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PubMed1998

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.

Anthranilate Synthase↗
PubMed2003

Complexity of coordinative bonding in thallium(I) anthranilates and salicylates.

An inventory of the structural chemistry of thallium(I) shows many unexpected, almost random coordination numbers and coordination geometries that appear erratic and inconsistent. This nonstandard behavior is often ascribed to the specific lone-pair characteristics originating from relativistic effects. To provide data on a set of closely related compounds from which simple general rules of coordinative bonding at Tl(+) can be established, three thallium(I) anthranilates and three thallium(I) salicylates have been prepared from Tl(2)CO(3) and the corresponding 2-amino- and 2-hydroxy-benzoic acids and crystallized from aqueous solutions. All six compounds, the simple anthranilate (1) and salicylate (4) and the 3- and 4-methyl-substituted homologues (2, 3 and 5, 6) show different structures with large variations in the coordination motif. The coordination by oxygen in a geometry which covers less than a coordination hemisphere is the only common feature, complemented (only in 1) by a nitrogen coordination and by eta(6)-coordination of one (in 1, 2, 3, 6) or two phenyl rings (in 4). Tl-Tl contacts for which "thallophilic" bonding between closed shell metal atoms could be invoked, are generally very long (close to 4.0 A) or even well beyond the limit of standard van der Waals contacts. Hydrogen bonding is only obvious for the internal contacts of the amino- or hydroxy-benzoate ligands and does not contribute significantly to the assembly of the supramolecular structure which is dominated by oxygen bridges between thallium atoms. With the exception of 5, the formula units Tl[O(2)C(2-R)(3-R')(4-R'')C(6)H(2)] are generally aggregated into dimers of various configurations depending on the relative orientation of the edge-sharing four-membered rings, and these dimers are further linked into strings or columns establishing N-Tl or Tl-O contacts and arene coordination. The drastic changes induced in the structures upon only small variations such as methyl substitution in 3- or 4-position of the ligand suggest that thallium(I) coordination is generally restricted to one hemisphere of nearest neighbors, but is extremely flexible in this realm. The open hemisphere may be partially capped by arene coordination (which is weak at a distance of ca. 3.1 A to the centroid of the ring) or feature very weak thallophilic contacts.

Journal Article↗
PubMed1987

Enhancement of Epstein-Barr virus membrane protein (LMP) expression by serum, TPA, or n-butyrate in latently infected Raji cells.

The BamHI Nhet region of the EBV DNA is known to code for two proteins. One is a membrane protein (LMP) with an apparent molecular weight of 60,000 on SDS-PAGE which is expressed in latently EBV infected cells. The second protein, so far unidentified, is presumably a late protein with a calculated molecular weight of 28,000 Da. Antisera against both proteins were generated by immunizing rabbits with either a fusion protein containing 155 amino acids of the C-terminus of LMP and a 37,000 mol wt piece of the bacterial anthranilate synthase or with a C-terminal synthetic peptide of 7 amino acids. These sera reacted with a protein varying in size between 60,000 and 65,000 mol wt on SDS-PAGE, found in all cell lines harboring EBV. In addition, these sera identified a second protein with an apparent molecular weight of 49,000 on SDS-PAGE in B95-8, P3HR-1, and M-ABA cells, which is presumably identical with the 28,000-Da protein mentioned above. Furthermore, with these sera a positive cytoplasmic immunofluorescence in 1 to 10% of the cells was obtained, depending on the cell line examined. Analyzing the nonproducer Raji cell line, the number of immunofluorescence-positive cells and the amount of the 60,000 protein, as judged by immunoblotting, was rapidly increased by addition of fresh medium with 10% fetal calf serum as well as by the tumor promoter TPA or to an even higher extend by n-butyrate. The kinetics of induction reached a maximum 24 hr after addition of medium plus 10% fresh serum or TPA or n-butyrate and decreased after 24 to 48 hr. Since the induction of the EBV early antigen (EA) associated proteins by TPA or n-butyrate exhibits a diverse kinetic with a maximum at 72 hr, the regulation of the 60,000 protein synthesis appears to be different from known EA-associated proteins.

Antigens, Viral↗
PubMed2005

Design and synthesis of aromatic inhibitors of anthranilate synthase.

