Prediction of quaternary structure by analysis of hot spot residues in protein-protein interfaces: the case of anthranilate phosphoribosyltransferases.

It is an important goal of computational biology to correctly predict the association state of a protein based on its amino acid sequence and the structures of known homologues. We have pursued this goal on the example of anthranilate phosphoribosyltransferase (AnPRT), an enzyme that is involved in the biosynthesis of the amino acid tryptophan. Firstly, known crystal structures of naturally occurring homodimeric AnPRTs were analyzed using the Protein Interfaces, Surfaces, and Assemblies (PISA) service of the European Bioinformatics Institute (EBI). This led to the identification of two hydrophobic "hot spot" amino acids in the protein-protein interface that were predicted to be essential for self-association. Next, in a comprehensive multiple sequence alignment (MSA), naturally occurring AnPRT variants with hydrophilic or charged amino acids in place of hydrophobic residues in the two hot spot positions were identified. Representative variants were characterized in terms of thermal stability, enzymatic activity, and quaternary structure. We found that AnPRT variants with charged residues in both hot spot positions exist exclusively as monomers in solution. Variants with hydrophilic amino acids in one hot spot position occur in both forms, monomer and dimer. The results of the present study provide a detailed characterization of the determinants of the AnPRT monomer-dimer equilibrium and show that analysis of hot spots in combination with MSAs can be a valuable tool in prediction of protein quaternary structures.

AGeNNT: annotation of enzyme families by means of refined neighborhood networks.

BACKGROUND: Large enzyme families may contain functionally diverse members that give rise to clusters in a sequence similarity network (SSN). In prokaryotes, the genome neighborhood of a gene-product is indicative of its function and thus, a genome neighborhood network (GNN) deduced for an SSN provides strong clues to the specific function of enzymes constituting the different clusters. The Enzyme Function Initiative ( http://enzymefunction.org/ ) offers services that compute SSNs and GNNs. RESULTS: We have implemented AGeNNT that utilizes these services, albeit with datasets purged with respect to unspecific protein functions and overrepresented species. AGeNNT generates refined GNNs (rGNNs) that consist of cluster-nodes representing the sequences under study and Pfam-nodes representing enzyme functions encoded in the respective neighborhoods. For cluster-nodes, AGeNNT summarizes the phylogenetic relationships of the contributing species and a statistic indicates how unique nodes and GNs are within this rGNN. Pfam-nodes are annotated with additional features like GO terms describing protein function. For edges, the coverage is given, which is the relative number of neighborhoods containing the considered enzyme function (Pfam-node). AGeNNT is available at https://github.com/kandlinf/agennt . CONCLUSIONS: An rGNN is easier to interpret than a conventional GNN, which commonly contains proteins without enzymatic function and overly specific neighborhoods due to phylogenetic bias. The implemented filter routines and the statistic allow the user to identify those neighborhoods that are most indicative of a specific metabolic capacity. Thus, AGeNNT facilitates to distinguish and annotate functionally different members of enzyme families.

Novel stand-alone RAM domain protein-mediated catalytic control of anthranilate phosphoribosyltransferase in tryptophan biosynthesis in Thermus thermophilus.

Regulation of amino acid metabolism (RAM) domains are widely distributed among prokaryotes. In most cases, a RAM domain fuses with a DNA-binding domain to act as a transcriptional regulator. The extremely thermophilic bacterium, Thermus thermophilus, only carries a single gene encoding a RAM domain-containing protein on its genome. This protein is a stand-alone RAM domain protein (SraA) lacking a DNA-binding domain. Therefore, we hypothesized that SraA, which senses amino acids through its RAM domain, may interact with other proteins to modify its functions. In the present study, we identified anthranilate phosphoribosyltransferase (AnPRT), the second enzyme in the tryptophan biosynthetic pathway, as a partner protein that interacted with SraA in T. thermophilus. In the presence of tryptophan, SraA was assembled to a decamer and exhibited the ability to form a stable hetero-complex with AnPRT. An enzyme assay revealed that AnPRT was only inhibited by tryptophan in the presence of SraA. This result suggests a novel feedback control mechanism for tryptophan biosynthesis through an inter-RAM domain interaction in bacteria.

