Glutamine Amidotransferase Subunit Research Articles

Structure and inhibition of subunit I of the anthranilate synthase complex of Mycobacterium tuberculosis and expression of the active complex.

The tryptophan-biosynthesis pathway is essential for Mycobacterium tuberculosis (Mtb) to cause disease, but not all of the enzymes that catalyse this pathway in this organism have been identified. The structure and function of the enzyme complex that catalyses the first committed step in the pathway, the anthranilate synthase (AS) complex, have been analysed. It is shown that the open reading frames Rv1609 (trpE) and Rv0013 (trpG) encode the chorismate-utilizing (AS-I) and glutamine amidotransferase (AS-II) subunits of the AS complex, respectively. Biochemical assays show that when these subunits are co-expressed a bifunctional AS complex is obtained. Crystallization trials on Mtb-AS unexpectedly gave crystals containing only AS-I, presumably owing to its selective crystallization from solutions containing a mixture of the AS complex and free AS-I. The three-dimensional structure reveals that Mtb-AS-I dimerizes via an interface that has not previously been seen in AS complexes. As is the case in other bacteria, it is demonstrated that Mtb-AS shows cooperative allosteric inhibition by tryptophan, which can be rationalized based on interactions at this interface. Comparative inhibition studies on Mtb-AS-I and related enzymes highlight the potential for single inhibitory compounds to target multiple chorismate-utilizing enzymes for TB drug discovery.

Differences in the Roles of a Glutamine Amidotransferase Subunit of Pyridoxal 5'-Phosphate Synthase betweenBacillus circulansandBacillus subtilis

BtrC2 of the butirosin producer Bacillus circulans is a non-catalytic subunit of 2-deoxy-scyllo-inosose (DOI) synthase that is involved in butirosin biosynthesis, and also a homolog of glutamine amidotransferase subunit (PdxT) of pyridoxal 5'-phosphate (PLP) synthase of Bacillus subtilis. BtrC2 has been found to have functions in B. circulans both in primary and secondary metabolism. In this study, we investigated the properties of PdxT of B. subtilis in order to determine whether the property of enzyme stabilization is universal among PdxT homologs. Complementation with PdxT in the btrC2 disruptant of B. circulans restored the growth and short-term production of antibiotics, but long-term production of antibiotics cannot be restored. Additionally, PdxT did not bind physically with or stabilize BtrC. Our results indicate that the function of BtrC2 in secondary metabolism is specific properties, not universal among PdxT homologs.

Solution Nuclear Magnetic Resonance Structure of the GATase Subunit and Structural Basis of the Interaction between GATase and ATPPase Subunits in a two-subunit-type GMPS from Methanocaldococcus jannaschii

The solution structure of the monomeric glutamine amidotransferase (GATase) subunit of the Methanocaldococcus janaschii (Mj) guanosine monophosphate synthetase (GMPS) has been determined using high-resolution nuclear magnetic resonance methods. Gel filtration chromatography and ¹⁵N backbone relaxation studies have shown that the Mj GATase subunit is present in solution as a 21 kDa (188-residue) monomer. The ensemble of 20 lowest-energy structures showed root-mean-square deviations of 0.35 ± 0.06 Å for backbone atoms and 0.8 ± 0.06 Å for all heavy atoms. Furthermore, 99.4% of the backbone dihedral angles are present in the allowed region of the Ramachandran map, indicating the stereochemical quality of the structure. The core of the tertiary structure of the GATase is composed of a seven-stranded mixed β-sheet that is fenced by five α-helices. The Mj GATase is similar in structure to the Pyrococcus horikoshi (Ph) GATase subunit. Nuclear magnetic resonance (NMR) chemical shift perturbations and changes in line width were monitored to identify residues on GATase that were responsible for interaction with magnesium and the ATPPase subunit, respectively. These interaction studies showed that a common surface exists for the metal ion binding as well as for the protein-protein interaction. The dissociation constant for the GATase-Mg(2+) interaction has been found to be ∼1 mM, which implies that interaction is very weak and falls in the fast chemical exchange regime. The GATase-ATPPase interaction, on the other hand, falls in the intermediate chemical exchange regime on the NMR time scale. The implication of this interaction in terms of the regulation of the GATase activity of holo GMPS is discussed.

