Cloning of the Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase gene in Escherichia coli. Nucleotide sequence determination and properties of the plasmid-encoded enzyme

The Bacillus subtilis gene encoding glutamine phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase) was cloned in pBR322. This gene is designated purF by analogy with the corresponding gene in Escherichia coli. B. subtilis purF was expressed in E. coli from a plasmid promoter. The plasmid-encoded enzyme was functional in vivo and complemented an E. coli purF mutant strain. The nucleotide sequence of a 1651-base pair B. subtilis DNA fragment was determined, thus localizing the 1428-base pair structural gene. A primary translation product of 476 amino acid residues was deduced from the DNA sequence. Comparison with the previously determined NH2-terminal amino acid sequence indicates that 11 residues are proteolytically removed from the NH2 terminus, leaving a protein chain of 465 residues having an NH2-terminal active site cysteine residue. Plasmid-encoded B. subtilis amidophosphoribosyltransferase was purified from E. coli cells and compared to the enzymes from B. subtilis and E. coli. The plasmid-encoded enzyme was similar in properties to amidophosphoribosyltransferase obtained from B. subtilis. Enzyme specific activity, immunological reactivity, in vitro lability to O2, Fe-S content, and NH2-terminal processing were virtually identical with amidophosphoribosyltransferase purified from B. subtilis. Thus E. coli correctly processed the NH2 terminus and assembled [4Fe-4S] centers in B. subtilis amidophosphoribosyltransferase although it does not perform these maturation steps on its own enzyme. Amino acid sequence comparison indicates that the B. subtilis and E. coli enzymes are homologous. Catalytic and regulatory domains were tentatively identified based on comparison with E. coli amidophosphoribosyltransferase and other phosphoribosyltransferase (Argos, P., Hanei, M., Wilson, J., and Kelley, W. (1983) J. Biol. Chem. 258, 6450-6457).     

Mutagenesis of ligands to the [4 Fe-4S] center of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase

Several mutations were constructed in residues thought to provide ligands for a [4Fe-4S] cluster in Bacillus subtilis amidophosphoribosyltransferase using site-directed mutagenesis of cloned purF. These replacements confirm the identification of cysteinyl ligands to the Fe-S center. Of five mutant enzymes, two had no activity, two less than 25% of the wild type activity, and one was lethal and could not be studied. The Fe content of the two mutant enzymes with partial activity was similar to that of the wild type. Results of partial characterization suggest that the [4Fe-4S] cluster is not involved in allosteric regulation and does not play a specific role in the ammonia- or glutamine-dependent reactions of the enzyme. At least partial enzymatic activity is required for NH2-terminal processing. Pulse labeling experiments suggest that processing is a slow post-translational process which is dependent upon cellular factors. A relationship between Fe-S centers and NH2-terminal processing of an undecapeptide leader suggests a functional connection between these two structural elements in amidophosphoribosyltransferase.     

Avian glutamine phosphoribosylpyrophosphate amidotransferase propeptide processing and activity are dependent upon essential cysteine residues.

Avian glutamine phosphoribosylpyrophosphate amidotransferase contains an NH2-terminal propetide-like sequence. NH2-terminal sequence analysis of immunoaffinity purified enzyme from chicken liver indicates that the propeptide is processed and the mature enzyme starts with Cys. Propeptide processing was investigated by site-directed mutagenesis using a system for expression in HeLa cells. Glutamine-dependent activity and processing were abolished by replacement of the conserved cysteine at position 1, whereas NH3-dependent activity was retained. Cys1 is thus inferred to have a role in glutamine-dependent activity and in propeptide processing. Inactive, insoluble enzymes in which the propeptide was not processed were obtained as a result of replacements of cysteines 415 and 488. Cysteine residues at positions 415 and 488 are inferred to be ligands to an Fe-S cluster on the basis of sequence similarity to the enzyme from Bacillus subtilis. Mutation of Cys269 and Cys295 led to loss of enzyme activity and propeptide processing, although solubility was unchanged. The results suggest that incorporation of an Fe-S cluster is needed for native structure, resultant propeptide processing, and glutamine-dependent activity.     