YbiB from Escherichia coli, the Defining Member of the Novel TrpD2 Family of Prokaryotic DNA-binding Proteins.

We present the crystal structure and biochemical characterization of Escherichia coli YbiB, a member of the hitherto uncharacterized TrpD2 protein family. Our results demonstrate that the functional diversity of proteins with a common fold can be far greater than predictable by computational annotation. The TrpD2 proteins show high structural homology to anthranilate phosphoribosyltransferase (TrpD) and nucleoside phosphorylase class II enzymes but bind with high affinity (KD = 10-100 nM) to nucleic acids without detectable sequence specificity. The difference in affinity between single- and double-stranded DNA is minor. Results suggest that multiple YbiB molecules bind to one longer DNA molecule in a cooperative manner. The YbiB protein is a homodimer that, therefore, has two electropositive DNA binding grooves. But due to negative cooperativity within the dimer, only one groove binds DNA in in vitro experiments. A monomerized variant remains able to bind DNA with similar affinity, but the negative cooperative effect is eliminated. The ybiB gene forms an operon with the DNA helicase gene dinG and is under LexA control, being induced by DNA-damaging agents. Thus, speculatively, the TrpD2 proteins may be part of the LexA-controlled SOS response in bacteria.

The substrate capture mechanism of Mycobacterium tuberculosis anthranilate phosphoribosyltransferase provides a mode for inhibition.

Anthranilate phosphoribosyltransferase (AnPRT, EC 2.4.2.18) is a homodimeric enzyme that catalyzes the reaction between 5'-phosphoribosyl 1'-pyrophosphate (PRPP) and anthranilate, as part of the tryptophan biosynthesis pathway. Here we present the results of the first chemical screen for inhibitors against Mycobacterium tuberculosis AnPRT (Mtb-AnPRT), along with crystal structures of Mtb-AnPRT in complex with PRPP and several inhibitors. Previous work revealed that PRPP is bound at the base of a deep cleft in Mtb-AnPRT and predicted two anthranilate binding sites along the tunnel leading to the PRPP binding site. Unexpectedly, the inhibitors presented here almost exclusively bound at the entrance of the tunnel, in the presumed noncatalytic anthranilate binding site, previously hypothesized to have a role in substrate capture. The potencies of the inhibitors were measured, yielding Ki values of 1.5-119 µM, with the strongest inhibition displayed by a bianthranilate compound that makes hydrogen bond and salt bridge contacts with Mtb-AnPRT via its carboxyl groups. Our results reveal how the substrate capture mechanism of AnPRT can be exploited to inhibit the enzyme's activity and provide a scaffold for the design of improved Mtb-AnPRT inhibitors that may ultimately form the basis of new antituberculosis drugs with a novel mode of action.

Experimental assessment of the importance of amino acid positions identified by an entropy-based correlation analysis of multiple-sequence alignments.

The analysis of a multiple-sequence alignment (MSA) with correlation methods identifies pairs of residue positions whose occupation with amino acids changes in a concerted manner. It is plausible to assume that positions that are part of many such correlation pairs are important for protein function or stability. We have used the algorithm H2r to identify positions k in the MSAs of the enzymes anthranilate phosphoribosyl transferase (AnPRT) and indole-3-glycerol phosphate synthase (IGPS) that show a high conn(k) value, i.e., a large number of significant correlations in which k is involved. The importance of the identified residues was experimentally validated by performing mutagenesis studies with sAnPRT and sIGPS from the archaeon Sulfolobus solfataricus. For sAnPRT, five H2r mutant proteins were generated by replacing nonconserved residues with alanine or the prevalent residue of the MSA. As a control, five residues with conn(k) values of zero were chosen randomly and replaced with alanine. The catalytic activities and conformational stabilities of the H2r and control mutant proteins were analyzed by steady-state enzyme kinetics and thermal unfolding studies. Compared to wild-type sAnPRT, the catalytic efficiencies (k(cat)/K(M)) were largely unaltered. In contrast, the apparent thermal unfolding temperature (T(M)(app)) was lowered in most proteins. Remarkably, the strongest observed destabilization (ΔT(M)(app) = 14 °C) was caused by the V284A exchange, which pertains to the position with the highest correlation signal [conn(k) = 11]. For sIGPS, six H2r mutant and four control proteins with alanine exchanges were generated and characterized. The k(cat)/K(M) values of four H2r mutant proteins were reduced between 13- and 120-fold, and their T(M)(app) values were decreased by up to 5 °C. For the sIGPS control proteins, the observed activity and stability decreases were much less severe. Our findings demonstrate that positions with high conn(k) values have an increased probability of being important for enzyme function or stability.

