Knockout of Tyrosine Aminotransferase Gene by Homologous Recombination Arrests Growth and Disrupts Redox Homeostasis in Leishmania Parasite.

Tyrosine aminotransferase is a well-characterized enzyme in the Leishmania parasite, but the role of TAT in the parasite functioning remains largely unknown. In this study, we attempt to gain a better understanding of the enzyme's role in the parasite by gene knockout and overexpression of the TAT gene. The overexpression of TAT protein was well tolerated by the parasites in two independent repeats. Single knockout of TAT gene by homologous recombination, LdTAT+/- displayed distinct retardation in the proliferation rates and entered the death phase immediately. Morphology of LdTAT+/- parasites had important structural defects as they rounded up with elongated flagella. Gene regulation studies suggested the upregulation of key apoptotic and redox metabolism genes in LdTAT+/-. Moreover, LdTAT+/- cells accumulated higher ROS, thiols, intracellular Ca2+ concentrations, and mitochondrial membrane depolarization signifying the onset of apoptosis. Tocopherol levels were reduced by 50% in LdTAT+/- suggesting the involvement of TAT in tocopherol biosynthesis in the parasite. Overall, our results provide the first evidence that gene knockout of TAT results in apoptosis and that TAT is required for the survival and viability of Leishmania donovani.

Flavones reversibly inhibit Leishmania donovani tyrosine aminotransferase by binding to the catalytic pocket: An integrated in silico-in vitro approach.

The current drugs for treating Leishmaniasis are toxic, non-economical and with the emergence of drug resistance makes the need for novel therapeutics urgent and necessary. In the current study, we report the identification of compounds TI 1-5 against tyrosine aminotransferase of L. donovani from a curated ZINC15 database containing 183,659 compounds. These flavonoid compounds had binding energies < -8 kcal/mol and interacted with the active site residues S151, K286, C290, and P291. Assessment of physicochemical descriptors and ADMET properties established the drug likeliness of these compounds. The all-atom molecular dynamic simulations of the TAT-TI complexes exhibited stable geometrical properties and further trajectory analysis revealed the high-affinity interactions of TI 1, 3, 4, and 5 with the active site residues. DFT calculations reported the high electrophilic nature of TI 2 while other TI compounds demonstrated good kinetic stability and reactivity. From in vitro studies, TI 3 and TI 4 had the highest inhibition with Ki values of 0.9 ± 0.2 µM and 0.30 ± 0.1 µM, respectively. Taken together, the results from this study indicate the potentiality of TI 1, 3, 4, and 5 as anti-leishmanial leads, and these compounds can be exploited to manage the growing Leishmaniasis crisis in the world.

Mapping N- and C-terminals of Leishmania donovani tyrosine aminotransferase by gene truncation strategy: a functional study using in vitro and in silico approaches.

Tyrosine aminotransferase (TAT) catalyzes the transamination of amino acids in Leishmania sp.. TAT from Leishmania donovani has been found to be extremely stable at extreme temperatures and pH conditions. This study was conceived to map the functions of the non-conserved N-terminal and conserved C-terminal domain of TAT. N-terminal (NTAT) and C-terminal (CTAT) domain of TAT was truncated and cloned into the pET28a(+) vector. The truncated proteins were expressed, purified, and biochemically characterized. The Km of NTAT and CTAT for the tyrosine-pyruvate pair was determined to be 3.468 ± 0.796 mM and 4.581 ± 0.627 mM, repectively. Temperature and pH stability studies found NTAT to be stable like TAT but CTAT was extremely susceptible to temperature and pH changes. Upon docking and simulation for 100 ns, NTAT had lower SASA values. From UV spectroscopic study, PLP bound better to CTAT than NTAT because of the reduced SASA of NTAT. The sensitivity of CTAT was reasoned when the urea denaturation studies showed two-state denaturation which differed from NTAT's and TAT's biphasic folding mechanism. From this study, the authors hypothesize that the N-terminal is responsible for PLP stabilization and C-terminal protects the active site from extreme conditions.

Concerted motion of structure and active site charge is required for tyrosine aminotransferase activity in Leishmania parasite.

