The evolution of function within the Nudix homology clan.

The Nudix homology clan encompasses over 80,000 protein domains from all three domains of life, defined by homology to each other. Proteins with a domain from this clan fall into four general functional classes: pyrophosphohydrolases, isopentenyl diphosphate isomerases (IDIs), adenine/guanine mismatch-specific adenine glycosylases (A/G-specific adenine glycosylases), and nonenzymatic activities such as protein/protein interaction and transcriptional regulation. The largest group, pyrophosphohydrolases, encompasses more than 100 distinct hydrolase specificities. To understand the evolution of this vast number of activities, we assembled and analyzed experimental and structural data for 205 Nudix proteins collected from the literature. We corrected erroneous functions or provided more appropriate descriptions for 53 annotations described in the Gene Ontology Annotation database in this family, and propose 275 new experimentally-based annotations. We manually constructed a structure-guided sequence alignment of 78 Nudix proteins. Using the structural alignment as a seed, we then made an alignment of 347 "select" Nudix homology domains, curated from structurally determined, functionally characterized, or phylogenetically important Nudix domains. Based on our review of Nudix pyrophosphohydrolase structures and specificities, we further analyzed a loop region downstream of the Nudix hydrolase motif previously shown to contact the substrate molecule and possess known functional motifs. This loop region provides a potential structural basis for the functional radiation and evolution of substrate specificity within the hydrolase family. Finally, phylogenetic analyses of the 347 select protein domains and of the complete Nudix homology clan revealed general monophyly with regard to function and a few instances of probable homoplasy. Proteins 2017; 85:775-811. © 2016 Wiley Periodicals, Inc.

Substrate specificity characterization for eight putative nudix hydrolases. Evaluation of criteria for substrate identification within the Nudix family.

The nearly 50,000 known Nudix proteins have a diverse array of functions, of which the most extensively studied is the catalyzed hydrolysis of aberrant nucleotide triphosphates. The functions of 171 Nudix proteins have been characterized to some degree, although physiological relevance of the assayed activities has not always been conclusively demonstrated. We investigated substrate specificity for eight structurally characterized Nudix proteins, whose functions were unknown. These proteins were screened for hydrolase activity against a 74-compound library of known Nudix enzyme substrates. We found substrates for four enzymes with kcat /Km values >10,000 M-1  s-1 : Q92EH0_LISIN of Listeria innocua serovar 6a against ADP-ribose, Q5LBB1_BACFN of Bacillus fragilis against 5-Me-CTP, and Q0TTC5_CLOP1 and Q0TS82_CLOP1 of Clostridium perfringens against 8-oxo-dATP and 3'-dGTP, respectively. To ascertain whether these identified substrates were physiologically relevant, we surveyed all reported Nudix hydrolytic activities against NTPs. Twenty-two Nudix enzymes are reported to have activity against canonical NTPs. With a single exception, we find that the reported kcat /Km values exhibited against these canonical substrates are well under 105 M-1  s-1 . By contrast, several Nudix enzymes show much larger kcat /Km values (in the range of 105 to >107 M-1  s-1 ) against noncanonical NTPs. We therefore conclude that hydrolytic activities exhibited by these enzymes against canonical NTPs are not likely their physiological function, but rather the result of unavoidable collateral damage occasioned by the enzymes' inability to distinguish completely between similar substrate structures. Proteins 2016; 84:1810-1822. © 2016 Wiley Periodicals, Inc.

Probing the informational and regulatory plasticity of a transcription factor DNA-binding domain.