Stabilization of a metabolic enzyme by library selection in Thermus thermophilus.

The anthranilate phosphoribosyl transferase from the hyperthermophilic archaeon Sulfolobus solfataricus (sAnPRT, encoded by strpD), which catalyzes the third step in tryptophan biosynthesis, is a thermostable homodimer with low enzymatic activity at room temperature. We have combined two mutations leading to the monomerization and two mutations leading to the activation of sAnPRT. The resulting "activated monomer" sAnPRT-I36E-M47D+D83G-F149S, which is much more labile than wild-type sAnPRT, was stabilized by a combination of random mutagenesis and metabolic library selection using the extremely thermophilic bacterium Thermus thermophilus as host. This approach led to the identification of five mutations that individually increased the thermal stability of sAnPRT-I36E-M47D+D83G-F149S by 1 to 8 °C, and by 13 °C when combined. The beneficial exchanges were located in different parts of the protein structure, but none of them led to the "re-dimerization" of the enzyme. We observed a negative correlation between thermal stability and catalytic activity of the mutants; this suggests that conformational flexibility is required for catalysis by sAnPRT.

Members of the YjgF/YER057c/UK114 family of proteins inhibit phosphoribosylamine synthesis in vitro.

The YjgF/YER057c/UK114 family of proteins is highly conserved across all three domains of life and currently lacks a consensus biochemical function. Analysis of Salmonella enterica strains lacking yjgF has led to a working model in which YjgF functions to remove potentially toxic secondary products of cellular enzymes. Strains lacking yjgF synthesize the thiamine precursor phosphoribosylamine (PRA) by a TrpD-dependent mechanism that is not present in wild-type strains. Here, PRA synthesis was reconstituted in vitro with anthranilate phosphoribosyltransferase (TrpD), threonine dehydratase (IlvA), threonine, and phosphoribosyl pyrophosphate. TrpD-dependent PRA formation in vitro was inhibited by S. enterica YjgF and the human homolog UK114. Thus, the work herein describes the first biochemical assay for diverse members of the highly conserved YjgF/YER057c/UK114 family of proteins and provides a means to dissect the cellular functions of these proteins.

A rationally designed monomeric variant of anthranilate phosphoribosyltransferase from Sulfolobus solfataricus is as active as the dimeric wild-type enzyme but less thermostable.

The anthranilate phosphoribosyltransferase from Sulfolobus solfataricus (ssAnPRT) forms a homodimer with a hydrophobic subunit interface. To elucidate the role of oligomerisation for catalytic activity and thermal stability of the enzyme, we loosened the dimer by replacing two apolar interface residues with negatively charged residues (mutations I36E and M47D). The purified double mutant I36E+M47D formed a monomer with wild-type catalytic activity but reduced thermal stability. The single mutants I36E and M47D were present in a monomer-dimer equilibrium with dissociation constants of about 1 microM and 20 microM, respectively, which were calculated from the concentration-dependence of their heat inactivation kinetics. The monomeric form of M47D, which is populated at low subunit concentrations, was as thermolabile as monomeric I36E+M47D. Likewise, the dimeric form of I36E, which was populated at high subunit concentrations, was as thermostable as dimeric wild-type ssAnPRT. These findings show that the increased stability of wild-type ssAnPRT compared to the I36E+M47D double mutant is not caused by the amino acid exchanges per se but by the higher intrinsic stability of the dimer compared to the monomer. In accordance with the negligible effect of the mutations on catalytic activity and stability, the X-ray structure of M47D contains only minor local perturbations at the dimer interface. We conclude that the monomeric double mutant resembles the individual wild-type subunits, and that ssAnPRT is a dimer for stability but not for activity reasons.