Leishmania donovani tyrosine aminotransferase (LdTAT) is an essential enzyme that catalyzes the first step of amino acid catabolism. To understand LdTAT activity at different pH, molecular dynamics simulations were performed and trajectory and T-pad analysis pad were conducted. Fluorescence spectroscopy of LdTAT at various pH was measured to understand structural stability. UV studies on PLP were performed to determine the binding of the enzyme to cofactor PLP at different pH. The MD simulations showed that the structure of LdTAT was stable and no structural denaturation was observed at pH 2, 7 and 12. LdTAT exhibited the highest activity at pH -8 and fluorescent spectroscopy also corroborated by exhibiting the highest intensity at pH -8. Moreover, no structural denaturation was observed during the pH gradient. UV studies concluded that the aldimine bond forms only around neutral pH and redshift was observed on enzyme binding. From our observation, we hypothesize that the activity of LdTAT is a close interplay between the structure and charges of K286 and PLP. This study may provide significant insight into understanding parasitic enzymes like LdTAT during the life-cycle of Leishmania parasite. Knowledge of such enzyme mechanisms can pave the way for the design and delivery of enzyme-specific inhibitors.

Biochemical and structural characterization of tyrosine aminotransferase suggests broad substrate specificity and a two-state folding mechanism in Leishmania donovani.

Tyrosine aminotransferase (TAT) is an aminotransferase with broad substrate specificity that catalyzes the transamination of aromatic amino acids in Leishmania donovani and plays a crucial role in the survival and pathogenicity of the parasite. In this study, we have biochemically characterized tyrosine aminotransferase from Leishmania donovani using in vitro and in silico techniques. Leishmania donovani tyrosine aminotransferase (LdTAT) was cloned into the pET28a(+) vector and expressed in the BL21 strain of Escherichia coli. The Ni-NTA-purified protein was then characterized biochemically, and its various kinetic parameters were investigated. The apparent Km value for the tyrosine-pyruvate pair was determined to be 3.5 ± 0.9 mm, and Vmax was analyzed to be at 11.7 ± 1.5 µm·min.µg-1 . LdTAT was found to exhibit maximum activity at 50 °C and at a pH of 8.0. Cofactor identification for LdTAT showed that pyridoxal-5-phosphate (PLP) binds with a Km value of 23.59 ± 3.99 µm and that the phosphate group is vital for the activity of the enzyme. Sequence analysis revealed that S151, Y256, K286, and P291 are conserved residues and form hydrogen bonds with PLP. Urea-based denaturation studies revealed a biphasic folding mechanism involving N→X→D states. Molecular dynamic simulations of modeled LdTAT at various conditions were performed to understand enzyme behavior and interactions at the molecular level. The biochemical and structural divergence between host and parasite TAT suggests the LdTAT has evolved to utilize pyruvate rather than α-ketoglutarate as co-substrate. Furthermore, our data suggest that LdTAT may be a potential drug target due to its divergence in structure and substrate specificity from the host.

A tyrosine aminotransferase involved in rosmarinic acid biosynthesis in Prunella vulgaris L.

Rosmarinic acid (RA) and its derivants are medicinal compounds that comprise the active components of several therapeutics. We isolated and characterised a tyrosine aminotransferase of Prunella vulgaris (PvTAT). Deduced PvTAT was markedly homologous to other known/putative plant TATs. Cytoplasmic localisation of PvTAT was observed in tobacco protoplasts. Recombinantly expressed and purified PvTAT had substrates preference for L-tyrosine and phenylpyruvate, with apparent K m of 0.40 and 0.48 mM, and favoured the conversion of tyrosine to 4-hydroxyphenylpyruvate. In vivo activity was confirmed by functional restoration of the Escherichia coli tyrosine auxotrophic mutant DL39. Agrobacterium rhizogenes-mediated antisense/sense expression of PvTAT in hairy roots was used to evaluate the contribution of PvTAT to RA synthesis. PvTAT were reduced by 46-95% and RA were decreased by 36-91% with low catalytic activity in antisense transgenic hairy root lines; furthermore, PvTAT were increased 0.77-2.6-fold with increased 1.3-1.8-fold RA and strong catalytic activity in sense transgenic hairy root lines compared with wild-type counterparts. The comprehensive physiological and catalytic evidence fills in the gap in RA-producing plants which didn't provide evidence for TAT expression and catalytic activities in vitro and in vivo. That also highlights RA biosynthesis pathway in P. vulgaris and provides useful information to engineer natural products.