Structure of Aminodeoxychorismate Synthase from Stenotrophomonas maltophilia

PabB, aminodeoxychorismate synthase, is the chorismic acid binding component of the heterodimeric PabA-PabB complex that converts chorismic acid to 4-amino-4-deoxychorismate, a precursor of p-aminobenzoate and folic acid in microorganisms. The second component, a glutamine amidotransferase subunit, PabA, generates ammonia that is channeled to the PabB active site where it attacks C4 of a chorismate-derived intermediate that is covalently bound, through C2, to an active site lysine residue. The presence of a PIKGT motif was, until recently, believed to allow discrimination of PabB enzymes from the closely related enzyme anthranilate synthase, which typically contains a PIAGT active site motif and does not form a covalent enzyme-substrate intermediate with chorismate. A subclass of PabB enzymes that employ an alternative mechanism requiring 2 equiv of ammonia from glutamine and that feature a noncovalently bound 2-amino-2-deoxyisochorismate intermediate was recently identified. Here we report the 2.25 Å crystal structure of PabB from the emerging pathogen Stenotrophomonas maltophilia. It is the first reported structure of a PabB that features the PIAGT motif. Surprisingly, no dedicated pabA is evident in the genome of S. maltophilia, suggesting that another cellular amidotransferase is able to fulfill the role of PabA in this organism. Evaluation of the ammonia-dependent aminodeoxychorismate synthase activity of S. maltophilia PabB alone revealed that it is virtually inactive. However, in the presence of a heterologous PabA surrogate, typical levels of activity were observed using either glutamine or ammonia as the nitrogen source. Additionally, the structure suggests that a key segment of the polypeptide can remodel itself to interact with a nonspecialized or shared amidotransferase partner in vivo. The structure and mass spectral analysis further suggest that S. maltophilia PabB, like Escherichia coli PabB, binds tryptophan in a vestigial regulatory site. The observation that the binding site is unoccupied in the crystal structure, however, suggests the affinity may be low relative to that of E. coli PabB.

1H, 13C, 15N assignment and secondary structure determination of glutamine amido transferase subunit of gaunosine monophosphate synthetase from Methanocaldococcus jannaschii

Sequence specific resonance assignments have been obtained for (1)H, (13)C and (15)N nuclei of the 21 kDa (188 residues long) glutamine amido transferase subunit of guanosine monophosphate synthetase from Methanocaldococcus jannaschii. From an analysis of (1)H and (13)C(α), (13)C(β) secondary chemical shifts, (3) JH(N)H(α) scalar coupling constants and sequential, short and medium range (1)H-(1)H NOEs, it was deduced that the glutamine amido transferase subunit has eleven strands and five helices as the major secondary structural elements in its tertiary structure.

Crystal Structure of the ATPPase Subunit and Its Substrate-Dependent Association with the GATase Subunit: A Novel Regulatory Mechanism for a Two-Subunit-Type GMP Synthetase from Pyrococcus horikoshii OT3

Guanosine 5′-monophosphate synthetase(s) (GMPS) catalyzes the final step of the de novo synthetic pathway of purine nucleotides. GMPS consists of two functional units that are present as domains or subunits: glutamine amidotransferase (GATase) and ATP pyrophosphatase (ATPPase). GATase hydrolyzes glutamine to yield glutamate and ammonia, while ATPPase utilizes ammonia to convert adenyl xanthosine 5′-monophosphate (adenyl-XMP) into guanosine 5′-monophosphate. Here we report the crystal structure of PH-ATPPase (the ATPPase subunit of the two-subunit-type GMPS from the hyperthermophilic archaeon Pyrococcus horikoshii OT3). PH-ATPPase consists of two domains (N-domain and C-domain) and exists as a homodimer in the crystal and in solution. The N-domain contains an ATP-binding platform called P-loop, whereas the C-domain contains the xanthosine 5'-monophosphate (XMP)-binding site and also contributes to homodimerization. We have also demonstrated that PH-GATase (the glutamine amidotransferase subunit of the two-subunit-type GMPS from the hyperthermophilic archaeon P . horikoshii OT3) alone is inactive, and that all substrates of PH-ATPPase except for ammonia (Mg 2+, ATP and XMP) are required to stabilize the active complex of PH-ATPPase and PH-GATase subunits.

Translation Control of trpG from Transcripts Originating from the Folate Operon Promoter of Bacillus subtilis Is Influenced by Translation-Mediated Displacement of Bound TRAP, While Translation Control of Transcripts Originating from a Newly Identified trpG Promoter Is Not