Nucleotide sequence of Escherichia coli purF and deduced amino acid sequence of glutamine phosphoribosylpyrophosphate amidotransferase

The Escherichia coli gene purF, coding for 5-phosphoribosylamine:glutamine pyrophosphate phosphoribosyltransferase (amidophosphoribosyltransferase) was subcloned from a ColE1-purF plasmid into pBR322. Amidophosphoribosyltransferase levels were elevated more than 5-fold in the ColE1-purF plasmid-bearing strain compared to the wild type control, and a further 10- to 13-fold elevation was observed in several pBR322 derivatives. The nucleotide sequence of a 2478-base pair PvuI-HinfI fragment encoding purF was determined. The purF45 structural gene codes for a 56,395 Mr protein chain having 504 amino acid residues. Methionine-1 is removed by processing in vivo leaving cysteine as the NH2-terminal residue. The deduced amino acid sequence was confirmed by comparisons with the NH2-terminal amino acid sequence determined by automated Edman degradation (Tso, J. Y., Hermodson, M. A., and Zalkin, H. (1982) J. Biol. Chem. 257, 3532-3536) and amino acid analyses of CNBr peptides including a 4-residue peptide from the CO2H terminus of the enzyme. Nucleotide sequences characteristic of bacterial promoter-operator regions were identified in the 5' flanking region. The coding region appears to be preceded by a 277-297 nucleotide mRNA leader. A deletion removing the putative promoter-operator region results in defective purF expression.     

The glutamine-utilizing site of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase

Reaction of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase with 6-diazo-5-oxo-L-norleucine resulted in complete loss of its ability to catalyze glutamine-dependent phosphoribosylamine formation and its glutaminase activity, whereas its ability to catalyze ammonia-dependent phosphoribosylamine formation and to hydrolyze phosphoribosylpyrophosphate was increased. The site of reaction with 6-diazo-5-oxo-L-norleucine was the NH2-terminal cysteine residue. The NH2-terminal sequence of the B. subtilis enzyme was homologous with that of the corresponding amidotransferase from Escherichia coli, for which the NH2-terminal cysteine is also essential for glutamine utilization (Tso, J. Y., Hermodson, M. A., and Zalkin, H. (1982) J. Biol. Chem. 257, 3532-3536). The fact that the metal-free E. coli amidotransferase contains a glutamine-utilizing structure that is very similar to that found in B. subtilis amidotransferase, which contains an essential [4Fe-4S] center, indicates that the iron-sulfur center probably plays no role in glutamine utilization.     

Catalytic domains of carbamyl phosphate synthetase. Glutamine-hydrolyzing site of Escherichia coli carbamyl phosphate synthetase

We present evidence that cysteine 269 of the small subunit of Escherichia coli carbamyl phosphate synthetase is essential for the hydrolysis of glutamine. When cysteine 269 is replaced with glycine or with serine by site-directed mutagenesis of the carA gene, the resulting enzymes are unable to catalyze carbamyl phosphate synthesis with glutamine as nitrogen donor. Even though the glycine 269, and particularly the serine 269 enzyme bind significant amounts of glutamine, neither glycine 269 nor serine 269 can hydrolyze glutamine. The mutations at cysteine 269 do not affect carbamyl phosphate synthesis with NH3 as substrate. The NH3-dependent activity of the mutant enzymes was equal to that of wild-type. Measurements of Km indicate that the enzyme uses unionized NH3 rather than ammonium ion as substrate. The apparent Km for NH3 of the wild-type enzyme is calculated to be about 5 mM, independent of pH. The substitution of cysteine 269 with glycine or with serine results in a decrease of the apparent Km value for NH3 from 5 mM with the wild-type to 3.9 mM with the glycine, and 2.9 mM with the serine enzyme. Neither the glycine nor the serine mutation at position 269 affects the ability of the enzyme to catalyze ATP synthesis from ADP and carbamyl phosphate. Allosteric properties of the large subunit are also unaffected. However, substitution of cysteine 269 with glycine or with serine causes an 8- and 18-fold stimulation of HCO-3 -dependent ATPase activity, respectively. The increase in ATPase activity and the decrease in apparent Km for NH3 provide additional evidence for an interaction of the glutamine binding domain of the small subunit with one of the two known ATP sites of the large subunit.     

