Expression,purification, and crystallization of glutamyl-tRNA(Gln) specific amidotransferase from Bacillus stearothermophilus

Although the genes that encode the glutamyl-tRNA(Gln) (Glu-tRNA(Gln)) specific amidotransferase (Glu-AdTase) from various bacteria and eukaryotic organelles are known, the precise mechanism of the enzyme is still unclear. One of the reasons is that there is no information on the three-dimensional structure of the complex, the Glu-AdTase:Glu-tRNA(Gln):ATP:amino group donor. To obtain the crystals of Glu-AdTase, the Glu-AdTase of Bacillus stearothermophilus was overexpressed and purified after cloning of the gene that encodes the enzyme. The cloned DNA contained the full-length gene cluster that represented the Glu-AdTase of B. stearothermophilus, and was organized as an operon that consisted of three open-reading frames (ORFs). The order of the genes was gatCAB, as shown in Bacillus subtilis. The ORFs showed a high amino-acid homology to those of B. subtilis (A subunit, 73.2%; B subunit, 81.6%; C subunit, 69.5%) and Staphylococcus aureus (A subunit, 61.9%; B subunit, 71.8%; C subunit, 45.9%). The ORFs were re-cloned on the overexpression vector, pTrc99a, and a recombinant pTrcgatCABBST was obtained. The Glu-AdTase that was overexpressed with pTrcgatCABBST in Escherichia coli retained transamidation activity on the mischarged glutamic acid on the tRNA(Gln). It also produced correctly-charged Gln-tRNA(Gln) at 37, 42, and 50 degrees C. Although Glu-AdTases from both B. subtilis and B. stearothermophilus were subjected to crystallization, the micro-crystals were only obtained from the B. stearothermophilus enzyme.     

Growth inhibition of Escherichia coli during heterologous expression of Bacillus subtilis glutamyl-tRNA synthetase that catalyzes the formation of mischarged glutamyl-tRNA1 Gln

It is known that Bacillus subtilis glutamyl-tRNA synthetase (GluRS) mischarges E. coli tRNA1 Gln with glutamate in vitro. It has also been established that the expression of B. subtilis GluRS in Escherichia coli results in the death of the host cell. To ascertain whether E. coli growth inhibition caused by B. subtilis GluRS synthesis is a consequence of Glu-tRNA1 Ghn formation, we constructed an in vivo test system, in which B. subtilis GluRS gene expression is controlled by IPTG. Such a system permits the investigation of factors affecting E. coli growth. Expression of E. coli glutaminyl-tRNA synthetase (GlnRS) also ameliorated growth inhibition, presumably by competitively preventing tRNA1 Gln misacylation. However, when amounts of up to 10 mM L-glutamine, the cognate amino acid for acylation of tRNA1 Gln, were added to the growth medium, cell growth was unaffected. Overexpression of the B. subtilis gatCAB gene encoding Glu-tRNAGln amidotransferase (Glu-AdT) rescued cells from toxic effects caused by the formation of the mischarging GluRS. This result indicates that B. subtilis Glu-AdT recognizes the mischarged E. coli GlutRNA1 Gln, and converts it to the cognate Gln-tRNA1 Gln species. B. subtilis GluRS-dependent Glu-tRNA1 Gln formation may cause growth inhibition in the transformed E. coli strain, possibly due to abnormal protein synthesis.     

In vivo formation of glutamyl-tRNA(Gln) in Escherichia coli by heterologous glutamyl-tRNA synthetases

Two types of glutamyl-tRNA synthetase exist: the discriminating enzyme (D-GluRS) forms only Glu-tRNA(Glu), while the non-discriminating one (ND-GluRS) also synthesizes Glu-tRNA(Gln), a required intermediate in protein synthesis in many organisms (but not in Escherichia coli). Testing the capacity to complement a thermosensitive E. coli gltX mutant and to suppress an E. coli trpA49 missense mutant we examined the properties of heterologous gltX genes. We demonstrate that while Acidithiobacillus ferrooxidans GluRS1 and Bacillus subtilis Q373R GluRS form Glu-tRNA(Glu), A. ferrooxidans and Helicobacter pylori GluRS2 form Glu-tRNA(Gln) in E. coli in vivo.     

Glutamyl-gamma-boronate inhibitors of bacterial Glu-tRNA(Gln) amidotransferase.

