Identification of thepurA gene encoding adenylosuccinate synthetase inThiobacillus ferrooxidans

ThepurA gene ofThiobacillus ferrooxidans encoding adenylosuccinate synthetase [EC 6.3.4.4] was identified in the upstream region of theiro gene encoding Fe(II)-oxidase (J. Biol. Chem 267:11242–11247, 1992). ThepurA gene consisted of 1290 base-pairs, which translated into a 29-amino-acid protein. The gene is functionally active, because it is able to complement anEscherichia coli purA-deficient strain. The deduced gene product has a high degree (60.9%) of sequence identity with that (432 aa) ofE. coli purA gene, and both the products share GDEGKGK-DETG-TKLD sequences which are supposed to be GTP-binding domain. The downstream region of theiro gene contained another open-reading frame (ORF) of 1218 bp, and this showed high homlogy (56.6% over 249 bp) withE. coli ORF-II, which is found as a second ORF and truncated form in the downstream region of thepurA gene. Comparison of the gene organization in the flanking region ofpurA gene betweenT. ferrooxidans andE. coli is also described.     

Nucleotide mutations in purA gene and pur operon promoter discovered in guanosine- and inosine-producing Bacillus subtilis strains

The promoter region of the pur operon, which contains 12 genes for inosine monophosphate biosynthesis from phosphoribosylpyrophosphate, and the purA gene, encoding the adenylosuccinate synthetase, were compared among wild-type and three purine-producing Bacillus subtilis strains. A single nucleotide deletion at position 55 (relative to translation start site) in purA gene was found in a high inosine-producing strain and in a high guanosine-producing strain, which correlates with the absence of adenylosuccinate synthetase activity in these strains. Within the pur operon promoter of high guanosine-producing strain, in addition to a single nucleotide deletion in PurBox1 and a single nucleotide substitution in PurBox2, there were 4 substitutions in the flanking region of the PurBoxes and 32 nucleotide mutations in the 5′ untranslated region. These mutations may explain the purine accumulation in purine-producing strains and be helpful to the rational design of high-yield recombinant strains.     

Site-directed mutagenesis of the phosphate-binding consensus sequence in Escherichia coli adenylosuccinate synthetase.

Adenylosuccinate synthetases from different sources contain an N-terminal glycine-rich sequence GDEGKGK, which is homologous to the conserved sequence GXXXXGK found in many other guanine nucleotide-binding proteins or enzymes. To determine the role of this sequence in the structure and function of Escherichia coli adenylosuccinate synthetase, site-directed mutagenesis was performed to generate five mutant enzymes: G12V (Gly12----Val), G15V (Gly15----Val), G17V (Gly17----Val), K18R (Lys18----Arg), and I19T (Ile19----Thr). Comparison of the kinetic properties of the wild-type enzyme and those of the mutant enzymes revealed that the sequence is critical for enzyme activity. Replacement of Gly12, Gly15, or Gly17 with Val, or replacement of Lys18 with Arg, resulted in significant decreases in the kcat/Km values of the enzyme. Because the consensus sequence GXXXXGK(T/S) has been found in many GTP-binding proteins, isoleucine at position 19 in the E. coli adenylosuccinate synthetase was changed to threonine to produce the sequence GDEGKGKT. This mutation, which more closely resembles the consensus sequence, resulted in a 160-fold increase in the Km value for substrate GTP; however, there were no great changes for the other two substrates, IMP and aspartate. Based on these data, we suggest that the N-terminal glycinerich sequence in E. coli adenylosuccinate synthetase plays a more important role in enzyme catalysis than in substrate binding. In addition, a hydrophobic amino acid residue such as isoleucine, leucine, or valine, rather than threonine, may play a critical role in GTP binding in adenosuccinate synthetase. These findings suggest that the glycine-rich sequence in adenylosuccinate synthetase functions differently relative to those in other GTP binding proteins or enzymes.     