Mutations in the tryptophan operon allow PurF-independent thiamine synthesis by altering flux in vivo.

Phosphoribosyl amine (PRA) is an intermediate in purine biosynthesis and also required for thiamine biosynthesis in Salmonella enterica. PRA is normally synthesized by phosphoribosyl pyrophosphate amidotransferase, a high-turnover enzyme of the purine biosynthetic pathway encoded by purF. However, PurF-independent PRA synthesis has been observed in strains having different genetic backgrounds and growing under diverse conditions. Genetic analysis has shown that the anthranilate synthase-phosphoribosyltransferase (AS-PRT) enzyme complex, involved in the synthesis of tryptophan, can play a role in the synthesis of PRA. This work describes the in vitro synthesis of PRA in the presence of the purified components of the AS-PRT complex. Results from in vitro assays and in vivo studies indicate that the cellular accumulation of phosphoribosyl anthranilate can result in nonenzymatic PRA formation sufficient for thiamine synthesis. These studies have uncovered a mechanism used by cells to redistribute metabolites to ensure thiamine synthesis and may define a general paradigm of metabolic robustness.

Tryptophan biosynthesis in stramenopiles: eukaryotic winners in the diatom complex chloroplast.

Tryptophan is an essential amino acid that, in eukaryotes, is synthesized either in the plastids of photoautotrophs or in the cytosol of fungi and oomycetes. Here we present an in silico analysis of the tryptophan biosynthetic pathway in stramenopiles, based on analysis of the genomes of the oomycetes Phytophthora sojae and P. ramorum and the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum. Although the complete pathway is putatively located in the complex chloroplast of diatoms, only one of the involved enzymes, indole-3-glycerol phosphate synthase (InGPS), displays a possible cyanobacterial origin. On the other hand, in P. tricornutum this gene is fused with the cyanobacteria-derived hypothetical protein COG4398. Anthranilate synthase is also fused in diatoms. This fusion gene is almost certainly of bacterial origin, although the particular source of the gene cannot be resolved. All other diatom enzymes originate from the nucleus of the primary host (red alga) or secondary host (ancestor of chromalveolates). The entire pathway is of eukaryotic origin and cytosolic localization in oomycetes; however, one of the enzymes, anthranilate phosphoribosyl transferase, was likely transferred to the oomycete nucleus from the red algal nucleus during secondary endosymbiosis. This suggests possible retention of the complex plastid in the ancestor of stramenopiles and later loss of this organelle in oomycetes.

PurF-independent phosphoribosyl amine formation in yjgF mutants of Salmonella enterica utilizes the tryptophan biosynthetic enzyme complex anthranilate synthase-phosphoribosyltransferase.

In Salmonella enterica, the biosynthetic pathways for the generation of purines and the essential cofactor thiamine pyrophosphate branch after sharing five enzymatic steps. Phosphoribosyl amine (PRA) is the first intermediate in the common portion of the pathway and is generated from phosphoribosylpyrophosphate and glutamine by the PurF enzyme (phosphoribosylpyrophosphate amidotransferase). A null mutation in yjgF allows PurF-independent PRA formation by an unknown mechanism. The tryptophan biosynthetic enzyme complex anthranilate synthase-phosphoribosyltransferase, composed of the TrpD and TrpE proteins, was shown to be essential for PRA formation in strains lacking both yjgF and purF. The activity generating PRA in a yjgF mutant background has features that distinguish it from the TrpDE-mediated PRA formation shown previously for this enzyme in strains with an active copy of yjgF. The data presented here are consistent with a model in which the absence of YjgF uncovers a new catalytic activity of TrpDE.

Structural and mutational analysis of substrate complexation by anthranilate phosphoribosyltransferase from Sulfolobus solfataricus.

The metabolic synthesis and degradation of essential nucleotide compounds are primarily carried out by phosphoribosyltransferases (PRT) and nucleoside phosphorylases (NP), respectively. Despite the resemblance of their reactions, five classes of PRTs and NPs exist, where anthranilate PRT (AnPRT) constitutes the only evolutionary link between synthesis and degradation processes. We have characterized the active site of dimeric AnPRT from Sulfolobus solfataricus by elucidating crystal structures of the wild-type enzyme complexed to its two natural substrates anthranilate and 5-phosphoribosyl-1-pyrophosphate/Mg(2+). These bind into two different domains within each protomer and are brought together during catalysis by rotational domain motions as shown by small angle x-ray scattering data. Steady-state kinetics of mutated AnPRT variants address the role of active site residues in binding and catalysis. Results allow the comparative analysis of PRT and pyrimidine NP families and expose related structural motifs involved in nucleotide/nucleoside recognition by these enzyme families.