Mutational analysis of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase propeptide processing

Glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis is a member of an N-terminal nucleophile hydrolase enzyme superfamily, several of which undergo autocatalytic propeptide processing to generate the mature active enzyme. A series of mutations was analyzed to determine whether amino acid residues required for catalysis are also used for propeptide processing. Propeptide cleavage was strongly inhibited by replacement of the cysteine nucleophile and two residues of an oxyanion hole that are required for glutaminase function. However, significant propeptide processing was retained in a deletion mutant with multiple defects in catalysis that was devoid of enzyme activity. Intermolecular processing of noncleaved mutant enzyme subunits by active wild-type enzyme subunits was not detected in hetero-oligomers obtained from a coexpression experiment. While direct in vitro evidence for autocatalytic propeptide cleavage was not obtained, the results indicate that some but not all of the amino acid residues that have a role in catalysis are also needed for propeptide processing.     

Mutational analysis of the glutamine phosphoribosylpyrophosphate amidotransferase pro-peptide

Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase is synthesized as a pro-enzyme having an 11-amino acid leader. Maturation requires insertion of a [4Fe-4S] cluster and processing of the pro-peptide to expose an NH2-terminal active site cysteine residue. Point and deletion mutations were constructed in the leader region. These mutations affect processing and enzyme activities. Processing of the leader is dependent upon glutamic acid residues at positions -2 and -1 as well as Cys1. In addition, processing requires a pro-peptide longer than 3 residues. Function of the active site cysteine is dependent on pro-peptide processing. Enzyme purified from a pro-peptide deletion strain has activity and iron content that is comparable to the wild type. These results establish that the pro-peptide is not essential for enzyme maturation, but they leave unanswered the question of pro-peptide function.     

Amino-terminal deletions define a glutamine amide transfer domain in glutamine phosphoribosylpyrophosphate amidotransferase and other PurF-type amidotransferases.

A series of deletions was constructed in cloned Escherichia coli purF encoding glutamine phosphoribosylpyrophosphate amidotransferase. These deletions extended into the NH2 terminus of the protein and removed amino acids that are required for glutamine-dependent enzyme activity. Enzyme function, ascribed to the NH3-dependent activity, was retained in deletions that removed up to 237 amino acids. This result supports a model in which PurF-type amidotransferases contain an NH2-terminal glutamine amide transfer domain of approximately 194 to 200 amino acids fused to an aminator domain with NH3-dependent function.     

Study of anthranilate synthase function by replacement of cysteine 84 using site-directed mutagenesis

Cysteine 84 was replaced by glycine in Serratia marcescens anthranilate synthase Component II using site-directed mutagenesis of cloned trpG. This replacement abolished the glutamine-dependent anthranilate synthase activity but not the NH3-dependent activity of the enzyme. The mutation provides further evidence for the role of active site cysteine 84 in the glutamine amide transfer function of anthranilate synthase Component II. By the criteria of circular dichroism, proteolytic inactivation, and feedback inhibition the mutant and wild type enzymes were structurally similar. The NH3-dependent anthranilate synthase activity of the mutant enzyme supported tryptophan synthesis in media containing a high concentration of ammonium ion.     

Evidence that the iron-sulfur cluster of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase determines stability of the enzyme to degradation in vivo

Bacillus subtilis glutamine P-Rib-PP amidotransferase contains a [4Fe-4S] cluster which is essential for activity. The enzyme also undergoes removal of 11 NH2-terminal residues from the primary translation product in vivo to form the active enzyme. It has been proposed that oxidative inactivation of the FeS cluster in vivo is the first step in degradation of the enzyme in starving cells. Four mutants of amidotransferases that alter cysteinyl ligands to the FeS cluster or residues adjacent to them have been prepared by site-directed mutagenesis, expressed in Escherichia coli, and characterized (Makaroff, C. A., Paluh, J. L., and Zalkin, H. (1986) J. Biol. Chem. 261, 11416-11423). These mutations were integrated into the B. subtilis chromosome in place of the normal purF gene. Inactivation and degradation in vivo of wild type and mutant amidotransferases were characterized in these integrants. Mutants FeS1 (C448S) and FeS2 (C451S) failed to form active enzyme, assemble FeS clusters, or undergo NH2-terminal processing. The immunochemically cross-reactive protein produced by both mutants was degraded rapidly (t1/2 = 16 min) in exponentially growing cells. In contrast the wild type enzyme was stable in growing cells, and activity and cross-reactive protein were lost from glucose-starved cells with a t1/2 of 57 min. Mutant FeS3 (F394V) contained an FeS cluster and was processed normally, but had only about 40% of normal specific activity. The FeS3 enzyme was also inactivated by reaction with O2 in vitro about twice as fast as the wild type. The amidotransferase produced by the FeS3 integrant was stable in growing cells but was inactivated and degraded in glucose-starved cells more rapidly (t1/2 = 35 min) than the wild type enzyme. Mutant FeS4 (C451S, D442C) also contained an FeS cluster and was processed; the enzyme had about 50% of wild type-specific activity and reacted with O2 in vitro at the same rate as the wild type. Inactivation and degradation of the FeS4 mutant in vivo in glucose-starved cells proceeded at a rate (t1/2 = 45 min) that was somewhat faster than normal. The correlation between absence of an FeS cluster or enhanced lability of the cluster to O2 and increased degradation rates in vivo supports the conclusions that stability of the enzyme in vivo requires an intact FeS cluster and that O2-dependent inactivation is the rate-determining step in degradation of the enzyme. The fact that mutant FeS3 was processed normally but degraded rapidly argues against a role for NH2-terminal processing in controlling degradation rates.     