Crystal Structures of Trypanosomal Histidyl-tRNA Synthetase Illuminate Differences between Eukaryotic and Prokaryotic Homologs

Crystal structures of histidyl-tRNA synthetase (HisRS) from the eukaryotic parasites Trypanosoma brucei and Trypanosoma cruzi provide a first structural view of a eukaryotic form of this enzyme and reveal differences from bacterial homologs. HisRSs in general contain an extra domain inserted between conserved motifs 2 and 3 of the Class II aminoacyl-tRNA synthetase catalytic core. The current structures show that the three-dimensional topology of this domain is very different in bacterial and archaeal/eukaryotic forms of the enzyme. Comparison of apo and histidine-bound trypanosomal structures indicates substantial active-site rearrangement upon histidine binding but relatively little subsequent rearrangement after reaction of histidine with ATP to form the enzyme's first reaction product, histidyladenylate. The specific residues involved in forming the binding pocket for the adenine moiety differ substantially both from the previously characterized binding site in bacterial structures and from the homologous residues in human HisRSs. The essentiality of the single HisRS gene in T. brucei is shown by a severe depression of parasite growth rate that results from even partial suppression of expression by RNA interference.     

Characterization of acetyl-CoA synthetase kinetics and ATP-binding

BackgroundThe superfamily of adenylating enzymes is a large family of enzymes broadly distributed from bacteria to humans. Acetyl-CoA synthetase (Acs), member of this family, is a metabolic enzyme with an essential role in Escherichia coli (E. coli) acetate metabolism, whose catalytic activity is regulated by acetylation/deacetylation in vivo.MethodsIn this study, the kinetics and thermodynamic parameters of deacetylated and acetylated E. coli Acs were studied for the adenylating step. Moreover, the role of the T264, K270, D500 and K609 residues in catalysis and ATP-binding was also determined by Isothermal titration calorimetry.ResultsThe results showed that native Acs enzyme binds ATP in an endothermic way. The dissociation constant has been determined and ATP-binding showed no significant differences between acetylated and deacetylated enzyme, although k_cat was much higher for the deacetylated enzyme. However, K609 lysine mutation resulted in an increase in ATP-Acs-affinity and in a total loss of enzymatic activity, while T264 and D500 mutant proteins showed a total loss of ATP-binding ability and a decrease in catalytic activity. K609 site-specified acetylation induced a change in Acs conformation which resulted in an exothermic and more energetic ATP-binding.ConclusionsThe differences in ATP-binding could explain the broadly conserved inactivation of Acs when K609 is acetylated.General SignificanceThe results presented in this study demonstrate the importance of the selected residues in Acs ATP-binding and represent an advance in our understanding of the adenylation step of the superfamily of adenylating enzymes and of their acetylation/deacetylation regulation.     

Recombinant mouse muscle adenylosuccinate synthetase: overexpression, kinetics, and crystal structure

Vertebrates possess two isozymes of adenylosuccinate synthetase. The acidic isozyme is similar to the synthetase from bacteria and plants, being involved in the de novo biosynthesis of AMP, whereas the basic isozyme participates in the purine nucleotide cycle. Reported here is the first instance of overexpression and crystal structure determination of a basic isozyme of adenylosuccinate synthetase. The recombinant mouse muscle enzyme purified to homogeneity in milligram quantities exhibits a specific activity comparable with that of the rat muscle enzyme isolated from tissue and K(m) parameters for GTP, IMP, and l-aspartate (12, 45, and 140 microm, respectively) similar to those of the enzyme from Escherichia coli. The mouse muscle and E. coli enzymes have similar polypeptide folds, differing primarily in the conformation of loops, involved in substrate recognition and stabilization of the transition state. Residues 65-68 of the muscle isozyme adopt a conformation not observed in any previous synthetase structure. In its new conformation, segment 65-68 forms intramolecular hydrogen bonds with residues essential for the recognition of IMP and, in fact, sterically excludes IMP from the active site. Observed differences in ligand recognition among adenylosuccinate synthetases may be due in part to conformational variations in the IMP pocket of the ligand-free enzymes.     