Structure of tyrosine aminotransferase from Leishmania infantum.

The trypanosomatid parasite Leishmania infantum is the causative agent of visceral leishmaniasis (VL), which is usually fatal unless treated. VL has an incidence of 0.5 million cases every year and is an important opportunistic co-infection in HIV/AIDS. Tyrosine aminotransferase (TAT) has an important role in the metabolism of trypanosomatids, catalyzing the first step in the degradation pathway of aromatic amino acids, which are ultimately converted into their corresponding L-2-oxoacids. Unlike the enzyme in Trypanosoma cruzi and mammals, L. infantum TAT (LiTAT) is not able to transaminate ketoglutarate. Here, the structure of LiTAT at 2.35 Å resolution is reported, and it is confirmed that the presence of two Leishmania-specific residues (Gln55 and Asn58) explains, at least in part, this specific reactivity. The difference in substrate specificity between leishmanial and mammalian TAT and the importance of this enzyme in parasite metabolism suggest that it may be a useful target in the development of new drugs against leishmaniasis.

A bacterial tyrosine aminomutase proceeds through retention or inversion of stereochemistry to catalyze its isomerization reaction.

ß-Amino acids are biologically active compounds of interest in medicinal chemistry. A class I lyase-like family of aminomutases isomerizes (S)-α-arylalanines to the corresponding ß-amino acids by exchange of the NH2/H pair. This family uses a 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) group within the active site to initiate the reaction. The absolute stereochemistry of the product is known for an MIO-dependent tyrosine aminomutase from Chondromyces crocatus (CcTAM) that isomerizes (S)-α-tyrosine to (R)-ß-tyrosine. To evaluate the cryptic stereochemistry of the CcTAM mechanism, (2S,3S)-[2,3-(2)H2]- and (2S,3R)-[3-(2)H]-α-tyrosine were stereoselectively synthesized from unlabeled (or [(2)H]-labeled) (4'-hydroxyphenyl)acrylic acids by reduction with D2 (or H2) gas and a chiral Rh-Prophos catalyst. GC/EIMS analysis of the [(2)H]-ß-tyrosine biosynthesized by CcTAM revealed that the α-amino group was transferred to Cß of the phenylpropanoid skeleton with retention of configuration. These labeled substrates also showed that the pro-(3S) proton exchanges with protons from the bulk media during its migration to Cα during catalysis. (1)H- and (2)H NMR analyses of the [(2)H]-ß-tyrosine derived from (2S)-[3,3-(2)H2]-α-tyrosine by CcTAM catalysis showed that the migratory proton attached to Cα of the product also with retention of configuration. CcTAM is stereoselective for (R)-ß-tyrosine (85%) yet also forms the (S)-ß-tyrosine enantiomer (15%) through inversion of configuration at both migration termini, as described herein. The proportion of the (S)-ß-isomer made by CcTAM during steady state interestingly increased with solvent pH, and this effect on the proposed reaction mechanism is also discussed.

Engineering homooligomeric proteins to detect weak intersite allosteric communication: aminotransferases, a case study.

The existence of low levels of intersubunit communication in homooligomeric enzymes is often difficult to discover, as the identical active sites cannot be probed individually to dissect their interdependent contributions. The homodimeric paralogs, E. coli aspartate- (AATase) and tyrosine aminotransferase (TATase), have not been demonstrated to show allostery. To address this question, we engineered a hybrid aminotransferase containing two distinct catalytic pockets: an AATase and a TATase site. The TATase/AATase hybrid was constructed by grafting an engineered TATase active site into one of the catalytic pockets of E. coli AATase. Each active site conserves its specific catalytic and inhibitor binding properties, and the hybrid catalyzes simultaneously each aminotransferase reaction at the respective site. Importantly, association of a selective inhibitor into one of the catalytic pockets decreases the activity of the second active site by up to 25%, thus proving unequivocally the existence of allosteric communication between active sites. The procedure may be applicable to other homologous sets of enzymes.