Anthranilate synthase catalyses the conversion of chorismate to anthranilate, a key step in tryptophan biosynthesis. A series of 3-(1-carboxy-ethoxy) benzoic acids were synthesised as chorismate analogues, with varying functionality at C-4, the position of the departing hydroxyl group in chorismate. Most of the compounds were moderate inhibitors of anthranilate synthase, with inhibition constants between 20-30 microM. The exception was 3-(1-carboxy-ethoxy) benzoic acid, (C-4 = H), for which K(I)= 2.4 microM. These results suggest that a hydrogen bonding interaction with the active site general acid (Glu309) is less important than previously assumed for inhibition of the enzyme by these aromatic chorismate analogues.

Anthranilate Synthase↗
PubMed1993

New mechanism-based inactivators of trypsin-like proteinases. Selective inactivation of urokinase by functionalized cyclopeptides incorporating a sulfoniomethyl-substituted m-aminobenzoic acid residue.

In order to obtain selective suicide substrates of trypsin-like proteases including plasminogen activators, plasmin, and thrombin, a series of cyclopeptides cyclo[Arg or Lys-aB(CH2X)-Gly4], in which a substituted o- or m-aminobenzoyl group constitutes a latent electrophile, have been prepared. Treatment of the corresponding phenyl ethers cyclo[P1-aB(CH2OC6H5)-Gly4] with HBr/HOAc or R1R2S/TFA gives the bromides (X = Br) or the sulfonium salts (X = +SR1R2 with R1 = R2 = Me or R1 = Me and R2 = C6H5), respectively. These water-soluble cyclopeptides behave as time-dependent inhibitors of bovine trypsin and human urokinase (u-PA) but have no effect on tissue plasminogen activator (t-PA) and no or poor effect on plasmin and thrombin. The compounds containing a m-aminobenzoic acid residue are more efficient inactivators than their anthranilic analogues. The kinetic criteria expected for a suicide inhibition are met. A mechanism of inhibition involving the formation of a quinonimmonium methide intermediate is proposed. The activity of the inhibitors is very sensitive to the nature of the X benzylic substituent. An increased efficiency for the inactivation of human urokinase is observed with the sulfonium salts. The selectivity of the inactivation of u-PA compared to t-PA could be of therapeutical significance in controlling cell proliferation and invasion.

Aminobenzoates↗
PubMed1984

Nucleotide sequence of Escherichia coli pabB indicates a common evolutionary origin of p-aminobenzoate synthetase and anthranilate synthetase.

Biochemical and immunological experiments have suggested that the Escherichia coli enzyme p-aminobenzoate synthetase and anthranilate synthetase are structurally related. Both enzymes are composed of two nonidentical subunits. Anthranilate synthetase is composed of proteins encoded by the genes trp(G)D and trpE, whereas p-aminobenzoate synthetase is composed of proteins encoded by pabA and pabB. These two enzymes catalyze similar reactions and produce similar products. The nucleotide sequences of pabA and trp(G)D have been determined and indicate a common evolutionary origin of these two genes. Here we present the nucleotide sequence of pabB and compare it with that of trpE. Similarities are 26% at the amino acid level and 40% at the nucleotide level. We propose that pabB and trpE arose from a common ancestor and hence that there is a common ancestry of genes encoding p-aminobenzoate synthetase and anthranilate synthetase.

Amino Acid Sequence↗
PubMed1999

Plasmid-encoded anthranilate synthase (TrpEG) in Buchnera aphidicola from aphids of the family pemphigidae.