Transcription factors have two functional constraints on their evolution: (1) their binding sites must have enough information to be distinguishable from all other sequences in the genome, and (2) they must bind these sites with an affinity that appropriately modulates the rate of transcription. Since both are determined by the biophysical properties of the DNA-binding domain, selection on one will ultimately affect the other. We were interested in understanding how plastic the informational and regulatory properties of a transcription factor are and how transcription factors evolve to balance these constraints. To study this, we developed an in vivo selection system in Escherichia coli to identify variants of the helix-turn-helix transcription factor MarA that bind different sets of binding sites with varying degrees of degeneracy. Unlike previous in vitro methods used to identify novel DNA binders and to probe the plasticity of the binding domain, our selections were done within the context of the initiation complex, selecting for both specific binding within the genome and for a physiologically significant strength of interaction to maintain function of the factor. Using MITOMI, quantitative PCR, and a binding site fitness assay, we characterized the binding, function, and fitness of some of these variants. We observed that a large range of binding preferences, information contents, and activities could be accessed with a few mutations, suggesting that transcriptional regulatory networks are highly adaptable and expandable.

Molecular function prediction for a family exhibiting evolutionary tendencies toward substrate specificity swapping: recurrence of tyrosine aminotransferase activity in the Iα subfamily.

The subfamily Iα aminotransferases are typically categorized as having narrow specificity toward carboxylic amino acids (AATases), or broad specificity that includes aromatic amino acid substrates (TATases). Because of their general role in central metabolism and, more specifically, their association with liver-related diseases in humans, this subfamily is biologically interesting. The substrate specificities for only a few members of this subfamily have been reported, and the reliable prediction of substrate specificity from protein sequence has remained elusive. In this study, a diverse set of aminotransferases was chosen for characterization based on a scoring system that measures the sequence divergence of the active site. The enzymes that were experimentally characterized include both narrow-specificity AATases and broad-specificity TATases, as well as AATases with broader-specificity and TATases with narrower-specificity than the previously known family members. Molecular function and phylogenetic analyses underscored the complexity of this family's evolution as the TATase function does not follow a single evolutionary thread, but rather appears independently multiple times during the evolution of the subfamily. The additional functional characterizations described in this article, alongside a detailed sequence and phylogenetic analysis, provide some novel clues to understanding the evolutionary mechanisms at work in this family.

A continuous fluorescence assay for the characterization of Nudix hydrolases.

The common substrate structure for the functionally diverse Nudix protein superfamily is nucleotide-diphosphate-X, where X is a large variety of leaving groups. The substrate specificity is known for less than 1% of the 29,400 known members. Most activities result in the release of an inorganic phosphate ion or of a product bearing a terminal phosphate moiety. Reactions have typically been monitored by a modification of the discontinuous Fiske-SubbaRow assay, which is relatively insensitive and slow. We report here the development of a continuous fluorescence assay that enables the rapid and accurate determination of substrate specificities in a 96-well format. We used this novel assay to confirm the reported substrate characterizations of MutT and NudD of Escherichia coli and to characterize DR_1025 of Deinococcus radiodurans and MM_0920 of Methanosarcina mazei. Novel findings enabled by the new assay include the following. First, in addition to the well-characterized hydrolysis of 8-oxo-dGTP at the α-ß position, MutT cleaves at the ß-γ phosphate bond at a rate of 3% of that recorded for hydrolysis at the α-ß position. Second, MutT also catalyzes the hydrolysis of 5-methyl-dCTP. Third, 8-oxo-dGTP was observed to be the best substrate for DR_1025 of the 41 compounds screened.

The fitness landscapes of cis-acting binding sites in different promoter and environmental contexts.