Analogues of glutamyl-gamma-boronate (1) were synthesized as mechanism-based inhibitors of bacterial Glu-tRNA(Gln) amidotransferase (Glu-AdT) and were designed to engage a putative catalytic serine nucleophile required for the glutaminase activity of the enzyme. Although 1 provides potent enzyme inhibition, structure-activity studies revealed a narrow range of tolerated chemical changes that maintained activity. Nonetheless, growth inhibition of organisms that require Glu-AdT by the most potent enzyme inhibitors appears to validate mechanism-based inhibitor design of Glu-AdT as an approach to antimicrobial development.     

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.     

Affinity chromatography of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase.

Purified glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis bound to affinity adsorbents containing immobilized adenine nucleotides. Although the enzyme probably bound via an allosteric site at which AMP acts most effectively, 50 times more enzyme was bound by N⁶-(aminohexyl)-ATP-agarose than by N⁶-(aminohexyl)-AMP-agarose. The enzyme could be efficiently and specifically eluted from N⁶-(aminohexyl)-ATP-agarose with the substrate phosphoribosylpyrophosphate, which antagonizes AMP inhibition in kinetic experiments. Elution could also be effected by 0.5 m KCl or by chelation of Mg²⁺ ions. The usefulness of these techniques in purification of partially purified amidotransferase was demonstrated.     

A model for the Bacillus subtilis formylglycinamide ribonucleotide amidotransferase multiprotein complex

Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) catalyzes the conversion of formylglycinamide ribonucleotide (FGAR), ATP, and glutamine to formylglycinamidine ribonucleotide (FGAM), ADP, P(i), and glutamate in the fourth step of the purine biosynthetic pathway. PurL exists in two forms: large PurL (lgPurL) is a single chain, multidomain enzyme of about 1300 amino acids, whereas small PurL (smPurL) contains about 800 amino acids but requires two additional gene products, PurS and PurQ, for activity. smPurL contains the ATP and FGAR binding sites, PurQ is a glutaminase, and the function of PurS is just now becoming understood. We determined the structure of Bacillus subtilis PurS in two different crystal forms P2(1) and C2 at 2.5 and 2.0 A resolution, respectively. PurS forms a tight dimer with a central six-stranded beta-sheet flanked by four helices. In both the P2(1) and the C2 crystal forms, the quaternary structure of PurS is a tetramer. The concave faces of the PurS dimers interact via the C-terminal region to form a twelve-stranded beta-barrel with a hydrophilic core. We used the structure of PurS together with the structure of lgPurL from Salmonella typhimurium to construct a model of the PurS/smPurL/PurQ complex. The HisH (glutaminase) domain of imidazole glycerol phosphate synthetase was used as an additional model of PurQ. The model shows stoichiometry of 2PurS/smPurL/PurQ using a PurS dimer or 4PurS/2smPurL/2PurQ using a PurS tetramer. Both models place key conserved residues at the ATP/FGAR binding site and at a structural ADP binding site. The homology model is consistent with biochemical studies on the reconstituted complex.     

Regulation of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase inactivation in vivo

Glutamine phosphoribosylpyrophosphate amidotransferase is stable in growing cells, but is inactivated in an oxygen-dependent process at various rates in starving or antibiotic-treated cells. On the basis of studies of the purified enzyme, we suggested (D.A. Bernlohr and R.L. Switzer, Biochemistry 20:5675-5681, 1981) that the inactivation in vivo was regulated by substrate stabilization and a competition between stabilizing (AMP) and destabilizing (GMP, GDP, and ADP) nucleotides. This proposal was tested by measuring the intracellular levels of these metabolites under cultural conditions in which the stability of the amidotransferase varied. The results established that the stability of amidotransferase in vivo cannot be explained by the simple interactions observed in vitro. Metabolite levels associated with stability of the enzyme in growing cells did not confer stability under other conditions, such as ammonia starvation or refeeding of glucose-starved cells. The data suggest that a previously unrecognized event, possibly a covalent modification of amidotransferase, is required to mark the enzyme for oxygen-dependent inactivation.     

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.     