[Fragmentation of the coding region chromatin of trp-dioxygenase (to) and tyr-aminotransferase (tat) genes in their active and repressed state by micrococcal nuclease].

The blot-hybridization technique-assisted we have studied the pattern of fragmentation by mirococcal nuclease (MNase) of DNA tyr-aminotransferase (tat) and trp-dioxygenase (to) genes in active (in rat cell liver nuclei) and repressed (in brain nuclei) states. It was provided, over a wide range of enzyme concentration two types of fragments are mainly produced: near full-size to- and tat-transcription unit (19,000 and 11,000 bp, respectively) and their large (from 1500 bp) heterogeneous in length. To-and tat-fragments of both kinds are preserved in hydrolyzates at limit of MNase digestion of total chromatin DNA when nuclease breaks occur in nearly all accessible sites of chromatin. This means that these fragments originate from two distinct subsets of transcription units coexistent in liver nuclei. The first of them do not contain MNase accessible sites over their entire length, whereas in other resistant regions alternate with rare irregular located MNase-sensitive segments. We presume that resistance to MNase within transcription units is peculiar to the competent genes. As a result of transcription MNase-sensitive areas arise which possibly flank elongating RNA polymerases.

Tyrosine aminotransferase: biochemical and structural properties and molecular dynamics simulations.

Tyrosine aminotransferase (TAT) catalyzes the transamination of tyrosine and other aromatic amino acids. The enzyme is thought to play a role in tyrosinemia type II, hepatitis and hepatic carcinoma recovery. The objective of this study is to investigate its biochemical and structural characteristics and substrate specificity in order to provide insight regarding its involvement in these diseases. Mouse TAT (mTAT) was cloned from a mouse cDNA library, and its recombinant protein was produced using Escherichia coli cells and purified using various chromatographic techniques. The recombinant mTAT is able to catalyze the transamination of tyrosine using α-ketoglutaric acid as an amino group acceptor at neutral pH. The enzyme also can use glutamate and phenylalanine as amino group donors and p-hydroxy-phenylpyruvate, phenylpyruvate and alpha-ketocaproic acid as amino group acceptors. Through macromolecular crystallography we have determined the mTAT crystal structure at 2.9 Å resolution. The crystal structure revealed the interaction between the pyridoxal-5'-phosphate cofactor and the enzyme, as well as the formation of a disulphide bond. The detection of disulphide bond provides some rational explanation regarding previously observed TAT inactivation under oxidative conditions and reactivation of the inactive TAT in the presence of a reducing agent. Molecular dynamics simulations using the crystal structures of Trypanosoma cruzi TAT and human TAT provided further insight regarding the substrate-enzyme interactions and substrate specificity. The biochemical and structural properties of TAT and the binding of its cofactor and the substrate may help in elucidation of the mechanism of TAT inhibition and activation.

Functional characterization of stage-specific aminotransferases from trypanosomatids.

As part of a study on aminotransferases, genes coding for putative enzymes from Trypanosoma brucei and Leishmania major (alanine aminotransferases: ALATs, Tb927.1.3950 and LmjF12.0630; kynurenine aminotransferase: KAT, Tb10.389.1810; and tyrosine aminotransferase: TAT, LmjF36.2360) were cloned and functionally expressed in Escherichia coli. The putative T. brucei KAT, in fact coded for a glutamine aminotransferase (GlnAT), which exhibited a notably high affinity (in the micromolar range) towards glutamine and cysteine; in addition, like bacterial GlnATs and mammalian KATs, it was able to utilize different 2-oxoacids as amino acceptors. L. major TAT resembled T. cruzi TAT in substrate specificity, although the leishmanial enzyme did not exhibit ALAT activity. On the other hand, T. brucei ALAT, shortened by the first 65 amino acids assigned in the data bases, was functional and actively transaminated the substrate pair l-alanine and 2-oxoglutarate. Moreover in Western blots, the molecular size of the protein detected in crude extracts of T. brucei procyclics was identical to the value of the recombinant enzyme. Like T. brucei and T. cruzi orthologues, L. major ALAT displayed narrow substrate specificity. The leishmanial ALAT, like the T. cruzi enzyme, exhibited a dual subcellular localization, in the cytosol and in the mitochondrion. In line with the findings of comparative proteomic analyses of insect and mammalian stages of T. brucei and Leishmania parasites, our results also showed that T. cruzi ALAT is constitutively expressed, with remarkably higher levels being detected in amastigotes than in epimastigotes. ALATs are expressed in the clinically important stages of TriTryps, probably fulfilling an essential role, which deserves further studies.