Homology Modeling of Adenylosuccinate Synthetase from Saccharomyces Cerevisiae Reveals a Possible Binding Region for Single-Stranded ARS Sequences

Abstract

Adenylosuccinate synthetase from Saccharomyces cerevisiae was investigated in order to find a structural explanation for its ability to bind specifically to single-stranded ARS elements (autonomously replicating sequences). Using the E. coli enzyme as template, a model for the structure of adenylosuccinate synthetase from S. cerevisiae was generated and subsequently refined by molecular dynamics techniques.

The resulting three-dimensional structure offers an explanation for the DNA binding activity of the yeast enzyme by revealing a distinct basic region that is not present in the homologous enzymes from other organisms.

The model is also in good agreement with biochemical data available for a mutant protein in which Glycine 252 is replaced by Aspartate. On the basis of the model a significant structural distortion near the catalytic center was predicted for this mutant, corresponding well to the enzymatic inactivity observed. The mutant enzyme shows larger structural fluctuations than the wild-type protein according to the results of two independent molecular dynamics simulations.     

Studies on active site mutants of P. falciparum adenylosuccinate synthetase: Insights into enzyme catalysis and activation

Adenylosuccinate synthetase catalyzes a reversible reaction utilizing IMP, GTP and aspartate in the presence of Mg²⁺ to form adenylosuccinate, GDP and inorganic phosphate. Comparison of similarly liganded complexes of Plasmodium falciparum, mouse and Escherichia coli AdSS reveals H-bonding interactions involving nonconserved catalytic loop residues (Asn429, Lys62 and Thr307) that are unique to the parasite enzyme. Site-directed mutagenesis has been used to examine the role of these interactions in catalysis and structural organization of P. falciparum adenylosuccinate synthetase (PfAdSS). Mutation of Asn429 to Val, Lys62 to Leu and Thr307 to Val resulted in an increase in K_m values for IMP, GTP and aspartate, respectively along with a 5 fold drop in the k_cat value for N429V mutant suggesting the role of these residues in ligand binding and/or catalysis. We have earlier shown that the glycolytic intermediate, fructose 1,6 bisphosphate, which is an inhibitor of mammalian AdSS is an activator of the parasite enzyme. Enzyme kinetics along with molecular docking suggests a mechanism for activation wherein F16BP seems to be binding to the Asp loop and inducing a conformation that facilitates aspartate binding to the enzyme active site. Like in other AdSS, a conserved arginine residue (Arg155) is involved in dimer crosstalk and interacts with IMP in the active site of the symmetry related subunit of PfAdSS. We also report on the biochemical characterization of the arginine mutants (R155L, R155K and R155A) which suggests that unlike in E. coli AdSS, Arg155 in PfAdSS influences both ligand binding and catalysis.     

Cloning and sequence analysis ofglnB-like gene from nitrogen-fixingPaenibacillus polymyxa G2

A 240 bpglnB-like gene fragment was PCR amplified fromPaenibacillus polymyxa G2 using universal degenerate primers. These degenerate primers, based on all knownglnB-like genes including thenrgB ofBacillus subtilis at the time of design, were chosen from highly conserved amino acid sequences. The GlnB-like sequence ofP. polymyxa G2 had relatively high identities (more than 70%) to other bacteria GlnB, e.g.Escherichia coli (78%) andKlebsiella pneuomoniae (79%). However, the identity of theP. polymyxa GlnB-like sequence to a GlnK homologue (NrgB) of B.subtilis was low (46%). This is the first report of the sequence of theglnB-like gene from the genusPaenibacillus. Knowledge of theglnB-like gene sequence ofP. polymyxa will make us study deeply the function ofglnB in the genusPaenibacillus.     