Bacillus subtilis trpG encodes a glutamine amidotransferase subunit that participates in the biosynthesis of both tryptophan and folic acid. TRAP inhibits translation of trpG in response to tryptophan by binding to a site that overlaps the trpG Shine-Dalgarno sequence, thereby blocking ribosome binding. Similar mechanisms regulate trpP and ycbK translation. The equilibrium binding constants of tryptophan-activated TRAP for the trpG, ycbK, and trpP transcripts were determined to be 8, 3, and 50 nM, respectively. Despite TRAP having a higher affinity for the trpG transcript, TRAP exhibited the least control of trpG expression. The trpG Shine-Dalgarno sequence overlaps the stop codon of the upstream pabB gene, while six of nine triplet repeats within the TRAP binding site are located upstream of the pabB stop codon. Thus, ribosomes translating the upstream pabB cistron could be capable of reducing TRAP-dependent control of TrpG synthesis by displacing bound TRAP. Expression studies using pabB-trpG'-'lacZ fusions in the presence or absence of an engineered stop codon within pabB suggest that translation-mediated displacement of bound TRAP reduces TRAP-dependent inhibition of TrpG synthesis from transcripts originating from the folate operon promoter (P(pabB)). A new trpG promoter (P(trpG)) was identified in the pabB coding sequence that makes a larger contribution to trpG expression than does P(pabB). We found that TRAP-dependent regulation of trpG expression is more extensive for a transcript originating from P(trpG) and that transcripts originating from P(trpG) are not subject to translation-mediated displacement of bound TRAP.

The pab1 gene of Coprinus cinereus encodes a bifunctional protein for para-aminobenzoic acid (PABA) synthesis: implications for the evolution of fused PABA synthases.

The pab1 gene of the basidiomycete Coprinus cinereus encodes PABA synthase, necessary for para-aminobenzoic acid production. The C. cinereus protein is bifunctional with an N-terminal glutamine amidotransferase domain and a C-terminal chorismate amination domain. In most bacteria, these two functions are encoded in separate genes (e.g., pabA and pabB of E. coli). Fused PABA synthases have so far been detected in actinomycetes, Plasmodium falciparum, fungi and Arabidopsis thaliana. Phylogenetic analysis shows that the fused PAB sequences form a tight group that also includes uncharacterized PabB homologues from several bacteria. Unfused bacterial PabA proteins group with the glutamine amidotransferase subunits of bacterial anthranilate synthases, independent of organismal systematics, indicating a complex and perhaps independent evolutionary origin. In contrast, unfused PabB group and fused PabA/B proteins form a monophyletic group on a branch separate from the chorismate amination subunits of anthranilate synthases, probably reflecting a need for recognition of different positions in the common substrate chorismate.

On the Structure of hisH: Protein Structure Prediction in the Context of Structural and Functional Genomics

We predict a structure of the glutamine amidotransferase subunit (hisH) of imidazole glycerol phosphate synthase (IGPS) which catalyzes the fifth step of the histidine biosynthesis in Escherichia coli. The model is constructed using an energy-based threading program augmented by a multiple sequence to structure profile analysis. In developing our model we identified a conserved core region within hisH and a variable domain which is the likely site of interaction with the synthase subunit (hisF) of IGPS. Information available from structural and functional genomics studies was used to improve the structure prediction, to discuss parallels between histidine biosynthesis and other amino acid and nucleotide metabolic pathways, and to better understand the protein–protein interactions between the hisH and hisF domains of IGPS. This work allows us to develop a preliminary model for the structure of the entire IGPS holoenzyme.

The crystal structure of anthranilate synthase from Sulfolobus solfataricus: functional implications.

Anthranilate synthase catalyzes the synthesis of anthranilate from chorismate and glutamine and is feedback-inhibited by tryptophan. The enzyme of the hyperthermophile Sulfolobus solfataricus has been crystallized in the absence of physiological ligands, and its three-dimensional structure has been determined at 2.5-A resolution with x-ray crystallography. It is a heterotetramer of anthranilate synthase (TrpE) and glutamine amidotransferase (TrpG) subunits, in which two TrpG:TrpE protomers associate mainly via the TrpG subunits. The small TrpG subunit (195 residues) has the known "triad" glutamine amidotransferase fold. The large TrpE subunit (421 residues) has a novel fold. It displays a cleft between two domains, the tips of which contact the TrpG subunit across its active site. Clusters of catalytically essential residues are located inside the cleft, spatially separated from clustered residues involved in feedback inhibition. The structure suggests a model in which chorismate binding triggers a relative movement of the two domain tips of the TrpE subunit, activating the TrpG subunit and creating a channel for passage of ammonia toward the active site of the TrpE subunit. Tryptophan presumably blocks this rearrangement, thus stabilizing the inactive states of both subunits. The structure of the TrpE subunit is a likely prototype for the related enzymes 4-amino 4-deoxychorismate synthase and isochorismate synthase.

Regulation of an anthranilate synthase gene in Streptomyces venezuelae by a trp attenuator.