Clinical and mutational investigations of tyrosinemia type II in Northern Tunisia: identification and structural characterization of two novel TAT mutations.

Tyrosinemia type II or Richner-Hanhart Syndrome (RHS) is an autosomal recessive disorder characterized by keratitis, palmoplantar keratosis, mental retardation, and elevated blood tyrosine levels. The disease is due to a deficiency of hepatic cytosolic tyrosine aminotransferase (TATc), an enzyme involved in the tyrosine catabolic pathway. Because of the high rate of consanguinity this disorder seems to be relatively common among the Arab and Mediterranean populations. RHS is characterized by inter and intrafamilial phenotypic variability. A large spectrum of mutations within TATc gene has been shown to be responsible for RHS. In the present study, we report the clinical features and the molecular investigation of RHS in three unrelated consanguineous Tunisian families including 7 patients with confirmed biochemical diagnosis of tyrosinemia type II. Mutation analyses were performed and two novel missense mutations were identified (C151Y) and (L273P) within exon 5 and exon 8, respectively. The 3D-structural characterization of these mutations provides evidence of defective folding of the mutant proteins, and likely alteration of the enzymatic activity. Phenotype variability was observed even among individuals sharing the same pathogenic mutation.

Enzymatic resolution for the preparation of enantiomerically enriched D-beta-heterocyclic alanine derivatives using Escherichia coli aromatic L-amino acid transaminase.

An enzymatic resolution was carried out for the preparation of enriched beta-heterocyclic D-alanine derivatives using Escherichia coli aromatic L-amino acid transaminase. The excess of pyrazole, imidazole, or 1,2,4-triazole reacted with methyl-2-acetamidoacrylate in acetonitrile in the presence of potassium carbonate at 60 degrees C, directly leading to make the potassium salt of the corresponding N-acetyl-beta-heterocyclic alanine derivatives. After the acidic deprotection of the N-acetyl group, 10 mM of racemic pyrazolylalanine, triazolylalanine, and imidazolylalanine were resolved to D-pyrazolylalanine, D-triazolylalanine, and D-imidazolylalanine with 46% (85% ee), 42% (72% ee), and 48% (95% ee) conversion yield in 18 h, respectively, using E. coli aromatic L-amino acid transaminase (EC 2.6.1.5). Although the three beta-heterocyclic L-alanine derivatives have similar molecular structures, they showed different reaction rates and enantioselectivities. The relative reactivities of the transaminase toward the beta-heterocyclic L-alanine derivatives could be explained by the relationship between the substrate binding energy (E, kcal/mol) to the enzyme active site and the distance (delta, A) from the nitrogen of alpha-amino group of the substrates to the C4' carbon of PLP-Lys258 Schiff base. As the ratio of the substrate binding energy (E) to the distance (delta) becomes indicative value of k(cat)/K(M) of the enzyme to the substrate, the relative reactivities of the beta-heterocyclic L-alanine derivatives were successfully correlated with E/delta, and the relationship was confirmed by our experiments.

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.

Improving protein pharmacokinetics by genetic fusion to simple amino acid sequences.