Peptidyl-tRNA hydrolase from Sulfolobus solfataricus

An enzyme capable of liberating functional tRNA^Lys from Escherichia coli diacetyl-lysyl-tRNA^Lys was purified from the archae Sulfolobus solfataricus. Contrasting with the specificity of peptidyl- tRNA hydrolase (PTH) from E.coli, the S.solfataricus enzyme readily accepts E.coli formyl-methionyl-tRNA^fMet as a substrate. N-terminal sequencing of this enzyme identifies a gene that has homologs in the whole archaeal kingdom. Involvement of this gene (SS00175) in the recycling of peptidyl-tRNA is supported by its capacity to complement an E.coli strain lacking PTH activity. The archaeal gene, the product of which appears markedly different from bacterial PTHs, also has homologs in all the available eukaryal genomes. Since most of the eukaryotes already display a bacterial-like PTH gene, this observation suggests the occurrence in many eukaryotes of two distinct PTH activities, either of a bacterial or of an archaeal type. Indeed, the bacterial- and archaeal-like genes encoding the two full-length PTHs of Saccharomyces cerevisiae, YHR189w and YBL057c, respectively, can each rescue the growth of an E.coli strain lacking endogeneous PTH. In vitro assays confirm that the two enzymes ensure the recycling of tRNA^Lys from diacetyl-lysyl-tRNA^Lys. Finally, the growth of yeast cells in which either YHR189w or YBL057c has been disrupted was compared under various culture conditions. Evidence is presented that YHR189w, the gene encoding a bacterial-like PTH, should be involved in mitochondrial function.     

Micrococcus luteus homolog of theEscherichia coli uvrA gene: Identification of a mutation in the UV-sensitive mutant DB7

Summary Restriction fragments ofMicrococcus luteus DNA containing the gene affected by a mutation in the UV-sensitive mutant DB7 were cloned both from the wild type and from the mutant in anEscherichia coli host-vector system. The wild-type fragment was able to reverse the multiple sensitivity of the mutant to UV, mitomycin C, and 4-nitroquinoline 1-oxide by a one-step transformation. Determination of the nucleotide sequences revealed a potential open reading frame coding for a protein of 992 (tentative) amino acid residues, within which the DB7 mutation was identified as a CG-to-TA transition causing a translation termination. The putative product of the open reading frame shares an extensive amino acid sequence homology with theE. coli UvrA protein comprising 940 residues. The homology extends over the greater part of both polypeptides except for two extra sequences of 31 and 24 amino acid residues located at the amino-terminal and in the interior, respectively, of theM. luteus protein. In the homologous region, 56.7% and 16.7% of the 933 pairs of the aligned amino acids were accounted for by conserved residues and conservative substitutions, respectively. These results indicate that the gene defined by the mutation in DB7 represents a homolog of theE. coli uvrA gene. Hence, it has to be concluded that DB7, known for its deficiency in UV endonuclease (pyrimidine dimer DNA glycosylase/apurinic-apyrimidinic endonuclease) activity, is a double mutant which is also defective in an enzyme complex similar to theE. coli UvrABC excinuclease. Dedicated to the memory of Shunzo Okubo (1930–1978), who considered the possibility of a double-mutant status for the mutant DB7 most seriously     

Control of feedback repression of the enzymes of purine biosynthesis in Aerobacter aerogenes

Studies have been made of the regulation of the synthesis of six purine biosynthetic enzymes: P-ribosyl-PP amidotransferase (I), P-ribosyl glycinamide synthetase (II), P-ribosyl formyl glycinamide amidotransferase (IV), adenylosuccinate lyase (VIII-IIA), adenylosuccinate synthetase (IA), and IMP dehydrogenase (IG). Wild type Aerobacter aerogenes and two purine requiring mutants derived from it, were grown with limiting or excess adenine or guanine, cell extracts prepared, and enzyme activities measured.     

The phylogenetic distribution of bacterial ribonucleases

Ribonucleases play key, often essential, roles in cellular metabolism. Nineteen ribonuclease activities, from 22 different proteins, have so far been described in bacteria, the majority of them from either Escherichia coli or Bacillus subtilis. Here we examine the phylogenetic distribution of all of these ribonucleases in 50 eubacterial and archaeal species whose genomes have been completely sequenced, with particular emphasis on the endoribonucleases. Although some enzymes are very highly conserved throughout evolution, there appears to be no truly universal ribonuclease. While some organisms, like E.coli, have a large selection of ribonucleases, many with overlapping functions, others seem to have relatively few or have many that remain to be discovered.     