The biophysical nature of the interaction between a transcription factor and its target sequences in vitro is sufficiently well understood to allow for the effects of DNA sequence alterations on affinity to be predicted. But even in relatively simple in vivo systems, the complexities of promoter organization and activity have made it difficult to predict how altering specific interactions between a transcription factor and DNA will affect promoter output. To better understand this, we measured the relative fitness of nearly all Escherichia coli sigma(70) -35 binding sites in different promoter and environmental contexts by competing four randomized -35 promoter libraries controlling the expression of the tetracycline resistance gene (tet)against each other in increasing concentrations of drug. We sequenced populations after competition to determine the relative enrichment of each -35 sequence. We observed a consistent relationship between the frequency of recovery of each -35 binding site and its predicted affinity for sigma(70) that varied depending on the sequence context of the promoter and drug concentration. Overall the relative fitness of each promoter could be predicted by a simple thermodynamic model of transcriptional regulation, in which the rate of transcriptional initiation (and hence fitness) is dependent upon the overall stability of the initiation complex, which in turn is dependent upon the energetic contributions of all sites within the complex. As implied by this model, a decrease in the free energy of association at one site could be compensated for by an increase in the binding energy at another to produce a similar output. Furthermore, these data show that a large and continuous range of transcriptional outputs can be accessed by merely changing the -35, suggesting that evolved or engineered mutations at this site could allow for subtle and precise control over gene expression.

Specificity and cooperativity at ß-lactamase position 104 in TEM-1/BLIP and SHV-1/BLIP interactions.

Establishing a quantitative understanding of the determinants of affinity in protein-protein interactions remains challenging. For example, TEM-1/ß-lactamase inhibitor protein (BLIP) and SHV-1/BLIP are homologous ß-lactamase/ß-lactamase inhibitor protein complexes with disparate K(d) values (3 nM and 2 µM, respectively), and a single substitution, D104E in SHV-1, results in a 1000-fold enhancement in binding affinity. In TEM-1, E104 participates in a salt bridge with BLIP K74, whereas the corresponding SHV-1 D104 does not in the wild type SHV-1/BLIP co-structure. Here, we present a 1.6 Å crystal structure of the SHV-1 D104E/BLIP complex that demonstrates that this point mutation restores this salt bridge. Additionally, mutation of a neighboring residue, BLIP E73M, results in salt bridge formation between SHV-1 D104 and BLIP K74 and a 400-fold increase in binding affinity. To understand how this salt bridge contributes to complex affinity, the cooperativity between the E/K or D/K salt bridge pair and a neighboring hot spot residue (BLIP F142) was investigated using double mutant cycle analyses in the background of the E73M mutation. We find that BLIP F142 cooperatively stabilizes both interactions, illustrating how a single mutation at a hot spot position can drive large perturbations in interface stability and specificity through a cooperative interaction network.

Active site prediction using evolutionary and structural information.

MOTIVATION: The identification of catalytic residues is a key step in understanding the function of enzymes. While a variety of computational methods have been developed for this task, accuracies have remained fairly low. The best existing method exploits information from sequence and structure to achieve a precision (the fraction of predicted catalytic residues that are catalytic) of 18.5% at a corresponding recall (the fraction of catalytic residues identified) of 57% on a standard benchmark. Here we present a new method, Discern, which provides a significant improvement over the state-of-the-art through the use of statistical techniques to derive a model with a small set of features that are jointly predictive of enzyme active sites. RESULTS: In cross-validation experiments on two benchmark datasets from the Catalytic Site Atlas and CATRES resources containing a total of 437 manually curated enzymes spanning 487 SCOP families, Discern increases catalytic site recall between 12% and 20% over methods that combine information from both sequence and structure, and by >or=50% over methods that make use of sequence conservation signal only. Controlled experiments show that Discern's improvement in catalytic residue prediction is derived from the combination of three ingredients: the use of the INTREPID phylogenomic method to extract conservation information; the use of 3D structure data, including features computed for residues that are proximal in the structure; and a statistical regularization procedure to prevent overfitting.

The partially folded homodimeric intermediate of Escherichia coli aspartate aminotransferase contains a "molten interface" structure.