The nucleotide sequence of a 2-4 kb BamHI-SalI fragment of Streptomyces venezuelae ISP5230 DNA that complements trpE and trpG mutations in Escherichia coli contains two ORFs. The larger of these (ORF2) encodes a 624 amino acid sequence similar to the overall sequence of the two subunits of anthranilate synthase. The two-thirds nearest the amino terminus resembles the aminase subunit; the remaining one-third resembles the glutamine amidotransferase subunit. Upstream of ORF2 is a small ORF encoding 18 amino acids that include three adjacent Trp residues; in addition the ORF contains inverted repeats with sequence and positional similarity to the products of attenuator (trpL) regions that regulate tryptophan biosynthesis in other bacteria. In cultures of a trpC mutant of S. venezuelae, increasing the concentration of exogenous tryptophan decreased the formation of anthranilate synthase; similar evidence of endproduct repression was obtained in a trpCER mutant of E. coli transformed with a vector containing the cloned DNA fragment from S. venezuelae. The anthranilate synthase activity in S. venezuelae cell extracts was inhibited by tryptophan, although only at high concentrations of the amino acid. A two-base deletion introduced into the cloned S. venezuelae DNA fragment prevented complementation of a trpE mutation in E. coli. However, S. venezuelae transformants in which the two-base deletion had been introduced by replacement of homologous chromosomal DNA did not exhibit a Trp- phenotype. The result implies that S. venezuelae has one or more additional genes for anthranilate synthase. In alignments with anthranilate synthase genes from other organisms, ORF2 from S. venezuelae most closely resembled genes for phenazine biosynthesis in Pseudomonas. The results bear on the function of the gene in S. venezuelae.

The trp RNA-binding attenuation protein regulates TrpG synthesis by binding to the trpG ribosome binding site of Bacillus subtilis.

The trpG gene of Bacillus subtilis encodes a glutamine amidotransferase subunit which is involved in the biosynthesis of L-tryptophan and folic acid. The trp RNA-binding attenuation protein (TRAP) is involved in controlling expression of trpG at the level of translation in response to changes in the intracellular concentration of tryptophan. We performed in vitro experiments using purified TRAP to elucidate the mechanism of TRAP-dependent trpG regulation. A TRAP-trpG RNA footprint analysis showed that tryptophan-activated TRAP interacts with one UAG, one AAG, and seven GAG repeats present in the trpG transcript. Results from ribosome and TRAP toeprint experiments indicated that the ribosome and TRAP binding sites overlap. Experiments with a B. subtilis cell-free translation system demonstrated that TRAP inhibits TrpG synthesis. Thus, TRAP regulates translation of trpG by blocking ribosome access to the trpG ribosome binding site. Our results are consistent with a model in which each tryptophan-activated TRAP subunit interacts with one trinucleotide repeat in an RNA target, thereby wrapping the transcript around the periphery of the TRAP complex.

Translation of trpG in Bacillus subtilis is regulated by the trp RNA-binding attenuation protein (TRAP)

trpG of Bacillus subtilis encodes a glutamine amidotransferase subunit that is involved in the synthesis of both folic acid and L-tryptophan. Expression of trpG is negatively regulated by tryptophan even though this gene is located within a folic acid biosynthetic operon. Examination of both transcriptional and translational gene fusions to lacZ involving trpG and direct measurements of trpG mRNA levels and TrpG polypeptide accumulation demonstrated that translation of trpG is regulated by tryptophan whereas transcription is not. these studies also show that this regulation is mediated by the trp RNA-binding attenuation protein. Deletion and point mutations indicated that regulation is dependent on a series of G/UAG trinucleotide repeats surrounding the putative ribosome-binding site for trpG. Our results are consistent with a model in which the tryptophan-activated trp RNA-binding attenuation protein and ribosomes compete for binding to trpG mRNA.

The predicted secondary structure of the G-type glutamine amidotransferase is compatible with TIM-barrel topology.

Glutamine amidotransferase (GAT) subunits or domains catalyze an important partial reaction in many complex biosynthetic reactions. The structure of one member of the F-type GATs is known, but the structure of the unrelated G-type is still unknown. Because many protein sequences are available for anthranilate synthase component II (product of the trpG gene), we have predicted its average secondary structure by a joint prediction method [Niermann and Kirschner (1991a) Protein Engng, 4, 359-370]. The predicted eight beta-strands and seven alpha-helices follow an 8-fold cyclic repetition of a beta-strand-loop-alpha-helix-loop module with helix alpha 7 missing. This pattern of secondary structure suggests that the G-type GAT domain has an 8-fold beta alpha-barrel topology, as found first in triose phosphate isomerase (TIM-barrel). This model is supported by the location of known catalytically essential residues in loops between beta-strands and alpha-helices. Evidence from published sequencing and mutational studies on selected members of the GAT superfamily (carbamoyl phosphate, imidazoleglycerol phosphate, GMP and CTP synthases) support both the secondary structure prediction and the TIM-barrel topology.

