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1.  Escherichia coli purB gene: cloning, nucleotide sequence, and regulation by purR. 
Journal of Bacteriology  1992;174(1):130-136.
Escherichia coli purB encodes adenylosuccinate lyase (ASL), the enzyme that catalyzes step 8 in the pathway for de novo synthesis of IMP and also the final reaction in the two-step sequence from IMP to AMP. Gene purB was cloned and found to encode an ASL protein of 435 amino acids having a calculated molecular weight of 49,225. E. coli ASL is homologous to the corresponding enzymes from Bacillus subtilis and chickens and also to fumarase from B. subtilis. Gene phoP is 232 bp downstream of purB. Gene purB is regulated threefold by the purine pool and purR. Transcriptional regulation of purB involves binding of the purine repressor to the 16-bp conserved pur regulon operator. The purB operator is 224 bp downstream of the transcription start site and overlaps codons 62 to 67 in the protein-coding sequence.
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PMCID: PMC205686  PMID: 1729205
2.  Genetic Screen Reveals the Role of Purine Metabolism in Staphylococcus aureus Persistence to Rifampicin 
Antibiotics  2015;4(4):627-642.
Chronic infections with Staphylococcus aureus such as septicemia, osteomyelitis, endocarditis, and biofilm infections are difficult to treat because of persisters. Despite many efforts in understanding bacterial persistence, the mechanisms of persister formation in S. aureus remain elusive. Here, we performed a genome-wide screen of a transposon mutant library to study the molecular mechanisms involved in persistence of community-acquired S. aureus. Screening of the library for mutants defective in persistence or tolerance to rifampicin revealed many genes involved in metabolic pathways that are important for antibiotic persistence. In particular, the identified mutants belonged to metabolic pathways involved in carbohydrate, amino acid, lipid, vitamin and purine biosynthesis. Five mutants played a role in purine biosynthesis and two mutants, purB, an adenylosuccinate lyase, and purM, a phosphoribosylaminoimidazole synthetase, were selected for further confirmation. Mutants purB and purM showed defective persistence compared to the parental strain USA300 in multiple stress conditions including various antibiotics, low pH, and heat stress. The defect in persistence was restored by complementation with the wildtype purB and purM gene in the respective mutants. These findings provide new insights into the mechanisms of persistence in S. aureus and provide novel therapeutic targets for developing more effective treatment for persistent infections due to S. aureus.
doi:10.3390/antibiotics4040627
PMCID: PMC4790316  PMID: 27025643
Staphylococcus aureus; persisters; purines; rifampicin
3.  Structural and biochemical characterization of N 5-­carboxyaminoimidazole ribonucleotide synthetase and N 5-carboxyaminoimidazole ribonucleotide mutase from Staphylococcus aureus  
Purine biosynthesis is considered to be a promising new antibiotic target; in this article, the structural and biochemical characterization of S. aureus PurK and PurE are described.
With the rapid rise of methicillin-resistant Staphylococcus aureus infections, new strategies against S. aureus are urgently needed. De novo purine biosynthesis is a promising yet unexploited target, insofar as abundant evidence has shown that bacteria with compromised purine biosynthesis are attenuated. Fundamental differences exist within the process by which humans and bacteria convert 5-aminoimidazole ribonucleotide (AIR) to 4-carboxy-5-aminoimidazole ribo­nucleotide (CAIR). In bacteria, this transformation occurs through a two-step conversion catalyzed by PurK and PurE; in humans, it is mediated by a one-step conversion catalyzed by class II PurE. Thus, these bacterial enzymes are potential targets for selective antibiotic development. Here, the first comprehensive structural and biochemical characterization of PurK and PurE from S. aureus is presented. Structural analysis of S. aureus PurK reveals a nonconserved phenylalanine near the AIR-binding site that occupies the putative position of the imidazole ring of AIR. Mutation of this phenylalanine to isoleucine or tryptophan reduced the enzyme efficiency by around tenfold. The K m for bicarbonate was determined for the first time for a PurK enzyme and was found to be ∼18.8 mM. The structure of PurE is described in comparison to that of human class II PurE. It is confirmed biochemically that His38 is essential for function. These studies aim to provide foundations for future structure-based drug-discovery efforts against S. aureus purine biosynthesis.
doi:10.1107/S0907444911023821
PMCID: PMC3144853  PMID: 21795812
purine biosynthesis; Staphylococcus aureus; enzyme kinetics; structure-based drug discovery; antimicrobials
4.  Purine Salvage Pathways among Borrelia Species▿  
Infection and Immunity  2007;75(8):3877-3884.