Buchnera aphidicola is an obligate intracellular symbiont of aphids. One of its proposed functions is the synthesis of essential amino acids, nutrients required by aphids but deficient in their diet of plant phloem sap. The genetic organization of the tryptophan pathway in Buchnera from proliferous aphids of the family Aphididae has previously been shown to reflect a capacity to overproduce this essential amino acid (C.-Y. Lai, L. Baumann, and P. Baumann, Proc. Natl. Acad. Sci. USA 91:3819-3823, 1994). This involved amplification of the genes for the first enzyme in the pathway, anthranilate synthase (TrpEG), on a low-copy-number plasmid. Here we report on the finding and molecular characterization of TrpEG-encoding plasmids in Buchnera from aphids of the distantly related family Pemphigidae. Buchnera from Tetraneura caerulescens contained a 3.0-kb plasmid (pBTc2) that carried a single copy of trpEG and resembled trpEG plasmids of Buchnera from the Aphididae. The second plasmid (pBPs2), isolated from Buchnera of Pemphigus spyrothecae, contained a different replicon. It consisted of a putative origin of replication containing iterons and an open reading frame, designated repAC, which showed a high similarity to the gene encoding the replication initiation protein RepA of the RepA/C replicon from the broad-host-range IncA/C group of plasmids. The plasmid population was heterogeneous with respect to the number of tandem repeats of a 1.8-kb unit carrying repAC1, trpG, and remnants of trpE. The two principal forms consisted of either five or six copies of this repeat and a single-copy region carrying repAC2, the putative origin of replication, and trpE. The unexpected finding of elements of the RepA/C replicon in previously characterized trpEG plasmids from Buchnera of the Aphididae suggests that a replacement of replicons has occurred during the evolution of these plasmids, which may point to a common ancestry for all Buchnera trpEG amplifications.

Amino Acid Sequence↗
PubMed2001

Anthranilate synthase without an LLES motif from a hyperthermophilic archaeon is inhibited by tryptophan.

Tk-trpE and Tk-trpG, the genes that encode the two subunits of anthranilate synthase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1, have been expressed independently in Escherichia coli. The anthranilate synthase complex (Tk-AS complex) was obtained by heat-treatment of the mixture of cell-free extracts containing each recombinant protein, Tk-TrpE (alpha subunit) and Tk-TrpG (beta subunit), at 85 degrees C for 10 min. Further purification of Tk-AS complex was carried out by anion-exchange chromatography followed by gel-filtration. Molecular mass estimations from gel-filtration chromatography indicated that Tk-AS complex was a heterodimer (alphabeta). The complex displayed both ammonia- and glutamine-dependent anthranilate synthase activities, and could not utilize asparagine as an ammonia donor. The optimal pH was pH 10.0 and the optimal temperature was 85 degrees C in both cases. Mg2+ was necessary for the anthranilate synthase activity. At 75 degrees C, the K(m) values of chorismate for ammonia- and glutamine-dependent activities were 13.8 and 3.4 microM, respectively. The K(m) value of Mg2+ was 20.5 microM. The K(m) values of glutamine and NH4Cl were 88 microM and 5.6 mM, respectively. Although Tk-TrpE displayed 47.6% similarity with TrpE of Salmonella typhimurium, conserved amino acid residues proven to be essential for inhibition of enzyme activity by L-tryptophan were not present in Tk-TrpE. Namely, residues corresponding to Glu39, Met293, and Cys465 in the enzyme from S. typhimurium were replaced by Arg28, Thr221, and Ala384 in Tk-TrpE. Nevertheless, significant inhibition by L-tryptophan was observed, with K(i) values of 5.25 and 74 microM for ammonia and glutamine-dependent activities, respectively. The inhibition was competitive with respect to chorismate. The results suggest that the amino acid residues involved in the feedback inhibition by L-tryptophan in the case of Tk-AS complex are distinct from previously reported anthranilate synthases.

Amino Acid Motifs↗
PubMed1985

Replacement by site-directed mutagenesis indicates a role for histidine 170 in the glutamine amide transfer function of anthranilate synthase.

Anthranilate synthase is a glutamine amidotransferase that catalyzes the first reaction in tryptophan biosynthesis. Conserved amino acid residues likely to be essential for glutamine-dependent activity were identified by alignment of the glutamine amide transfer domains in four different enzymes: anthranilate synthase component II (AS II), p-aminobenzoate synthase component II, GMP synthetase, and carbamoyl-P synthetase. Conserved amino acids were mainly localized in three clusters. A single conserved histidine, AS II His-170, was replaced by tyrosine using site-directed mutagenesis. Glutamine-dependent enzyme activity was undetectable in the Tyr-170 mutant, whereas the NH3-dependent activity was unchanged. Affinity labeling of AS II active site Cys-84 by 6-diazo-5-oxonorleucine was used to distinguish whether His-170 has a role in formation or in breakdown of the covalent glutaminyl-Cys-84 intermediate. The data favor the interpretation that His-170 functions as a general base to promote glutaminylation of Cys-84. Reversion analysis was consistent with a proposed role of His-170 in catalysis as opposed to a structural function. These experiments demonstrate the application of combining sequence analyses to identify conserved, possibly functional amino acids, site-directed mutagenesis to replace candidate amino acids, and protein chemistry for analysis of mutationally altered proteins, a regimen that can provide new insights into enzyme function.