Structural analysis of two enzymes catalysing reverse metabolic reactions implies common ancestry.

The crystal structure of the dimeric anthranilate phosphoribosyltransferase (AnPRT) reveals a new category of phosphoribosyltransferases, designated as class III. The active site of this enzyme is located within the flexible hinge region of its two-domain structure. The pyrophosphate moiety of phosphoribosylpyrophosphate is co-ordinated by a metal ion and is bound by two conserved loop regions within this hinge region. With the structure of AnPRT available, structural analysis of all enzymatic activities of the tryptophan biosynthesis pathway is complete, thereby connecting the evolution of its enzyme members to the general development of metabolic processes. Its structure reveals it to have the same fold, topology, active site location and type of association as class II nucleoside phosphorylases. At the level of sequences, this relationship is mirrored by 13 structurally invariant residues common to both enzyme families. Taken together, these data imply common ancestry of enzymes catalysing reverse biological processes--the ribosylation and deribosylation of metabolic pathway intermediates. These relationships establish new links for enzymes involved in nucleotide and amino acid metabolism.

Replacement of the yeast TRP4 3' untranslated region by a hammerhead ribozyme results in a stable and efficiently exported mRNA that lacks a poly(A) tail.

The mRNA poly(A) tail serves different purposes, including the facilitation of nuclear export, mRNA stabilization, efficient translation, and, finally, specific degradation. The posttranscriptional addition of a poly(A) tail depends on sequence motifs in the 3' untranslated region (3' UTR) of the mRNA and a complex trans-acting protein machinery. In this study, we have replaced the 3' UTR of the yeast TRP4 gene with sequences encoding a hammerhead ribozyme that efficiently cleaves itself in vivo. Expression of the TRP4-ribozyme allele resulted in the accumulation of a nonpolyadenylated mRNA. Cells expressing the TRP4-ribozyme mRNA showed a reduced growth rate due to a reduction in Trp4p enzyme activity. The reduction in enzyme activity was not caused by inefficient mRNA export from the nucleus or mRNA destabilization. Rather, analyses of mRNA association with polyribosomes indicate that translation of the ribozyme-containing mRNA is impaired. This translational defect allows sufficient synthesis of Trp4p to support growth of trp4 cells, but is, nevertheless, of such magnitude as to activate the general control network of amino acid biosynthesis.

A counterselection for the tryptophan pathway in yeast: 5-fluoroanthranilic acid resistance.

The ability to counterselect, as well as to select for, a genetic marker has numerous applications in microbial genetics. Described here is the use of 5-fluoroanthranilic acid for the counterselection of TRP1, a commonly used genetic marker in the yeast Saccharomyces cerevisiae. Counterselection using 5-fluoroanthranilic acid involves antimetabolism by the enzymes of the tryptophan biosynthetic pathway, such that trp1, trp3, trp4 or trp5 strains, which lack enzymes required for the conversion of anthranilic acid to tryptophan, are resistant to 5-fluoroanthranilic acid. Commonly used genetic procedures, such as selection for loss of a chromosomally integrated plasmid, and a replica-plating method to rapidly assess genetic linkage in self-replicating shuttle vectors, can now be carried out using the TRP1 marker gene. In addition, novel tryptophan auxotrophs can be selected using 5-fluoroanthranilic acid.

Introns act post-transcriptionally to increase expression of the Arabidopsis thaliana tryptophan pathway gene PAT1.