Genome sequencing projects on two relapsing fever spirochetes, Borrelia hermsii and Borrelia turicatae, revealed differences in genes involved in purine metabolism and salvage compared to those in the Lyme disease spirochete Borrelia burgdorferi. The relapsing fever spirochetes contained six open reading frames that are absent from the B. burgdorferi genome. These genes included those for hypoxanthine-guanine phosphoribosyltransferase (hpt), adenylosuccinate synthase (purA), adenylosuccinate lyase (purB), auxiliary protein (nrdI), the ribonucleotide-diphosphate reductase alpha subunit (nrdE), and the ribonucleotide-diphosphate reductase beta subunit (nrdF). Southern blot assays with multiple Borrelia species and isolates confirmed the presence of these genes in the relapsing fever group of spirochetes but not in B. burgdorferi and related species. TaqMan real-time reverse transcription-PCR demonstrated that the chromosomal genes (hpt, purA, and purB) were transcribed in vitro and in mice. Phosphoribosyltransferase assays revealed that, in general, B. hermsii exhibited significantly higher activity than did the B. burgdorferi cell lysate, and enzymatic activity was observed with adenine, hypoxanthine, and guanine as substrates. B. burgdorferi showed low but detectable phosphoribosyltransferase activity with hypoxanthine even though the genome lacks a discernible ortholog to the hpt gene in the relapsing fever spirochetes. B. hermsii incorporated radiolabeled hypoxanthine into RNA and DNA to a much greater extent than did B. burgdorferi. This complete pathway for purine salvage in the relapsing fever spirochetes may contribute, in part, to these spirochetes achieving high cell densities in blood.
doi:10.1128/IAI.00199-07
PMCID: PMC1952022  PMID: 17502392
5.  Structural Biology of the Purine Biosynthetic Pathway 
Purine biosynthesis requires ten enzymatic transformations to generate inosine monophosphate. PurF, PurD, PurL, PurM, PurC, and PurB are common to all pathways, while PurN or PurT, PurK/PurE-I or PurE-II, PurH or PurP, and PurJ or PurO catalyze the same steps in different organisms. X-ray crystal structures are available for all 15 purine biosynthetic enzymes, including seven ATP-dependent enzymes, two amidotransferases and two tetrahydrofolate-dependent enzymes. Here we summarize the structures of the purine biosynthetic enzymes, discuss similarities and differences, and present arguments for pathway evolution. Four of the ATP-dependent enzymes belong to the ATP-grasp superfamily and two to the PurM superfamily. The amidotransferases are unrelated with one utilizing an NTN-glutaminase and the other utilizing a triad glutaminase. Likewise the tetrahydrofolate-dependent enzymes are unrelated. Ancestral proteins may have included a broad specificity enzyme instead of PurD, PurT, PurK, PurC, and PurP, and a separate enzyme instead of PurM and PurL.
doi:10.1007/s00018-008-8295-8
PMCID: PMC2596281  PMID: 18712276
purine biosynthesis; protein evolution; ATP-grasp superfamily; PurM superfamily; amidotransferases