Anthranilate Synthase↗
PubMed1978

Glucuronic acid conjugates of bilirubin-IXalpha in normal bile compared with post-obstructive bile. Transformation of the 1-O-acylglucuronide into 2-, 3-, and 4-O-acylglucuronides.

Structures have been determined for bilirubin-IXalpha conjugates in freshly collected bile of normal rats, dogs and man and in post-obstructive bile of man and rats. The originally secreted conjugate has been characterized as azopigment (I), i.e. a 1-O-acyl-beta-d-glucopyranuronic acid glycoside. Conversion of the acetylated methyl ester of azopigment (I) into methyl 2,3,4-tri-O-acetyl-1-bromo-1-deoxy-beta-d-glucopyranuronate (V) indicates the pyranose ring structure for the carbohydrate and a C-1 attachment for the bilirubin-IXalpha acyl group. Alternative procedures for deconjugation of azopigment (I) and its derivatives are also described. In post-obstructive bile, the 1-O-acylglucuronide is converted into 2-, 3- and 4-O-acylglucuronides via sequential intramolecular migrations of the bilirubin acyl group. The following approach was utilized. (1) The tetrapyrrole conjugates were cleaved to dipyrrolic aniline and ethyl anthranilate azopigments, and the azopigments were separated as the acids or methyl esters. (2) The isomeric methyl esters were characterized by mass spectral analysis of the acetates and silyl ethers. (3) The free glycosidic function was demonstrated by 1-oxime and 1-methoxime derivative formation. (4) The position of the dipyrrolic O-acyl group was determined for the methyl esters by protecting the free hydroxyl groups of the glucuronic acid moieties as the acetals formed with ethyl vinyl ether and by further conversion of the carbohydrates into partially methylated alditol acetates. These were analysed by using g.l.c.-mass spectrometry. The relevance of the present results with regard to previous reports on disaccharidic conjugates is discussed. Details of procedures for the formation of chemical derivatives for g.l.c. and mass spectrometry have been deposited as Supplementary Publication SUP 50081 (15 pages) at the British Library Lending Division, Boston Spa, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978), 169, 5.

Animals↗
PubMed1999

Purification and cDNA cloning of isochorismate synthase from elicited cell cultures of Catharanthus roseus.

Isochorismate is an important metabolite formed at the end of the shikimate pathway, which is involved in the synthesis of both primary and secondary metabolites. It is synthesized from chorismate in a reaction catalyzed by the enzyme isochorismate synthase (ICS; EC 5.4.99.6). We have purified ICS to homogeneity from elicited Catharanthus roseus cell cultures. Two isoforms with an apparent molecular mass of 64 kD were purified and characterized. The Km values for chorismate were 558 and 319 microM for isoforms I and II, respectively. The isoforms were not inhibited by aromatic amino acids and required Mg2+ for enzyme activity. Polymerase chain reaction on a cDNA library from elicited C. roseus cells with a degenerated primer based on the sequence of an internal peptide from isoform II resulted in an amplification product that was used to screen the cDNA library. This led to the first isolation, to our knowledge, of a plant ICS cDNA. The cDNA encodes a protein of 64 kD with an N-terminal chloroplast-targeting signal. The deduced amino acid sequence shares homology with bacterial ICS and also with anthranilate synthases from plants. Southern analysis indicates the existence of only one ICS gene in C. roseus.

Amino Acid Sequence↗
PubMed1978

Rearranged glucuronic acid conjugates of bilirubin-IX alpha in post-obstructive bile. Structure elucidation of azopigments beta and gamma as ethyl anthranilate N-glycosides derived from 2-, 3- and 4-o-acyl glucuronides.