The expression of the Arabidopsis thaliana PAT1 gene, which encodes the tryptophan biosynthetic enzyme phosphoribosylanthranilate transferase, was investigated using translational fusions of the PAT1 promoter to the GUS reporter gene. Independent stably transformed A. thaliana lines containing a single copy of a fusion that includes the entire plastid transit peptide and the first two introns of PAT1 had on average 30 times more GUS enzyme activity than plants transformed with a construct in which GUS was fused a short distance downstream of the PAT1 start codon. Plants containing the construct without introns or leader peptide accumulated undetectable amounts of PAT1-GUS fusion protein and mRNA, even though the transcriptional rate of both fusion constructs was comparable. Fusions containing the entire transit peptide and either of the first two introns yield as much GUS activity as constructs containing both introns, but constructs containing the transit peptide but no introns give rise to much lower levels. Therefore, introns greatly enhance the expression of PAT1-GUS fusions, and they act post-transcriptionally to increase the steady-state level of mRNA.

An allelic series of blue fluorescent trp1 mutants of Arabidopsis thaliana.

Nine blue fluorescent mutants of the flowering plant Arabidopsis thaliana were isolated by genetic selections and fluorescence screens. Each was shown to contain a recessive allele of trp1, a previously described locus that encodes the tryptophan biosynthetic enzyme phosphoribosylanthranilate transferase (PAT, called trpD in bacteria). The trp1 mutants consist of two groups, tryptophan auxotrophs and prototrophs, that differ significantly in growth rate, morphology, and fertility. The trp1 alleles cause plants to accumulate varying amounts of blue fluorescent anthranilate compounds, and only the two least severely affected of the prototrophs have any detectable PAT enzyme activity. All four of the trp1 mutations that were sequenced are G to A or C to T transitions that cause an amino acid change, but in only three of these is the affected residue phylogenetically conserved. There is an unusually high degree of sequence divergence in the single-copy gene encoding PAT from the wild-type Columbia and Landsberg erecta ecotypes of Arabidopsis.

Mutations in the trpD gene of Corynebacterium glutamicum confer 5-methyltryptophan resistance by encoding a feedback-resistant anthranilate phosphoribosyltransferase.

The trpD gene from tryptophan-hyperproducing Corynebacterium glutamicum ATCC 21850 was isolated on the basis of its ability to confer resistance to 5-methyltryptophan on wild-type C. glutamicum AS019. Comparative sequence analysis of the genes from the wild-type AS019 and ATCC 21850 trpD genes revealed two amino acid substitutions at the protein level. Further analysis demonstrated that the trpD gene product from ATCC 21850, anthranilate phosphoribosyltransferase, was more resistant to feedback inhibition by either tryptophan or 5-methyltryptophan than its wild-type counterpart. It is proposed that phosphoribosyltransferase insensitivity to tryptophan in ATCC 21850 contributes to an elevated level of tryptophan biosynthesis.

Regulation of tryptophan biosynthesis in Methanobacterium thermoautotrophicum Marburg.

A tryptophan-auxotrophic mutant of the archaeon Methanobacterium thermoautotrophicum Marburg was grown with growth-promoting and growth-limiting concentrations of tryptophan. The specific activities of anthranilate synthase (TrpEG) and tryptophan synthase (TrpB) increased 30- to 40-fold in tryptophan-starved cells. Levels of trpE-specific and trpD-specific mRNAs (transcripts of the first and the last genes, respectively, of the M. thermoautotrophicum Marburg trp gene cluster) increased about 10-fold upon starvation for tryptophan. Thus, the expression of the trp genes appears to be regulated primarily at the level of transcription. These data support transcription of trp genes as an operon and support a regulatory model involving a repressor. Anthranilate synthase was feedback inhibited by L-tryptophan, with a Ki of 3.0 microM. In a leucine-auxotrophic mutant starved for L-leucine, the level of alpha-isopropylmalate synthase (LeuA) was 10-fold higher than in cells grown with L-leucine. In addition to the finding of specific regulation of gene expression by the end products of their respective pathways, it was found that the levels of anthranilate synthase and alpha-isopropylmalate synthase were reduced upon growth in the presence of amino acids of other families, such as L-alanine, L-proline, or L-arginine. Conversely, starvation for tryptophan caused a slight elevation of alpha-isopropylmalate synthase and starvation for leucine caused a significant increase of anthranilate synthase and tryptophan synthase specific activities. The latter effect was also observed at the level of trp-specific mRNA and is reminiscent of general amino acid control.