Purification and functional characterization of the Glu-tRNA(Gln) amidotransferase from Chlamydomonas reinhardtii

The formation of glutaminyl-tRNA (Gln-tRNA) in Bacilli, chloroplasts, and mitochondria occurs in a two-step reaction. This involves misacylation of tRNA(Gln) with glutamate by glutamyl-tRNA synthetase and subsequent amidation of Glu-tRNA(Gln) to the correctly acylated Gln-tRNA(Gln) by a specific amidotransferase (Sch?n, A., Kannangara, C. G., Gough, S., and S?ll, D. (1988) Nature 331, 187-190). Here we demonstrate the existence of this pathway in green algae and describe the purification of the Glu-tRNA(Gln) amidotransferase from Chlamydomonas reinhardtii. The purified enzyme showed an Mr of approximately 120,000 when analyzed by glycerol gradient sedimentation and gel filtration. An apparent Mr of 63,000 of the denatured protein was demonstrated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. This indicates that the enzyme possesses an alpha 2 structure. The substrate for the purified enzyme is Glu-tRNA(Gln) but not Glu-tRNA(Glu). The enzyme requires ATP, Mg2+, and an amide donor for the conversion. Acceptable amide donors are glutamine, asparagine, and ammonia. Blocking of the glutamine-dependent reaction by alkylation of the protein with 6-diazo-5-oxonorleucine did not inhibit the ammonia-dependent reaction, suggesting that the enzyme has separate glutamine and ammonia binding sites. As suggested by Wilcox (Wilcox, M. (1969) Eur. J. Biochem. 11, 405-412) the amidation reaction may involve glutamyl-phosphate formation, since ATP is cleaved to ADP when the enzyme is incubated with Glu-tRNA(Gln) and ATP. In common with other glutamine amidotransferases, the enzyme also possesses low glutaminase activity. The purified Glu-tRNA(Gln) amidotransferase forms a stable complex with Glu-tRNA(Gln) in the presence of ATP and Mg2+ but in the absence of the amide donor as determined by gradient centrifugation.     

Spectroscopic characterization of the iron-sulfur cluster in Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase

The properties of the [4Fe-4S] cluster in glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis have been investigated using low temperature magnetic circular dichroism, electron paramagnetic resonance (EPR), and resonance Raman spectroscopies. The Raman spectra of the native enzyme in the Fe-S stretching region show a [4Fe-4S]2+ cluster that is structurally very similar to those in simple redox proteins. Photochemical reduction mediated by 5-deazaflavin with oxalate as the electron donor resulted in [4Fe-4S]+ clusters with a mixture of ground state spin multiplicities. Magnetic circular dichroism and EPR studies of samples ranging in concentration from 0.15 to 0.4 mM concur in finding S = 3/2 [4Fe-4S]+ clusters with predominantly axial and positive zero field splitting as the dominant species. The EPR studies also revealed minor contributions from S = 1/2 [4Fe-4S]+ centers and an S = 5/2 species. The latter becomes the dominant component in more concentrated samples (approximately 2 mM), and arguments are presented in favor of assignment to S = 5/2 [4Fe-4S]+ clusters rather than adventitiously bound high spin Fe(III) ions. The concentration-dependent spin state heterogeneity of the [4Fe-4S]+ cluster in glutamine phosphoribosylpyrophosphate amidotransferase is discussed in light of the magnetic and electronic properties of the [4Fe-4S]+ centers in other enzymes and proteins.     

Oxidation-reduction properties of the iron-sulfur cluster in Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase

Native Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase contains a [4Fe-4S] cluster in the diamagnetic (+2) state. The cluster is essential for catalytic function, even though amidotransferase does not catalyze a redox reaction. The ability of the Fe-S cluster to undergo oxidation and reduction reactions and the consequences of changes in the redox state of the cluster for enzyme activity were studied. Treatment of the enzyme with oxidants resulted in either no reaction or complete dissolution of the Fe-S cluster and loss of activity. A stable +3 oxidation state was not detected. A small amount of paramagnetic species, probably an oxidized 3Fe cluster, was formed transiently during oxidation. The native cluster was poorly reduced by dithionite, but it could be readily reduced to the +1 state by photoreduction with 5-deazaflavin and oxalate. The reduced enzyme did not display an EPR spectrum typical of [4Fe-4S] ferredoxins in the +1 state, unless it was prepared under denaturing conditions. M?ssbauer spectroscopy of reduced 57Fe-enriched amidotransferase confirmed that the cluster was in the +1 state, but the magnetic properties of the reduced cluster observed at 4.2 K indicated that it is characterized by a ground state spin S greater than or equal to 3/2. The midpoint potential of the +1/+2 couple was too low to measure accurately by conventional techniques, but it was below -600 mV, which is 100 mV more negative than reported for [4Fe-4S] clusters in bacterial ferredoxins. Fully reduced amidotransferase had about 40% of the activity of the native enzyme in glutamine-dependent phosphoribosylamine formation. The fact that both the +1 and +2 forms of the enzyme are active indicates that the cluster does not function as a site of reversible electron transfer during catalysis.     