Temperature-sensitive mutant of Bacillus subtilis glutamine synthetase obtained by random mutation

Random mutations were introduced into the B. subtilis glutamine synthetase gene by using nitrous acid, and a high temperature-sensitive mutant was selected. DNA sequencing of the restriction fragment containing the mutation revealed a single base-pair change resulting in the substitution of Leu 318 with Phe. The mutant enzyme was purified, and its kinetic and physical properties were characterized. The Mg2(+)-dependent activity and Mg2+ plus Mn2(+)-dependent activity of the mutant were less than 5% of those of the wild-type at 37 degrees C, and these activities decreased above 15 degrees C, whereas the Mn2(+)-dependent activity was nearly normal. Affinity of the mutant enzyme for glutamate was extremely decreased although the Km values for NH3 or ATP were almost the same as those of the wild-type. The mutant enzyme was more susceptible than the wild-type enzyme to digestion with chymotrypsin in the presence of glutamate, ATP, and Mg2+, although addition of glutamate, ATP, and Mn2+ completely protected both enzymes. These results and circular dichroism analyses suggested that Leu 318 is at the glutamate-binding site and that the substitution of Leu 318 for Phe reduces the ability of the enzyme to form the enzyme-substrate complex, probably supported by Mg2+.     

Glucosamine synthetase from Escherichia coli: purification, properties, and glutamine-utilizing site location

L-Glutamine:D-fructose-6-phosphate amidotransferase (glucosamine synthetase) has been purified to homogeneity from Escherichia coli. A subunit molecular weight of 70,800 was estimated by gel electrophoresis in sodium dodecyl sulfate. Pure glucosamine synthetase did not exhibit detectable NH3-dependent activity and did not catalyze the reverse reaction, as reported for more impure preparations [Gosh, S., Blumenthal, H. J., Davidson, E., & Roseman, S. (1960) J. Biol. Chem. 235, 1265]. The enzyme has a Km of 2 mM for fructose 6-phosphate, a Km of 0.4 mM for glutamine, and a turnover number of 1140 min-1. The amino-terminal sequence confirmed the identification of residues 2-26 of the translated E. coli glmS sequence [Walker, J. E., Gay, J., Saraste, M., & Eberle, N. (1984) Biochem. J. 224, 799]. Methionine-1 is therefore removed by processing in vivo, leaving cysteine as the NH2-terminal residue. The enzyme was inactivated by the glutamine analogue 6-diazo-5-oxo-L-norleucine (DON) and by iodoacetamide. Glucosamine synthetase exhibited half-of-the-sites reactivity when incubated with DON in the absence of fructose 6-phosphate. In its presence, inactivation with [6-14C]DON was accompanied by incorporation of 1 equiv of inhibitor per enzyme subunit. From this behavior, a dimeric structure was tentatively assigned to the native enzyme. The site of reaction with DON was the NH2-terminal cysteine residue as shown by Edman degradation.     

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.     