The role of intersubunit side chain-side chain interactions in the stability of the Escherichia coli aspartate aminotransferase (eAATase) homodimer was investigated by directed mutagenesis at 10 different interface contacts. The urea-mediated unfolding pathway of this enzyme proceeds through the formation of a dimeric intermediate, D*, that retains only 40% of the native enzyme secondary structure as judged by circular dichroism. Disruption of any single intersubunit interaction results in a >2.6 kcal mol(-1) decrease in native state stability, independent of its location or nature. However, the stability of D* with respect to U, the unfolded monomer, is the same for all mutants. The stability of the eAATase interface cannot be ascribed to the contribution of a few hot spots, or to the accumulation of a large number of weak interactions, but only to the presence of multiple important and interconnected interactions. It is proposed that a "molten interface" structure, flexible enough to accommodate point mutations, accounts for the stability of D*. Nuclei of tertiary structure, which are not involved in native intersubunit contacts, likely provide a scaffold for the unstructured interface of D*. Such a scaffold would account for the cooperative unfolding of the intermediate.

Free energies of protein-protein association determined by electrospray ionization mass spectrometry correlate accurately with values obtained by solution methods.

The advantages of electrospray ionization mass spectrometry (ESIMS) to measure relative solution-phase affinities of tightly bound protein-protein complexes are demonstrated with selected variants of the Bacillus amyloliquefaciens protein barstar (b*) and the RNAase barnase (bn), which form protein-protein complexes with a range of picomolar to nanomolar dissociation constants. A novel chemical annealing procedure rapidly establishes equilibrium in solutions containing competing b* variants with limiting bn. The relative ion abundances of the complexes and those of the competing unbound monomers are shown to reflect the relative solution-phase concentrations of those respective species. No measurable dissociation of the complexes occurs either during ESI or mass detection, nor is there any evidence for nonspecific binding at protein concentrations < 25 microM. Differences in DeltaDeltaG of dissociation between variants were determined with precisions < 0.1 kcal/mol. The DeltaDeltaG values obtained deviate on average by 0.26 kcal/mol from those measured with a solution-phase enzyme assay. It is demonstrated that information about the protein conformation and covalent modifications can be obtained from differences in mass and charge state distributions. This method serves as a rapid and precise means to interrogate protein-protein-binding surfaces for complexes that have affinities in the picomolar to nanomolar range.

The narrow substrate specificity of human tyrosine aminotransferase--the enzyme deficient in tyrosinemia type II.

Human tyrosine aminotransferase (hTATase) is the pyridoxal phosphate-dependent enzyme that catalyzes the reversible transamination of tyrosine to p-hydrophenylpyruvate, an important step in tyrosine metabolism. hTATase deficiency is implicated in the rare metabolic disorder, tyrosinemia type II. This enzyme is a member of the poorly characterized Igamma subfamily of the family I aminotransferases. The full length and truncated forms of recombinant hTATase were expressed in Escherichia coli, and purified to homogeneity. The pH-dependent titration of wild-type reveals a spectrum characteristic of family I aminotransferases with an aldimine pK(a) of 7.22. I249A mutant hTATase exhibits an unusual spectrum with a similar aldimine pK(a) (6.85). hTATase has very narrow substrate specificity with the highest enzymatic activity for the Tyr/alpha-ketoglutarate substrate pair, which gives a steady state k(cat) value of 83 s(-1). In contrast there is no detectable transamination of aspartate or other cosubstrates. The present findings show that hTATase is the only known aminotransferase that discriminates significantly between Tyr and Phe: the k(cat)/K(m) value for Tyr is about four orders of magnitude greater than that for Phe. A comparison of substrate specificities of representative Ialpha and Igamma aminotransferases is described along with the physiological significance of the discrimination between Tyr and Phe by hTATase as applied to the understanding of the molecular basis of phenylketonuria.

Structure of 1-aminocyclopropane-1-carboxylate synthase in complex with an amino-oxy analogue of the substrate: implications for substrate binding.