6.  Chromosomal mapping of a gene affecting enterotoxin A production in Staphylococcus aureus. 
In a previous study, transformation demonstrated that a gene governing enterotoxin A production (entA+) in Staphylococcus aureus strain S-6 was located on the chromosome between the purB110 and ilv-129 markers; in contrast, the entA+ gene of strain FRI-196E was shown not to be located in the same position. In the current study, 54 enterotoxin A-producing strains of S. aureus were examined to locate the entA+ gene. Conventional transformation procedures and a series of multiply marked derivatives of NCTC 8325 were used as recipients for chromosomal mapping. Of the 54 strains tested, 23 were found to contain the entA+ gene at the original locus between the purB110 and ilv-129 markers. Twenty-seven strains could not be analyzed either because their DNA was genetically ineffective in transforming strain 8325 (23 strains), or Pur+ Ilv+ transformants could not be recovered (four strains). Four other strains contained an entA+ gene that could not be located in any of the chromosomal linkage groups. A new insertion site for Tn551 was located within the hla+ gene involved in alpha-toxin production. It eliminated alpha-toxin production and was used to separate the entA+ gene from the hla+ marker in the purB110-ilv-129 region. This segment of the chromosome is shown to consist of the purB110, entA+, hla+, and ilv-129 markers in that order.
PMCID: PMC241838  PMID: 6277247
7.  The structure of phosphate-bound Escherichia coli adenylosuccinate lyase identifies His171 as a catalytic acid 
Here, the crystal structure of adenylosuccinate lyase from Escherichia coli was determined to 1.9 Å resolution.
Adenylosuccinate lyase (ASL) is an enzyme from the purine-biosynthetic pathway that catalyzes the cleavage of 5-aminoimidazole-4-(N-succinylcarboxamide) ribonucleotide (SAICAR) to 5-aminoimidazole-4-carboxamide ribo­nucleotide (AICAR) and fumarate. ASL is also responsible for the conversion of succinyladenosine monophosphate (SAMP) to adenosine monophosphate (AMP) and fumarate. Here, the crystal structure of adenylosuccinate lyase from Escherichia coli was determined to 1.9 Å resolution. The enzyme adopts a substrate-bound conformation as a result of the presence of two phosphate ions bound in the active site. Comparison with previously solved structures of the apoenzyme and an SAMP-bound H171A mutant reveals a conformational change at His171 associated with substrate binding and confirms the role of this residue as a catalytic acid.
doi:10.1107/S1744309109029674
PMCID: PMC2795585  PMID: 19724117
adenylosuccinate lyase; ASL; PurB; purine biosynthesis
8.  A Novel Function for the NTN Hydrolase Fold Demonstrated by the Structure of an Archeal Inosine Monophosphate Cyclohydrolase†,‡ 
Biochemistry  2007;46(17):5050-5062.
Inosine 5′-monophosphate (IMP) cyclohydrolase catalyzes the cyclization of 5-formaminoimidazole-4-carboxamide ribonucleotide (FAICAR) to IMP in the final step of de novo purine biosynthesis. Two major types of this enzyme have been discovered to date: PurH in Bacteria and Eukarya, and PurO in Archaea. The structure of the MTH1020 gene product from Methanothermobacter thermoautotrophicus was previously solved without functional annotation but shows high amino acid sequence similarity to other PurOs. We determined the crystal structure of the MTH1020 gene product in complex with either IMP or 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) at 2.0 Å and 2.6 Å resolution, respectively. Based on the sequence analysis, ligand-bound structures, and biochemical data, MTH1020 is confirmed as an archaeal IMP cyclohydrolase, thus designated as MthPurO. MthPurO has a four-layered αββα core structure, showing an N-terminal nucleophile (NTN) hydrolase fold. The active site is located at the deep pocket between two central β-sheets and contains residues strictly conserved within PurOs. Comparisons of the two types of IMP cyclohydrolase, PurO and PurH, revealed that there are no similarities in sequence, structure, or the active site architecture, suggesting that they are evolutionarily not related to each other. The MjR31K mutant of PurO from Methanocaldococcus jannaschii showed 76% decreased activity and MjE102Q mutation completely abolished enzymatic activity, suggesting that these highly conserved residues play critical roles in catalysis. Interestingly, green fluorescent protein (GFP), which has no structural homology to either PurO or PurH but catalyzes a similar intramolecular cyclohydrolase reaction required for chromophore maturation, utilizes Arg96 and Glu222 in a mechanism analogous to that of PurO.
doi:10.1021/bi061637j
PMCID: PMC2631436  PMID: 17407260
9.  In Vitro Evolution of an Archetypal Enteropathogenic Escherichia coli Strain 
Journal of Bacteriology  2013;195(19):4476-4483.