The role of primary amino acid sequences in protein pharmacokinetics, an issue of relevance in both basic knowledge and biotechnology, was addressed here using as a starting point two repetitive antigens from the hemoflagellate Trypanosoma cruzi that are known to stabilize their associated proteins in the bloodstream. A major drawback to their pharmacological application is that these repetitive sequences are highly immunogenic, being therefore the deletion of this characteristic desirable. Based on sequence homology and epitope mapping analyses, an artificial repetitive sequence (PSTAD) was engineered. This motif was tested by genetic fusion to the C terminus of both the trypanosomal trans-sialidase and the rat tyrosine aminotransferase and found to produce a 4.5-6-fold increase in the half-life of the associated proteins in blood while displaying significantly lower immunogenicity. Residues involved in the stabilizing properties of the novel peptide were mapped by a site-directed mutagenesis approach, allowing us to successfully identify another two motifs. Searching databases for sequences displaying some homology, embedded in proline frameworks and associated to shed virulence factors from unrelated microorganisms, resulted in the identification of four other protein extensions. Remarkably, three of them (from Streptococcus pneumoniae, Actinomyces viscosus, and Escherichia coli) revealed similar pharmacokinetic features, suggesting therefore an analogous evolutionarily acquired mechanism to ensure the biodistribution of their corresponding proteins. Our findings indicate that the insertion of defined motifs into a proline-rich framework constitutes a suitable alternative to construct a chimeric protein with extended half-life in blood.

Involvement of conserved asparagine and arginine residues from the N-terminal region in the catalytic mechanism of rat liver and Trypanosoma cruzi tyrosine aminotransferases.

Rat liver and Trypanosoma cruzi tyrosine aminotransferases (TATs) share over 40% sequence identity, but differ in their substrate specificities. To explore the molecular features related to these differences, comparative mutagenesis studies were conducted on full length T. cruzi TAT and N-terminally truncated rat TAT recombinant enzymes. The functionality of Arg315 and Arg417 in rat TAT was investigated for comparison with the conserved Arg292 and Arg386 in aspartate and bacterial aromatic aminotransferases (ASATs and ARATs). The rat TAT Arg315Lys variant remained fully active indicating that, as in T. cruzi TAT and contrary to subfamily Ialpha aminotransferases, this residue is not critical for activity. In contrast, the Arg417Gln variant was inactive. The catalytic relevance of the putative rat TAT active site residues Asn54 and Arg57, which are strictly conserved in TATs (Asn17 and Arg20 in T. cruzi TAT) but differ in ASATs and ARATs, was also explored. The substitutions Arg57Ala and Arg57Gln abolished enzymatic activity of these mutants. In both variants, spectral studies demonstrated that aromatic but not dicarboxylic substrates could efficiently bind in the active site. Thus, Arg57 appears to be functionally equivalent to Arg292 of ASATs and ARATs. Asn54 also appears to be involved in the catalytic mechanism of rat TAT since its exchange for Ser lowered the k(cat)/K(m) ratios towards its substrates. Mutation of the analogous residues in T. cruzi TAT also lowered the catalytic efficiencies (k(cat)/K(m)) of the variants substantially. The results imply that the mamalian TAT is more closely related to the T. cruzi TAT than to ASATs and ARATs.

How does an enzyme evolved in vitro compare to naturally occurring homologs possessing the targeted function? Tyrosine aminotransferase from aspartate aminotransferase.

Aspartate aminotransferase (AATase) and tyrosine aminotransferase (TATase) are Escherichia coli paralogs that share 43% sequence identity. A plausible model posits that TATase arose from a duplication of an ancestral AATase-like enzyme. Directed evolution of AATase to an enzyme having TATase activity was undertaken in order to compare the evolved AATase variants with homologous TATases. Eight rounds of DNA shuffling and in vivo selection followed by a backcross with WT AATase produced enzymes that exhibited 100-270-fold increases in k(cat)/K(m)(Phe) and had as much as 11% of the tyrosine aminotransferase activity of WT E.coli TATase. Amino acid substitutions in 11 clones from rounds 7 and 8 were compared with conserved residues in AATases and TATases. The findings are conveniently and compactly illustrated by the use of Venn diagrams and set theory notation. A statistically significant (0.001or=75% identical) in AATases and variable (<75% identical) in TATases. Very few mutations occur in the intersection (set AAT intersection TAT) of amino acid residues that are conserved in both enzyme types. Seven mutations from set AAT-TAT were combined by site-directed mutagenesis to give a construct that is 60% as active as the best round 8 enzyme, which has 13 amino acid replacements. The Venn diagrams may provide a generally useful tool to highlight the most important specificity determinants for rational redesign. Amino acid replacements were mapped onto the crystal structure of a hydrocinnamate complex of a designed TATase. Five of the seven positions most frequently substituted in the evolved clones are within 15 A of the phenyl side-chain, but only six of the 48 positions that were mutated once or twice are within that radius. Context dependence, neutral mutations, different selective pressures, and stochastic components provide explanations for the observation that many of the substitutions found in the directly evolved enzymes differ from the corresponding amino acids found in the modern natural TATases.