The crystal structure of 1-aminocyclopropane-1-carboxylate (ACC) synthase in complex with the substrate analogue [2-(amino-oxy)ethyl](5'-deoxyadenosin-5'-yl)(methyl)sulfonium (AMA) was determined at 2.01-A resolution. The crystallographic results show that a covalent adduct (oxime) is formed between AMA (an amino-oxy analogue of the natural substrate S-adenosyl-L-methionine (SAM)) and the pyridoxal 5'-phosphate (PLP) cofactor of ACC synthase. The oxime formation is supported by spectroscopic data. The ACC synthase-AMA structure provides reliable and detailed information on the binding mode of the natural substrate of ACC synthase and complements previous structural and functional work on this enzyme.

Structure of ACC synthase inactivated by the mechanism-based inhibitor L-vinylglycine.

L-Vinylglycine (L-VG) is both a substrate for and a mechanism-based inhibitor of 1-aminocyclopropane-1-carboxylate (ACC) synthase. The ratio of the rate constants for catalytic conversion to alpha-ketobutyrate and ammonia to inactivation is 500/1. The crystal structure of the covalent adduct of the inactivated enzyme was determined at 2.25 Angstroms resolution. The active site contains an external aldimine of the adduct of L-VG with the pyridoxal 5'-phosphate cofactor. The side chain gamma-carbon of L-VG is covalently bound to the epsilon-amino group of Lys273. This species corresponds to one of the two alternatives proposed by Feng and Kirsch [Feng, L. and Kirsch, J.F. (2000) L-Vinylglycine is an alternative substrate as well as a mechanism-based inhibitor of 1-aminocyclopropane-1-carboxylate synthase. Biochemistry 39, 2436-2444] and presumably results from Michael addition to a vinylglycine ketimine intermediate.

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.

Directed evolution relieves product inhibition and confers in vivo function to a rationally designed tyrosine aminotransferase.

The Escherichia coli aspartate (AATase) and tyrosine (TATase) aminotransferases share 43% sequence identity and 72% similarity, but AATase has only 0.08% and 0.01% of the TATase activities (k(cat)/K(m)) for tyrosine and phenylalanine, respectively. Approximately 5% of TATase activity was introduced into the AATase framework earlier both by rational design (six mutations, termed HEX) and by directed evolution (9-17 mutations). The enzymes realized from the latter procedure complement tyrosine auxotrophy in TATase deficient E. coli. HEX complements even more poorly than does wild-type AATase, even though the (k(cat)/K(m)) value for tyrosine exhibited by HEX is similar to those of the enzymes found from directed evolution. HEX, however, is characterized by very low values of K(m) and K(D) for dicarboxylic ligands, and by a particularly slow release for oxaloacetate, the product of the reaction with aspartate and a TCA cycle intermediate. These observations suggest that HEX exists largely as an enzyme-product complex in vivo. HEX was therefore subjected to a single round of directed evolution with selection for complementation of tyrosine auxotrophy. A variant with a single amino acid substitution, A293D, exhibited substantially improved TATase function in vivo. The A293D mutation alleviates the tight binding to dicarboxylic ligands as K(m)s for aspartate and alpha-ketoglutarate are >20-fold higher in the HEX + A293D construct compared to HEX. This mutation also increased k(cat)/K(m)(Tyr) threefold. A second mutation, I73V, elicited smaller but similar effects. Both residues are in close proximity to Arg292 and the mutations may function to modulate the arginine switch mechanism responsible for dual substrate recognition in TATases and HEX.

The role of the conserved Lys68*:Glu265 intersubunit salt bridge in aspartate aminotransferase kinetics: multiple forced covariant amino acid substitutions in natural variants.

