Buffers for the reconstitution of aspartate aminotransferase

The cofactor activation of the apoenzyme of pig heart cytosolic aspartate aminotransferase was studied in various buffers. Cationic buffers are shown to allow maximal reconstitution in the pH range of 5.0 to 9.0. Anionic buffers made up of mono- and dicarboxylates are found to affect reconstitution in a pH-dependent manner. At low pH, the carboxylates strongly inhibit reconstitution, but at high pH, they show less effect. In contrast, the more potent inhibitor Pi shows the opposite pH profile. Dicarboxylates are considerably more inhibitory than monocarboxylates. Substantial protection against inhibition by a number of carboxylates may be achieved by the addition of sodium chloride.     

Aromatic L-amino acid decarboxylase: conformational change in the flexible region around Arg334 is required during the transaldimination process

Aromatic L-amino acid decarboxylase (AADC) catalytic mechanism has been proposed to proceed through two consecutive intermediates (i.e., Michaelis complex and the external aldimine). Limited proteolysis of AADC that preferentially digested at the C-terminal side of Arg334 was slightly retarded in the presence of dihydroxyphenyl acetate that formed a stable Michaelis complex. On the contrary, AADC was scarcely digested in the presence of L-dopa methyl ester that formed a stable external aldimine. Similar protection by the substrate analogs was observed in the chemical modification experiment. From these results, we concluded that the region around Arg334 must be exposed and flexible in the unliganded state, and forming the Michaelis complex generated a subtle conformational change, then underwent marked conformational change during the subsequent transaldimination process prerequisite to forming the external aldimine. For further analyses, we constructed a mutant gene encoding in tandem the two peptides of AADC cleaved at the Asn327-Met328 bond inside the putative flexible region. The gene product, fragmentary AADC, was still active with L-dopa as substrate, but its k(cat) value was decreased 57-fold, and the Km value was increased 9-fold compared with those of the wild-type AADC. The absorption spectra of the fragmentary AADC in the presence of L-dopa methyl ester showed shift in the equilibrium of the transaldimination from the external aldimine to the Michaelis complex. Tryptic digestion of the fragmentary AADC removed seven amino acid residues, Met328-Arg334, and resulted in complete inactivation. Susceptibility of the fragmentary enzyme to trypsin was not changed by L-dopa methyl ester revealing the loss of appropriate conformational change in the flexible region induced by substrate binding. From these results we propose that the conformational change in the flexible region is required during the transaldimination process.     

Identification and functional analysis of a prokaryotic-type aspartate aminotransferase: implications for plant amino acid metabolism

In this paper, we report the identification of genes from pine (PpAAT), Arabidopsis (AtAAT) and rice (OsAAT) encoding a novel class of aspartate aminotransferase (AAT, EC 2.6.1.1) in plants. The enzyme is unrelated to other eukaryotic AATs from plants and animals but similar to bacterial enzymes. Phylogenetic analysis indicates that this prokaryotic-type AAT is closely related to cyanobacterial enzymes, suggesting it might have an endosymbiotic origin. Interestingly, most of the essential residues involved in the interaction with the substrate and the attachment of pyridoxal phosphate cofactor in the active site of the enzyme were conserved in the deduced polypeptide. The polypeptide is processed in planta to a mature subunit of 45 kDa that is immunologically distinct from the cytosolic, mitochondrial and chloroplastic isoforms of AAT previously characterized in plants. Functional expression of PpAAT sequences in Escherichia coli showed that the processed precursor is assembled into a catalytically active homodimeric holoenzyme that is strictly specific for aspartate. These atypical genes are predominantly expressed in green tissues of pine, Arabidopsis and rice, suggesting a key role of this AAT in nitrogen metabolism associated with photosynthetic activity. Moreover, immunological analyses revealed that the plant prokaryotic-type AAT is a nuclear-encoded chloroplast protein. This implies that two plastidic AAT co-exist in plants: a eukaryotic type previously characterized and the prokaryotic type described here. The respective roles of these two enzymes in plant amino acid metabolism are discussed.     

Narrowing substrate specificity in a directly evolved enzyme: the A293D mutant of aspartate aminotransferase

Several mutant Escherichia coli aspartate aminotransferases (eAATases) have been characterized in the attempt to evolve or rationally redesign the substrate specificity of eAATase into that of E. coli tyrosine aminotransferase (eTATase). These include HEX (designed), HEX + A293D (design followed by directed evolution), and SRHEPT (directed evolution). The A293D mutation realized from directed evolution of HEX is here imported into the SRHEPT platform by site-directed mutagenesis, resulting in an enzyme (SRHEPT + A293D) with nearly the same ratio of k(cat)/K(m)(Phe) to k(cat)/K(m)(Asp) as that of wild-type eTATase. The A293D substitution is an important specificity determinant; it selectively disfavors interactions with dicarboxylic substrates and inhibitors compared to aromatic ones. Context dependence analysis is generalized to provide quantitative comparisons of a common substitution in two or more different protein scaffolds. High-resolution crystal structures of ligand complexes of HEX + A293D, SRHEPT, and SRHEPT + A293D were determined. We find that in both SRHEPT + A293D and HEX + A293D, the additional mutation holds the Arg 292 side chain away from the active site to allow increased specificity for phenylalanine over aspartate. The resulting movement of Arg 292 allows greater flexibility of the small domain in HEX + A293D. While HEX is always in the closed conformation, HEX + A293D is observed in both the closed and a novel open conformation, allowing for more rapid product release.     

Redesign of the substrate specificity of Escherichia coli aspartate aminotransferase to that of Escherichia coli tyrosine aminotransferase by homology modeling and site-directed mutagenesis.