Azopigment analysis was performed on conjugates of bilirubin-IXalpha in bile of man and rats obtained after obstruction of the bile duct or in bile incubated under N2. The azopigments beta and gamma, formed by applying a pH 2.7 diazonium reagent containing an excess of ethyl anthranilate, correspond to rearranged ethyl athranilate N-glucuronides having the azodipyrrole acyl group on positions 2, 3 and 4 of the sugar. These assignments were verified, first by conversion of the structurally known 2-, 3- and 4-O-acyl glucuronide azopigments, unsubstituted at C-1, into ethyl anthranilate N-glucuronide reference compounds, and second, by mass spectrometry of trimethylsilyl ether methyl ester derivatives of unknown and reference compounds. The C-1 ethyl anthranilate group of the N-glucuronides triggers characteristics fragmentation reactions of the carbohydrate moiety revealing the position of the azodipyrrole O-acyl group.

Animals↗
PubMed1990

19F NMR studies on 8-fluoroflavins and 8-fluoro flavoproteins.

The 19F NMR spectra of the oxidized and reduced forms of 8-fluororiboflavin, 8-fluoro-FAD, and the 8-fluoroflavin-reconstituted flavoproteins flavodoxin, riboflavin binding protein, D-amino acid oxidase, p-hydroxybenzoate hydroxylase, Old Yellow Enzyme, anthranilate hydroxylase, general acyl-CoA dehydrogenase, glucose oxidase, and L-lactate oxidase were measured. For the proteins studied the oxidized resonances appeared over a 10.1-ppm range, while the reduced resonances were spread over 10.3 ppm. Reduction caused an upfield shift of about 27 ppm for the free 8-fluoroflavins and most of the 8-fluoro flavoproteins. The notable exception was 8-fluoro-FMN flavodoxin, which was shifted 37.6 ppm, indicating an unusually high electron density in the benzene ring. Ligand binding to the oxidized 8-fluoro flavoproteins caused either upfield or downfield shifts of 1.5-5 ppm, depending on the protein/ligand combination. The 8-fluoro-FAD anthranilate hydroxylase resonance was shifted downfield and split into two peaks in the presence of anthranilate. The 8-fluoro-FMN Old Yellow Enzyme resonance was shifted upfield upon complexation with charge-transfer-forming, para-substituted phenolates. The upfield shift increased from less than 1 to 5 ppm as the electron-donating capacity of the phenolate increased. Complexation of native Old Yellow Enzyme with 2,4-difluorophenol caused the fluorine resonances of the ligand to shift and split into two pairs of signals. Each pair of signals was associated with a different isozyme of Old Yellow Enzyme.

Acyl-CoA Dehydrogenases↗
PubMed1989

Identification and nucleotide sequence of the Leptospira biflexa serovar patoc trpE and trpG genes.

Leptospira biflexa is a representative of an evolutionarily distinct group of eubacteria. In order to better understand the genetic organization and gene regulatory mechanisms of this species, we have chosen to study the genes required for tryptophan biosynthesis in this bacterium. The nucleotide sequence of the region of the L. biflexa serovar patoc chromosome encoding the trpE and trpG genes has been determined. Four open reading frames (ORFs) were identified in this region, but only three ORFs were translated into proteins when the cloned genes were introduced into Escherichia coli. Analysis of the predicted amino acid sequences of the proteins encoded by the ORFs allowed us to identify the trpE and trpG genes of L. biflexa. Enzyme assays confirmed the identity of these two ORFs. Anthranilate synthase from L. biflexa was found to be subject to feedback inhibition by tryptophan. Codon usage analysis showed that there was a bias in L. biflexa towards the use of codons rich in A and T, as would be expected from its G + C content of 37%. Comparison of the amino acid sequences of the trpE gene product and the trpG gene product with corresponding gene products from other bacteria showed regions of highly conserved sequence.

Amino Acid Sequence↗
PubMed2002

The catalytic mechanism of indole-3-glycerol phosphate synthase: crystal structures of complexes of the enzyme from Sulfolobus solfataricus with substrate analogue, substrate, and product.