Site-directed mutagenesis of a single tryptophan near the middle of the channel-forming toxin aerolysin inhibits its transfer across the outer membrane of Aeromonas salmonicida

The channel-forming protein aerolysin must cross both the inner and outer bacterial membranes during its secretion from Aeromonas hydrophila or from Aeromonas salmonicida containing the cloned structural gene. We examined the fate of three mutant proteins in which Trp-227, near the middle of the amino acid chain, was replaced with glycine, leucine, or phenylalanine by site-directed mutagenesis. All three proteins crossed the inner membrane and entered the periplasm in the same way as wild-type, and in each case the signal sequence was removed correctly. Little or none of the proaerolysin substituted with glycine or leucine was released into the culture supernatant. Instead, significant amounts became associated with the outer membrane. The Phe-227 protoxin was secreted by the bacteria but at a reduced rate. The leucine and phenylalanine mutant proteins were purified and compared with native proaerolysin. They were processed correctly to the mature forms by treatment with trypsin, and like native aerolysin, both were resistant to further proteolysis. In each case, processing was followed by the formation of oligomers similar to those produced by native toxin. The hemolytic activity of the processed Phe-227 mutant was one-quarter that of wild-type toxin whereas Leu-227 aerolysin had less than one-hundredth the wild-type activity. These results are further evidence that aerolysin is secreted in at least two steps. As well, they show that the last step, crossing the outer membrane, can be blocked by an apparently small change in the structure of the protein.     

Evidence for the involvement of tyrosine-69 in the control of stereospecificity of porcine pancreatic phospholipase A2

We have studied the role of Tyr-69 of porcine pancreatic phospholipase A2 in catalysis and substrate binding, using site-directed mutagenesis. A mutant was constructed containing Phe at position 69. Kinetic characterization revealed that the Phe-69 mutant has retained enzymatic activity on monomeric and micellar substrates, and that the mutation has only minor effects on kcat and Km. This shows that Tyr-69 plays no role in the true catalytic events during substrate hydrolysis. In contrast, the mutation has a profound influence on the stereospecificity of the enzyme. Whereas the wild-type phospholipase A2 is only able to catalyse the degradation of sn-3 phospholipids, the Phe-69 mutant hydrolyses both the sn-3 isomers and, at a low (1-2%) rate, the sn-1 isomers. Despite the fact that the stereospecificity of the mutant phospholipase has been altered, Phe-69 phospholipase still requires Ca2+ ions as a cofactor and also retains its specificity for the sn-2 ester bond. Our data suggest that in porcine pancreatic phospholipase A2 the hydroxyl group of Tyr-69 serves to fix and orient the phosphate group of phospholipid monomers by hydrogen bonding. Because no such interaction can occur between the Phe-69 side-chain and the phosphate moiety of the substrate monomer, the mutant enzyme loses part of its stereospecificity but not its positional specificity.     

Glutamate synthase. Properties of the glutamine-dependent activity.

Properties of glutamine-dependent glutamate synthase have been investigated using homogeneous enzyme from Escherichia coli K-12. In contrast to results with enzyme from E. coli strain B (Miller, R. E., and Stadtman, E. R. (1972) J. Biol. Chem. 247, 7407-7419), this enzyme catalyzes NH3-dependent glutamate synthase activity. Selective inactivation of glutamine-dependent activity was obtained by treatment with the glutamine analog. L-2-amino-4-oxo-5-chloropentanoic acid (chloroketone). Inactivation by chloroketone exhibited saturation kinetics; glutamine reduced the rate of inactivation and exhibited competitive kinetics. Iodoacetamide, other alpha-halocarbonyl compounds, and sulfhydryl reagents gave similar selective inactivation of glutamine-dependent activity. Saturation kinetics were not obtained for inactivation by iodoacetamide but protection by glutamine exhibited competitive kinetics. The stoichiometry for alkylation by chloroketone and iodoacetamide was approximately 1 residue per protomer of molecular weight approximately 188,000. The single residue alkylated with iodo [1-14C]acetamide was identified as cysteine by isolation of S-carboxymethylcysteine. This active site cysteine is in the large subunit of molecular weight approximately 153,000. The active site cysteine was sensitive to oxidation by H2O2 generated by autooxidation of reduced flavin and resulted in selective inactivation of glutamine-dependent enzyme activity. Similar to other glutamine amidotransferases, glutamate synthase exhibits glutaminase activity. Glutaminase activity is dependent upon the functional integrity of the active site cysteine but is not wholly dependent upon the flavin and non-heme iron. Collectively, these results demonstrate that glutamate synthase is similar to other glutamine amidotransferases with respect to distinct sites for glutamine and NH3 utilization and in the obligatory function of an active site cysteine residue for glutamine utilization.     

Activation of human furin precursor processing endoprotease occurs by an intramolecular autoproteolytic cleavage.