The heterotrimeric Thermus thermophilus Asp-tRNA(Asn) amidotransferase can also generate Gln-tRNA(Gln)

Thermus thermophilus strain HB8 is known to have a heterodimeric aspartyl-tRNA(Asn) amidotransferase (Asp-AdT) capable of forming Asn-tRNA(Asn) [Becker, H.D. and Kern, D. (1998) Proc. Natl. Acad. Sci. USA 95, 12832-12837]. Here we show that, like other bacteria, T. thermophilus possesses the canonical set of amidotransferase (AdT) genes (gatA, gatB and gatC). We cloned and sequenced these genes, and constructed an artificial operon for overexpression in Escherichia coli of the thermophilic holoenzyme. The overproduced T. thermophilus AdT can generate Gln-tRNA(Gln) as well as Asn-tRNA(Asn). Thus, the T. thermophilus tRNA-dependent AdT is a dual-specific Asp/Glu-AdT resembling other bacterial AdTs. In addition, we observed that removal of the 44 carboxy-terminal amino acids of the GatA subunit only inhibits the Asp-AdT activity, leaving the Glu-AdT activity of the mutant AdT unaltered; this shows that Asp-AdT and Glu-AdT activities can be mechanistically separated.     

Involvement of the stringent response in degradation of glutamine phosphoribosylpyrophosphate amidotransferase in Bacillus subtilis

Glutamine phosphoribosylpyrophosphate amidotransferase, the first enzyme of purine biosynthesis, has previously been shown to be rapidly inactivated and degraded in Bacillus subtilis cells at the end of growth. The loss of enzyme activity appears to involve the oxidation of an iron-sulfur cluster in the enzyme. The degradation of the inactive enzyme involves some elements of the stringent response because it is inhibited in relA and relC mutants. Intracellular pools of guanosine tetra- and pentaphosphate were measured by an improved extraction procedure in cells that had been manipulated in various ways to induce or inhibit amidotransferase degradation. The results are consistent with the hypothesis that one or both of these nucleotides stimulates the synthesis of a protein involved in degradation. An elevated level of these nucleotides was not required for the continued degradation of amidotransferase once it had begun.     

Characterization of Bacillus subtilis bacteriophages

Brodetsky, Anna M. (University of California, Los Angeles), and W. R. Romig. Characterization of Bacillus subtilis bacteriophages. J. Bacteriol. 90:1655-1663. 1965.-A group of six phages, SP5, SP6, SP7, SP8, SP9, and SP13, which use the Marburg strain of Bacillus subtilis as host was characterized. These phages, referred to as group 1, were examined for the following properties: host range, plaque morphology, stability, adsorption kinetics, one-step growth characteristics, calcium requirements, serum neutralization, thermal inactivation, and inactivation by ultraviolet irradiation. Five unrelated B. subtilis phages, SP3, SP10, PBS1, SP alpha, and SP beta, were included in the studies. When first isolated, none of the group 1 phages was able to replicate efficiently on B. subtilis SB19, a mutant of the "transforming" B. subtilis 168. Host range mutants capable of growth in SB19 were isolated for all of the group 1 phages except SP13, and are designated the "star" phages (SP5* through SP9*). For characterization, SB19 was used as host for the star phages, and another B. subtilis mutant, 168B, was host for SP13.     