Indoleglycerol phosphate synthase catalyzes the ring closure of an N-alkylated anthranilate to a 3-alkyl indole derivative, a reaction requiring Lewis acid catalysis in vitro. Here, we investigated the enzymatic reaction mechanism through X-ray crystallography of complexes of the hyperthermostable enzyme from Sulfolobus solfataricus with the substrate 1-(o-carboxyphenylamino) 1-deoxyribulose 5-phosphate, a substrate analogue and the product indole-3-glycerol phosphate. The substrate and the substrate analogue are bound to the active site in a similar, extended conformation between the previously identified phosphate binding site and a hydrophobic pocket for the anthranilate moiety. This binding mode is unproductive, because the carbon atoms that are to be joined are too far apart. The indole ring of the bound product resides in a second hydrophobic pocket adjacent to that of the anthranilate moiety of the substrate. Although the hydrophobic moiety of the substrate moves during catalysis from one hydrophobic pocket to the other, the triosephosphate moiety remains rigidly bound to the same set of hydrogen-bonding residues. Simultaneously, the catalytically important residues Lys53, Lys110 and Glu159 maintain favourable distances to the atoms of the ligand undergoing covalent changes. On the basis of these data, the structures of two putative catalytic intermediates were modelled into the active site. This new structural information and the modelling studies provide further insight into the mechanism of enzyme-catalyzed indole synthesis. The charged epsilon-amino group of Lys110 is the general acid, and the carboxylate group of Glu159 is the general base. Lys53 guides the substrate undergoing conformational transitions during catalysis, by forming a salt-bridge to the carboxylate group of its anthranilate moiety.

Binding Sites↗
PubMed1976

Properties of anthranilate synthetase component II from Pseudomonas putida.

The interaction of Pseudomanas putida anthranilate synthetase Component II (AS II) with glutamine, glutamine analogs, and iodoacetamide has been investigated in order to clarify the initial steps in the mechanism for glutamine utilization. AS II is alkylated and irreversibly inactivated by covalent attachment of approximately 1 eg of L-2-amino-4-oxo-5-chloropentanoic acid (chloroketone) or 1 eq of iodoacetamide. Alkylation of AS II by chloroketone involves initial formation of an enzyme-inhibitor complex having a Ki of 28 muM. Alkylation of AS II by iodoacetamide occurs without initial formation of a reversible complex. In both cases glutamine protects against alkylation and exhibits competitive kinetics. When anthranilate synthetase Component I (AS I) is associated with AS II, the second substrate, chorismate, enhances alkylation of AS II by chloroketone. Alkylation of AS II by iodoacetamide is unaffected by AS I and chorismate. These results suggest a role of chorismate-AS I complex to promote binding of glutamine to AS II or to facilitate conversion of an AS II-glutamine complex to the covalent glutamyl-AS II intermediate. This conclusion is supported by the fact that glutaminase activity of AS II, which requires formation of the glutamyl-AS II intermediate, is stimulated by AS I and chorismate.

Amino Acid Sequence↗
PubMed1985

Identification of a trpG-related glutamine amide transfer domain in Escherichia coli GMP synthetase.

An improved method was developed to align related protein sequences and search for homology. A glutamine amide transfer domain was identified in an NH2-terminal segment of GMP synthetase from Escherichia coli. Amino acid residues 1-198 in GMP synthetase are homologous with the glutamine amide transfer domain in trpG X D-encoded anthranilate synthase component II-anthranilate phosphoribosyltransferase and the related pabA-encoded p-aminobenzoate synthase component II. This result supports a model for gene fusion in which a trpG-related glutamine amide transfer domain was recruited to augment the function of a primitive NH3-dependent GMP synthetase. Sequence analyses emphasize that glutamine amide transfer domains are thus far found only at the NH2 terminus of fused proteins. Two rules are formulated to explain trpG and trpG-related fusions. (i) trpG and trpG-related genes must have translocated immediately up-stream of genes destined for fusion in order to position a glutamine amide transfer domain at the NH2 terminus after fusion. (ii) trpG and trpG-related genes could not translocate adjacent to a regulatory region at the 5' end of an operon. These rules explain known trpG-like fusions and explain why trpG and pabA are not fused to trpE and pabB, respectively. Alignment searches of GMP synthetase with two other enzymes that bind GMP, E. coli amidophosphoribosyltransferase and human hypoxanthine-guanine phosphoribosyltransferase, suggest a structurally homologous segment which may constitute a GMP binding site.

Amidophosphoribosyltransferase↗