Quantitative chimeric analysis of six specificity determinants that differentiate Escherichia coli aspartate from tyrosine aminotransferase.

The six mutations, referred to as the Hex mutations, that together have been shown to convert Escherichia coli aspartate aminotransferase (AATase) specificity to be substantially like that of E. coli tyrosine aminotransferase (TATase) are dissected into two groups, (T109S/N297S) and (V39L/K41Y/T47I/N69L). The letters on the left and right of the numbers designate AATase and TATase residues, respectively. The T109S/N297S pair has been investigated previously. The latter group, the "Grease" set, is now placed in the AATase framework, and the retroGrease set (L39V/Y41K/I47T/L69N) is substituted into TATase. The Grease mutations in the AATase framework were found primarily to lower K(M)s for both aromatic and dicarboxylic substrates. In contrast, retroGrease TATase exhibits lowered k(cat)s for both substrates. The six retroHex mutations, combining retroGrease and S109T/S297N, were found to invert the substrate specificity of TATase, creating an enzyme with a nearly ninefold preference (k(cat)/K(M)) for aspartate over phenylalanine. The retroHex mutations perturb the electrostatic environment of the pyridoxal phosphate cofactor, as evidenced by a spectrophotometric titration of the internal aldimine, which uniquely shows two pK(a)s, 6.1 and 9.1. RetroHex was also found to have impaired dimer stability, with a K(D) for dimer dissociation of 350 nM compared with the wild type K(D) of 4 nM. Context dependence and additivity analyses demonstrate the importance of interactions of the Grease residues with the surrounding protein framework in both the AATase and TATase contexts, and with residues 109 and 297 in particular. Context dependence and cooperativity are particularly evident in the effects of mutations on k(cat)/K(M)(Asp). Effects on k(cat)/K(M)(Phe) are more nearly additive and context independent.

A general method for the quantitative analysis of functional chimeras: applications from site-directed mutagenesis and macromolecular association.

Two new parameters, I: and C:, are introduced for the quantitative evaluation of functional chimeras: I: (impact) and C: (context dependence) are the free energy difference and sum, respectively, of the effects on a given property measured in forward and retro chimeras. The forward chimera is made by substitution of a part "a" from ensemble A into the analogous position of homologous ensemble B (S:(B --> A)). The C: value is a measure of the interaction of the interrogated position with its surroundings, whereas I: is an expression of the quantitative importance of the probed position. Both I: and C: vary with the evaluated property, for example, kinetics, binding, thermostability, and so forth. The retro chimera is the reverse substitution of the analogous part "b" from B into A, S:(A --> B). The I: and C: values derived from original data for forward and retro mutations in aspartate and tyrosine aminotransferase, from literature data for quasi domain exchange in oncomodulin and for the interaction of Tat with bovine and human TAR are evaluated. The most salient derived conclusions are, first, that Thr 109 (AATase) or Ser 109 (TATase) is an important discriminator for dicarboxylic acid selectivity by these two enzymes (I: < -2.9 kcal/mol). The T109S mutation in AATase produces a nearly equal and opposite effect to S109T in TATase (C: < 0.4 kcal/mol). Second, an I: value of 5.5 kcal/mol describes the effects of mirror mutations D94S (site 1) and S55D (site 2) in the Ca(2+) binding sites of oncomodulin on Ca(2+) affinity. The second mirror set, G98D (site 1) and D59G (site 2), yields a smaller impact (I: = -3.4 kcal/mol) on Ca(2+) binding; however, the effect is significantly more nearly context independent (C: = -0.6 versus C: = -2.7 kcal/mol). Third, the stem and loop regions of HIV and BIV TAR are predominantly responsible for the species specific interaction with BIV Tat(65-81) (I: = -1.5 to -1.6 kcal/mol), whereas I: = 0.1 kcal/mol for bulge TAR chimeras. The C: values are from -0.3 to -1.2 kcal/mol. The analysis described should have important applications to protein design.