Human furin is a calcium-dependent serine endoprotease that can efficiently cleave many precursor proteins on the carboxyl side of the consensus cleavage sequence, -Arg-X-Lys/Arg-Arg-, both in vivo and in vitro. Analysis of furin proteins in extracts of cells infected with a vaccinia recombinant expressing human furin show that the enzyme is present as two prominent forms of 90 and 96 kDa. Because the structurally related bacterial subtilisins require endoproteolytic removal of the NH2-terminal pro-region by an autocatalytic intramolecular cleavage, we speculated that the size heterogeneity in the furin doublet similarly may result from a proteolytic removal of an NH2-terminal pro-region. Here we report identification of the 90-kDa furin NH2 terminus and, based on the reported sequence of the furin cDNA, demonstrate that this furin protein is derived from a larger precursor by an endoproteolytic cleavage on the COOH-terminal side of a consensus furin cleavage site, -Arg-Thr-Lys-Arg107-. Expression of mutant furin molecules containing an altered cleavage site (Arg104----Ala or Arg107----Gly) resulted in the production of only the 96-kDa furin protein. Assays of furin-dependent cleavage of a protein substrate in vitro showed that proteolytic activity was associated with the 90-kDa and not the 96-kDa furin protein, demonstrating that removal of the NH2-terminal pro-region is required for furin activity. Expression of a third furin construct containing a mutation of the active site aspartate (Asp153----Asn) similarly resulted in the expression of only the 96-kDa protein, suggesting that furin activation occurs by an autoproteolytic cleavage. Finally, the production of 90-kDa furin from either site-directed furin mutant could not be potentiated by overexpressing active furin, suggesting that the autoproteolytic activation was an intramolecular event.     

Autocatalytic processing of procathepsin E to cathepsin E and their structural differences

The processing of human gastric procathepsin E to its mature form, cathepsin E, was studied at pH 3.5. The results revealed the autocatalytic and apparently one-step conversion of procathepsin E to cathepsin E within 10 min of incubation at 14 degrees C under the conditions used. Analyses of the amino acid sequences of both procathepsin E and cathepsin E showed that cleavage occurred at the Met36-Ile37 bond to produce the mature form, cathepsin E. The NH2-terminal amino acid sequence of procathepsin E thus determined was identical with that predicted from the cDNA sequence by Azuma et al. except that the NH2-terminal glutamine residue in the latter was converted into a pyroglutamic acid residue in the former and that the glycine residue at position 2 in the latter sequence was deleted in the former. On the other hand, the NH2-terminal amino acid sequence of cathepsin E was identical with that reported previously by us.     

Chemical genetic identification of glutamine phosphoribosylpyrophosphate amidotransferase as the target for a novel bleaching herbicide in Arabidopsis

A novel phenyltriazole acetic acid compound (DAS734) produced bleaching of new growth on a variety of dicotyledonous weeds and was a potent inhibitor of Arabidopsis (Arabidopsis thaliana) seedling growth. The phytotoxic effects of DAS734 on Arabidopsis were completely alleviated by addition of adenine to the growth media. A screen of ethylmethanesulfonate-mutagenized Arabidopsis seedlings recovered seven lines with resistance levels to DAS734 ranging from 5- to 125-fold. Genetic tests determined that all the resistance mutations were dominant and allelic. One mutation was mapped to an interval on chromosome 4 containing At4g34740, which encodes an isoform of glutamine phosphoribosylamidotransferase (AtGPRAT2), the first enzyme of the purine biosynthetic pathway. Sequencing of At4g34740 from the resistant lines showed that all seven contained mutations producing changes in the encoded polypeptide sequence. Two lines with the highest level of resistance (125-fold) contained the mutation R264K. The wild-type and mutant AtGPRAT2 enzymes were cloned and functionally overexpressed in Escherichia coli. Assays of the recombinant enzyme showed that DAS734 was a potent, slow-binding inhibitor of the wild-type enzyme (I(50) approximately 0.2 microm), whereas the mutant enzyme R264K was not significantly inhibited by 200 microm DAS734. Another GPRAT isoform in Arabidopsis, AtGPRAT3, was also inhibited by DAS734. This combination of chemical, genetic, and biochemical evidence indicates that the phytotoxicity of DAS734 arises from direct inhibition of GPRAT and establishes its utility as a new and specific chemical genetic probe of plant purine biosynthesis. The effects of this novel GPRAT inhibitor are compared to the phenotypes of known AtGPRAT genetic mutants.