Molecular cloning and characterization of a novel muscle adenylosuccinate synthetase,AdSSL1, from human bone marrow stromal cells

Vertebrates have muscle and non-muscle isozymes of adenylosuccinate synthetase (AdSS, EC 6.3.4.4), which catalyzes the first committed step in AMP synthesis. A novel muscle isozyme of adenylosuccinate synthetase, human AdSSL1, is identified from human bone marrow stromal cells. AdSSL1 is 98% identical to mouse muscle type AdSS1 and contains conserved sequence and structural features of adenylosuccinate synthetase. Human AdSSL1 gene is mapped to chromosome 14p32.33. After stimulation, leukemia cells express AdSSL1 in a time-dependent manner different from that of non-muscle adenylosuccinate synthetase. The human AdSSL1 is predominantly expressed in skeletal muscle and cardiac tissue consistent with the potential role for the enzyme in muscle metabolism. Overexpressed AdSSL1 protein in COS-7 cells locates in cytoplasm. Recombinant AdSSL1 protein possesses typical enzymatic activity to catalyze adenylosuccinate formation. The identification of human AdSSL1 with predominant expression in muscle tissue will facilitate future genetic and biochemical analysis of the enzyme in muscle physiology. (Mol Cell Biochem 269: 85–94, 2005)     

The α-barrel tip region of Escherichia coli TolC homologs of Vibrio vulnificus interacts with the MacA protein to form the functional macrolide-specific efflux pump MacAB-TolC

TolC and its homologous family of proteins are outer membrane factors that are essential for exporting small molecules and toxins across the outer membrane in Gram-negative bacteria. Two open reading frames in the Vibrio vulnificus genome that encode proteins homologous to Escherichia coli TolC, designated TolCV1 and TolCV2, have 51.3% and 29.6% amino acid identity to TolC, respectively. In this study, we show that TolCV1 and TolCV2 functionally and physically interacted with the membrane fusion protein, MacA, a component of the macrolide-specific MacAB-TolC pump of E. coli. We further show that the conserved residues located at the aperture tip region of the α-hairpin of TolCV1 and TolCV2 played an essential role in the formation of the functional MacAB-TolC pump using site-directed mutational analyses. Our findings suggest that these outer membrane factors have conserved tip-to-tip interaction with the MacA membrane fusion protein for action of the drug efflux pump in Gramnegative bacteria.     

Molecular characterization of Arabidopsis thaliana cDNAs encoding three purine biosynthetic enzymes

Glycinamide ribonucleotide (GAR) synthetase, GAR transformylase and aminoimidazole ribonucleotide (AIR) synthetase are the second, third and fifth enzymes in the 10-step de novo purine biosynthetic pathway. From a cDNA library of Arabidopsis thaliana, cDNAs encoding the above three enzymes were cloned by functional complementation of corresponding Escherichia coli mutants. Each of the cDNAs encode peptides comprising the complete enzymatic domain of either GAR synthetase, GAR transformylase or AIR synthetase. Comparisons of the three Arabidopsis purine biosynthetic enzymes with corresponding enzymes/polypeptide-fragments from procaryotic and eucaryotic sources indicate a high degree of conserved homology at the amino acid level, in particular with procaryotic enzymes. Assays from extracts of E. coli expressing the complementing clones verified the specific enzymatic activity of Arabidopsis GAR synthetase and GAR transformylase. Sequence analysis, as well as Northern blot analysis indicate that Arabidopsis has single and monofunctional enzymes. In this respect the organization of these three plant purine biosynthesis genes is fundamentally different from the multifunctional purine biosynthesis enzymes characteristic of other eucaryotes and instead resembles the one gene, one enzyme relationship found in procaryotes.     