A novel amidotransferase required for lipoic acid cofactor assembly in Bacillus subtilis

In the companion paper we reported that Bacillus subtilis requires three proteins for lipoic acid metabolism, all of which are members of the lipoate protein ligase family. Two of the proteins, LipM and LplJ, have been shown to be an octanoyltransferase and a lipoate : protein ligase respectively. The third protein, LipL, is essential for lipoic acid synthesis, but had no detectable octanoyltransferase or ligase activity either in vitro or in vivo. We report that LipM specifically modifies the glycine cleavage system protein, GcvH, and therefore another mechanism must exist for modification of other lipoic acid requiring enzymes (e.g. pyruvate dehydrogenase). We show that this function is provided by LipL, which catalyses the amidotransfer (transamidation) of the octanoyl moiety from octanoyl‐GcvH to the E2 subunit of pyruvate dehydrogenase. LipL activity was demonstrated in vitro with purified components and proceeds via a thioester‐linked acyl‐enzyme intermediate. As predicted, ΔgcvH strains are lipoate auxotrophs. LipL represents a new enzyme activity. It is a GcvH:[lipoyl domain] amidotransferase that probably uses a Cys‐Lys catalytic dyad. Although the active site cysteine residues of LipL and LipB are located in different positions within the polypeptide chains, alignment of their structures show these residues occupy similar positions. Thus, these two homologous enzymes have convergent architectures.     

Glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis. A novel iron-sulfur protein.

Glutamine phosphoribosylpyrophosphate amidotransferase, purifed to better than 98% purity from derepressed Bacillus subtilis, exists as a tetramer and as a dimer of apparently identical subunits with a molecular weight of 50,000 each. The enzyme contains 3 atoms of iron and 2 atoms of inorganic sulfide per subunit and has a yellow-brown color. The absorption spectrum is not altered by dithionite, but exposure to oxygen causes inactivation and partial bleaching of the visible spectrum. Thus, the Bacillus amidotransferase exhibits novel structural features and a new reaction type of proteins of the iron-sulfur group.     

The formylglycinamide ribonucleotide amidotransferase complex from Bacillus subtilis: metabolite-mediated complex formation

Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) catalyzes the ATP- and glutamine-dependent formation of formylglycinamidine ribonucleotide, ADP, P(i), and glutamate in the fourth step of de novo purine biosynthesis. Like all amidotransferases (ATs), FGAR-AT is proposed to channel ammonia between a glutaminase and AT domain. In Gram-negative bacteria and eukaryotes, FGAR-AT is a single approximately 140 kDa protein. In archae and Gram-positive bacteria, the FGAR-AT is formed from three proteins: PurS (10 kDa), PurQ (25 kDa, a glutaminase), and smPurL (80 kDa, an AT). This is the only known AT to require a third structural component (PurS) for activity. Here we report the first purification and biochemical characterization of a three-component AT from Bacillus subtilis. Efforts to isolate an intact FGAR-AT focused initially on coexpression of PurS, smPurL, and PurQ. However, all attempts to purify the complex resulted in separation of the constituent proteins. PurS, smPurL, and PurQ were therefore separately expressed and purified to homogeneity. PurQ had a glutaminase activity of 0.002 s(-1), and smPurL had an ammonia-dependent AT activity of 0.044 s(-1). Reconstitution of PurS, smPurL, and PurQ at a ratio of 2:1:1 gave an activity of 2.49 s(-1), similar to that previously reported for the Escherichia coli 140 kDa FGAR-AT (5.00 s(-1)). PurS was essential for the glutamine-dependent FGAR-AT activity. Surprisingly, activity was found to be absolutely dependent on the presence of Mg2+ and ADP, and a stable FGAR-AT complex of 2PurS/1smPurL/1PurQ was detected only in the presence of Mg2+, ADP, and glutamine. The implications of these observations are discussed with respect to ammonia channeling.     

Characterization of the 5' subtilisin (aprE) regulatory region from Bacillus subtilis

The poly(ADP-ribose) polymerase-like thermozyme purified from Sulfolobus solfataricus was characterised with respect to some physico-chemical properties. The archaeal protein exhibited a scarce electrophoretic mobility at both pH 2.9 and pH 7.5. Determination of the isoelectric point (pI=7.0-7.2) allowed us to understand the reason for the limited migration at pH 7.5, while amino acid composition analysis showed a moderate content of basic residues, which reduced mobility at pH 2.9. With respect to the charge, the archaeal enzyme behaved differently from the eukaryotic thermolabile poly(ADP-ribose) polymerase, described as a basic protein (pI=9.5). Well known inhibitors of the mesophilic polymerase like Zn(2+), nicotinamide and 3-aminobenzamide exerted a smaller effect on the enzyme from S. solfataricus, reducing the activity by at most 50%. Mg(2+) was a positive effector, although in a dose-dependent manner. It influenced the fluorescence spectrum of the archaeal protein, whereas NaCl had no effect.