The role of the Lys68*:Glu265 intersubunit salt bridge that is conserved (Csb) in all known aspartate aminotransferases (AATases), except those of animal cytosolic, Ac (His68*:Glu265), and plant mitochondrial, Pm (Met68*:Gln265), origins, was evaluated in the Escherichia coli AATase. Two double-mutant cycles, to K68M/E265Q and the charge reversed K68E/E265K, were characterized with the context dependence (C) and impact (I) formalism, previously defined for functional chimeric analysis. Mutations of Lys68* with Glu265 fixed are generally more deleterious than the converse mutations of Glu265 with Lys68* fixed, showing that buried negative charges have greater effects than buried positive charges in this context. Replacement of the charged Lys68*:Glu265 with the K68M/E265Q neutral pair introduces relatively small effects on the kinetic parameters. The differential sensitivity of k(cat)/K(M, L-Asp) and k(cat)/K(M, alpha-KG) to salt bridge mutagenic replacements is shown by a linear-free energy relationship, in which the logarithms of the latter second order rate constants are generally decreased by a factor of two more than are those of the former. Thus, k(cat)/K(M, L-Asp) and k(cat)/K(M, alpha-KG) are 133 and 442 mM(-1)s(-1) for the wild-type (WT) enzyme, respectively, but their relative order is reversed in the more severely compromised mutants (14.8 and 5.3 mM(-1)s(-1) for K68E). A Venn diagram illustrates apparent forced covariances of groups of amino acids that accompany the naturally occurring salt bridge replacements in the Pm and Ac classes. The more deeply rooted tree indicates that the Csb variant was the ancestral specie.

How do two unrelated antibodies, HyHEL-10 and F9.13.7, recognize the same epitope of hen egg-white lysozyme?

The anti-hen egg-white lysozyme (HEWL) antibodies HyHEL-10 and F9.13.7 recognize a common epitope. The structures of the complexes differ, however, in the numbers of electrostatic and hydrogen-bond interactions and in the distributions of contacts between the light and heavy chains. The equilibria and kinetics characterizing the F9.13.7 complex formation were evaluated for both wild-type and mutant derivatives of HEWL to help to understand how the different contacts are effectively used in the complexes with the two antibodies. Three epitope hot spots, Y20, K96, and R73 (destabilization > 4 kcal/mole), were found by alanine scanning mutagenesis. The first two constitute two of the three hot spots in the HyHEL-10 complex. The hot spots of the HyHEL-10 paratope are centered on the HEWL epitope; whereas R73 (HEWL), the only important light-chain-contacting residue, is clearly separated from the other hot spots of the F9.13.7 complex. The larger number of epitope warm plus hot spots found in the F9.13.7 complex compared with that of HyHEL-10 shows that the specificity of the former is greater even though the K(D) value is 20-fold larger. Conservative mutations showed that the specificity enhancement is related to the greater number of functional polar and hydrogen bond interactions in the F9.13.7 complex. Alanine scanning mutagenesis would not have illuminated these distinctions. It is shown that the concept of antigen specificity, as defined by cross-reactivity with natural variant antigens, is flawed by phylogenetic bias, and that specificity can only be defined by the use of unbiased epitopes, which are conveniently accessed by site-directed mutagenesis.

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.

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.

Autoinhibition of human dicer by its internal helicase domain.

Dicer, a member of the ribonuclease III family of enzymes, processes double-stranded RNA substrates into approximately 21- to 27-nt products that trigger sequence-directed gene silencing by RNA interference. Although the mechanism of RNA recognition and length-specific cleavage by Dicer has been established, the way in which dicing activity is regulated is unclear. Here, we show that the N-terminal domain of human Dicer, which is homologous to DExD/H-box helicases, substantially attenuates the rate of substrate cleavage. Deletion or mutation of this domain activates human Dicer in both single- and multiple-turnover assays. The catalytic efficiency (k(cat)/K(m)) of the deletion construct is increased by 65-fold over that exhibited by the intact enzyme. Kinetic analysis shows that this activation is almost entirely due to an enhancement in k(cat). Modest stimulation of catalysis by the full-length Dicer enzyme was observed in the presence of the TAR-RNA binding protein, which physically interacts with the DExD/H-box domain. These results suggest that the DExD/H-box domain likely disrupts the functionality of the Dicer active site until a structural rearrangement occurs, perhaps upon assembly with its molecular partners.