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)     

Binding to lipid membrane induces conformational changes in RPE65: implications for its isomerohydrolase activity

The visual cycle is a multi-step pathway to recycle 11-cis retinal, the chromophore for both rod and cone visual pigments. The isomerohydrolase RPE65, a membrane-associated enzyme, converts atRE (all-trans-retinyl ester) to 11-cis-retinol, a key step in the visual cycle. Previously, it has been shown that membrane association of RPE65 is essential for its catalytic activity. Using purified recombinant chicken RPE65 and an in vitro liposome-based floatation assay, we present evidence that the RPE65 membrane-binding affinity was significantly facilitated by incorporation of atRE, the substrate of RPE65, into liposomal membrane. Using tryptophan emission fluorescence quenching and CD spectroscopy, we showed that, upon membrane binding, RPE65 undergoes conformational changes at both the tertiary and secondary structural levels. Specifically, tryptophan fluorescence quenching showed that the tertiary RPE65 structure became more open towards the hydrophilic environment upon its association with the membrane. Simultaneously, a decrease in the α-helix content of RPE65 was revealed upon binding with the lipid membrane containing atRE. These results demonstrated that RPE65's functional activity depends on its conformational changes caused by its association with the membrane.     

Microbiological oxidation of the branched C5-dicarboxylic acids

Summary The oxidation of branched C₅-dicarboxylates has been studied with microorganisms obtained from enrichement cultures with each of these acids. Bacteria grown on itaconate, mesaconate or methylsuccinate oxidize most of these compounds rapidly; (+) citramalate is oxidized 2–4 times as rapidly as (-) citramalate; and citraconate only feebly. Citraconate-grown cells do not oxidize itaconate and oxidize feebly mesaconate and methylsuccinate; they oxidize (-) citramalate 3–4 times faster than (+) citramalate. Cell-free extracts of various strains grown on different C₅-dicarboxylates contain pronounced mesaconase activity, varying amounts of citraconase and hardly any itaconase activity.Communication number 633 from National Chemical Laboratory.     

The structure of LL-diaminopimelate aminotransferase from Chlamydia trachomatis: implications for its broad substrate specificity

We have previously reported the structures of the native holo and substrate-bound forms of ll-diaminopimelate aminotransferase from Arabidopsis thaliana (AtDAP-AT). Here, we report the crystal and molecular structures of the ll-diaminopimelate aminotransferase from Chlamydia trachomatis (CtDAP-AT) in the apo-form and the pyridoxal-5′-phosphate-bound form. The molecular structure of CtDAP-AT shows that its overall fold is essentially identical with that of AtDAP-AT except that CtDAP-AT adopts an “open” conformation as opposed to the “closed” conformation of AtDAP-AT. Although AtDAP-AT and CtDAP-AT are approximately 40% identical in their primary sequence, they have major differences in their substrate specificities; AtDAP-AT is highly specific for LL-DAP, whereas CtDAP-AT accepts a wider range of substrates. Since all of the residues involved in substrate recognition are highly conserved between AtDAP-AT and CtDAP-AT, we propose that differences in flexibility of the loops lining the active-site region between the two enzymes likely account for the differences in substrate specificity.     

Role of arginine-292 in the substrate specificity of aspartate aminotransferase as examined by site-directed mutagenesis

X-ray crystallographic data have implicated Arg-292 as the residue responsible for the preferred side-chain substrate specificity of aspartate aminotransferase. It forms a salt bridge with the beta or gamma carboxylate group of the substrate [Kirsch, J. F., Eichele, G., Ford, G. C., Vincent, M. G., Jansonius, J. N., Gehring, H., & Christen, P. (1984) J. Mol. Biol. 174, 497-525]. In order to test this proposal and, in addition, to attempt to reverse the substrate charge specificity of this enzyme, Arg-292 has been converted to Asp-292 by site-directed mutagenesis. The activity (kcat/KM) of the mutant enzyme, R292D, toward the natural anionic substrates L-aspartate, L-glutamate, and alpha-ketoglutarate is depressed by over 5 orders of magnitude, whereas the activity toward the keto acid pyruvate and a number of aromatic and other neutral amino acids is reduced by only 2-9 fold. These results confirm the proposal that Arg-292 is critical for the rapid turnover of substrates bearing anionic side chains and show further that, apart from the desired alteration, no major perturbations of the remainder of the molecule have been made. The activity of R292D toward the cationic amino acids L-arginine, L-lysine, and L-ornithine is increased by 9-16-fold over that of wild type and the ratio (kcat/KM)cationic/(kcat/KM)anionic is in the range 2-40-fold for R292D, whereas this ratio has a range of [(0.3-6) x 10(-6)]-fold for wild type. Thus, the mutation has produced an inversion of the substrate charge specificity.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel, definitive test for substrate channeling illustrated with the aspartate aminotransferase/malate dehydrogenase system.

A novel method is presented that establishes definitively the existence or nonexistence of direct metabolite transfer between consecutive enzymes in a metabolic sequence. The procedure is developed with the specific example of channeling of oxaloacetate between Escherichia coli aspartate aminotransferase (AATase) and malate dehydrogenase (MDH). The assay is carried out in the presence of a large excess of inactive variants of AATase. These mutants would outcompete the much smaller quantities of wild-type AATase for any docking sites on MDH and thus decrease the rate of the coupled L-aspartate to oxaloacetate to malate sequence only if the direct metabolite transfer mechanism is operative. The results show that oxaloacetate is not transferred directly from AATase to MDH because no decrease in rate was observed in the presence of approximately 100 microM inactive mutants. This concentration is 10 times the physiological AATase concentration, which was determined in this work. The methodology can be applied generally.