Expression of Escherichia coli pabA

Escherichia coli pabA encodes the glutamine amidotransferase subunit of p-aminobenzoate synthase. p-Aminobenzoate synthase catalyzes the conversion of chorismate and glutamine to 4-amino-4-deoxychorismate, which is then converted to p-aminobenzoate by a 4-amino-4-deoxychorismate lyase. The 5'-terminal segment of pabA was previously shown to be transcribed from two different promoters, one near the pabA coding sequence (P1) and one preceding fic (P2). However, a pabA-lacZ translational fusion was expressed only from the mRNA originating at P1. We have determined that expression of a pabA-lacZ chromosomal fusion is not changed by p-aminobenzoate limitation, growth rate, catabolite repression, overexpression of either p-aminobenzoate synthase subunit, or gene dosage of pabA and pabB. The lack of pabA expression from P2 appears to be the result of a stable secondary structure in the intergenic space preceding pabA that sequesters the pabA ribosome binding site. Disruption of the secondary structure by mutation allowed expression of pabA from P2, as did translation of ribosomes into the fic-pabA intergenic region.

Chromosomal organization and expression of Escherichia coli pabA

The pabA gene in Escherichia coli and Salmonella typhimurium encodes the glutamine amidotransferase subunit of para-aminobenzoate synthase, which catalyzes the first reaction in the conversion of chorismate to para-aminobenzoate (PABA). We have determined the nucleotide sequences of 1,362 base pairs preceding E. coli pabA and of 981 base pairs preceding S. typhimurium pabA. The nucleotide sequences suggest the presence of two protein-coding regions immediately upstream of pabA, designated orf1 and fic. Transcription analysis indicates that E. coli pabA is encoded by two overlapping transcriptional units. The polycistronic transcriptional unit includes orf1-fic-pabA and is initiated by the promoter designated P2. The monocistronic unit includes only pabA and is initiated by the promoter designated P1, which is located in the fic-coding region. Both promoters transcribe pabA to about the same steady-state level. However, expression analysis using chromosomal pabA-lacZ translational fusions indicated that P1 expressed PabA at least 50-fold more efficiently than P2. pabA-dependent growth rate analysis indicates that P1 is essential and P2 is dispensable for PABA metabolism. In the absence of P1, growth was reduced as a result of insufficient PabA expressed from P2. The significance of these results and possible posttranscriptional control mechanisms which affect PabA expression from the P2-initiated polycistronic unit are discussed.

Evolution of glutamine amidotransferase genes: Nucleotide sequences of the pabA genes from Salmonella typhimurium, Klebsiella aerogenes and Serratia marcescens

The amide group of glutamine is a source of nitrogen in the biosynthesis of a variety of compounds. These reactions are catalyzed by a group of enzymes known as glutamine amidotransferases; two of these, the glutamine amidotransferase subunits of p-aminobenzoate synthase and anthranilate synthase have been studied in detail and have been shown to be structurally and functionally related. In some micro-organisms, p-aminobenzoate synthase and anthranilate synthase share a common glutamine amidotransferase subunit. We report here the primary DNA and deduced amino acid sequences of the p-aminobenzoate synthase glutamine amidotransferase subunits from Salmonella typhimurium, Klebsiella aerogenes and Serratia marcescens. A comparison of these glutamine amidotransferase sequences to the sequences of ten others, including some that function specifically in either the p-aminobenzoate synthase or anthranilate synthase complexes and some that are shared by both synthase complexes, has revealed several interesting features of the structure and organization of these genes, and has allowed us to speculate as to the evolutionary history of this family of enzymes. We propose a model for the evolution of the p-aminobenzoate synthase and anthranilate synthase glutamine amidotransferase subunits in which the duplication and subsequent divergence of the genetic information encoding a shared glutamine amidotransferase subunit led to the evolution of two new pathwayspecific enzymes.

Regulation of a glutamine amidotransferase subunit from Bacillus subtilis by a cloned trpE gene from Bacillus pumilus.

The influence of a cloned trpE (trpE+p) gene from Bacillus pumilus on the expression of the gat locus in Bacillus subtilis was examined. The trpE gene was regulated by tryptophan and the mtr locus, which specifies the presumed aporepressor. The specific activity of subunit G varied directly with the level of subunit Ep, and the heterologous EpG complex that was formed was stable to gel filtration.