Bacteria possessing two RelA/SpoT-like proteins have evolved a specific stringent response involving the acyl carrier protein-SpoT interaction

Bacteria respond to nutritional stress by producing (p)ppGpp, which triggers a stringent response resulting in growth arrest and expression of resistance genes. In Escherichia coli, RelA produces (p)ppGpp upon amino acid starvation by detecting stalled ribosomes. The SpoT enzyme responds to various other types of starvation by unknown mechanisms. We previously described an interaction between SpoT and the central cofactor of lipid synthesis, acyl carrier protein (ACP), which is involved in detecting starvation signals in lipid metabolism and triggering SpoT-dependent (p)ppGpp accumulation. However, most bacteria possess a unique protein homologous to RelA/SpoT (Rsh) that is able to synthesize and degrade (p)ppGpp and is therefore more closely related to SpoT function. In this study, we asked if the ACP-SpoT interaction is specific for bacteria containing two RelA and SpoT enzymes or if it is a general feature that is conserved in Rsh enzymes. By testing various combinations of SpoT, RelA, and Rsh enzymes and ACPs of E. coli, Pseudomonas aeruginosa, Bacillus subtilis and Streptococcus pneumoniae, we found that the interaction between (p)ppGpp synthases and ACP seemed to be restricted to SpoT proteins of bacteria containing the two RelA and SpoT proteins and to ACP proteins encoded by genes located in fatty acid synthesis operons. When Rsh enzymes from B. subtilis and S. pneumoniae are produced in E. coli, the behavior of these enzymes is different from the behavior of both RelA and SpoT proteins with respect to (p)ppGpp synthesis. This suggests that bacteria have evolved several different modes of (p)ppGpp regulation in order to respond to nutrient starvation.     

Solution structure and activation mechanism of ubiquitin-like small archaeal modifier proteins

In archaea, two ubiquitin-like small archaeal modifier protein (SAMPs) were recently shown to be conjugated to proteins in vivo. SAMPs display homology to bacterial MoaD sulfur transfer proteins and eukaryotic ubiquitin-like proteins, and they share with them the conserved C-terminal glycine-glycine motif. Here, we report the solution structure of SAMP1 from Methanosarcina acetivorans and the activation of SAMPs by an archaeal protein with homology to eukaryotic E1 enzymes. Our results show that SAMP1 possesses a β-grasp fold and that its hydrophobic and electrostatic surface features are similar to those of MoaD. M. acetivorans SAMP1 exhibits an extensive flexible surface loop between helix-2 and the third strand of the β-sheet, which contributes to an elongated surface groove that is not observed in bacterial ubiquitin homologues and many other SAMPs. We provide in vitro biochemical evidence that SAMPs are activated in an ATP-dependent manner by an E1-like enzyme that we have termed E1-like SAMP activator (ELSA). We show that activation occurs by formation of a mixed anhydride (adenylate) at the SAMP C-terminus and is detectable by SDS-PAGE and electrospray ionization mass spectrometry.     

Cloning and characterization of theNeisseria gonorrhoeae MS11folC gene

The gene coding for folylpoly-(γ)-glutamate synthetase (FPGS)-dihydrofolate synthetase (DHFS) ofNeisseria gonorrhoeae (Ngo) has been cloned by functional complementation of anEscherichia coli folC mutant (SF4). The sequence encodes a 224-residue protein of 46.4 kDa. It shows 46% identity to theE. coli FPGS-DHFS and 29% identity to the PFGS ofLactobacillus casei. Sequence comparisons between the three genes reveal regions of high homology, including ATP binding sites required for bifunctionality, all of which may be important for FPGS activity. In contrast toL. casei FPGS, theE. coli andNgo enzymes share some additional regions which may be essential for DHFS activity. The products ofNgo folC and flanking genes were monitored by T7 promoter expression. Interestingly, deletion of the upstreamfolI gene, which encodes a 16.5 kDa protein, abolishes the capacity offolC to complementE. coli SF4 to the wild-type phenotype. The ability to complement can be restored byfolI providedin trans. UnlikefolC mutants, gonococcalfolI mutants are viable and display no apparent phenotype. Thus, in contrast toE. coli, Ngo folC is expressed at a sufficiently high level from its own promoter, in the absence of FolI. This study provides the first insights into the genetic complexity of one-carbon metabolism inNgo.