Enteropathogenic Escherichia coli (EPEC) is a leading cause of infantile diarrhea in developing countries. EPEC strain E2348/69 is used worldwide as a prototype to study EPEC genetics and disease. However, isolates of E2348/69 differ phenotypically, reflecting a history of in vitro selection. To identify the genomic and phenotypic changes in the prototype strain, we sequenced the genome of the nalidixic acid-resistant (Nalr) E2348/69 clone. We also sequenced a recent nleF mutant derived by one-step PCR mutagenesis from the Nalr strain. The sequencing results revealed no unintended changes between the mutant and the parent strain. However, loss of the pE2348-2 plasmid and 3 nonsynonymous mutations were found in comparison to the published streptomycin-resistant (Strr) E2348/69 reference genome. One mutation is a conservative amino acid substitution in ftsK. Another, in gyrA, is a mutation known to result in resistance to nalidixic acid. The third mutation converts a stop codon to a tryptophan, predicted to result in the fusion of hflD, the lysogenization regulator, to purB. The purB gene encodes an adenylosuccinate lyase involved in purine biosynthesis. The Nalr clone has a lower growth rate than the Strr isolate when cultured in minimal media, a difference which is corrected upon addition of adenine or by genetic complementation with purB. Addition of adenine or genetic complementation also restored the invasion efficiency of the Nalr clone. This report reconciles longstanding inconsistencies in phenotypic properties of an archetypal strain and provides both reassurance and cautions regarding intentional and unintentional evolution in vitro.
doi:10.1128/JB.00704-13
PMCID: PMC3807458  PMID: 23913321
10.  Substrate and Product Complexes of Escherichia coli Adenylosuccinate Lyase Provide New Insights into the Enzymatic Mechanism 
Journal of molecular biology  2007;370(3):541-554.
Adenylosuccinate lyase (ADL) catalyzes the breakdown of 5-aminoimida-zole- (N-succinylocarboxamide) ribotide (SAICAR) to 5-aminoimidazole-4-carboxamide ribotide (AICAR) and fumarate, and of adenylosuccinate (ADS) to adenosine monophosphate (AMP) and fumarate in the de novo purine biosynthetic pathway. ADL belongs to the argininosuccinate lyase (ASL)/fumarase C superfamily of enzymes. Members of this family share several common features including: a mainly α-helical, homotetrameric structure; three regions of highly conserved amino acid residues; and a general acid-base catalytic mechanism with the overall β-elimination of fumarate as a product. The crystal structures of wild-type Escherichia coli ADL (ec-ADL), and mutant-substrate (H171A-ADS) and -product (H171N-AMP•FUM) complexes have been determined to 2.0, 1.85, and 2.0 Å resolution, respectively. The H171A-ADS and H171N-AMP•FUM structures provide the first detailed picture of the ADL active site, and have enabled the precise identification of substrate binding and putative catalytic residues. Contrary to previous suggestions, the ec-ADL structures implicate S295 and H171 in base and acid catalysis, respectively. Furthermore, structural alignments of ec-ADL with other superfamily members suggest for the first time a large conformational movement of the flexible C3 loop (residues 287–303) in ec-ADL upon substrate binding and catalysis, resulting in its closure over the active site. This loop movement has been observed in other superfamily enzymes, and has been proposed to be essential for catalysis. The ADL catalytic mechanism is re-examined in light of the results presented here.
doi:10.1016/j.jmb.2007.04.052
PMCID: PMC4113493  PMID: 17531264 CAMSID: cams4571
adenylosuccinate lyase; purine biosynthesis; β-elimination; argininosuccinate lyase/fumarase C superfamily
11.  Regulation of Escherichia coli purA by purine repressor, one component of a dual control mechanism. 
Journal of Bacteriology  1994;176(4):1009-1013.
Escherichia coli purA encodes adenylosuccinate synthetase, one of two enzymes required for synthesis of AMP from IMP. purA is subject to two- to threefold regulation by purR and about twofold regulation by a purR-independent mechanism. The 5'-flanking region of purA confers purR-dependent transcriptional regulation of purA but not the purR-independent regulation. Two operator sites in the 5'-flanking region which bind purine repressor in vitro and are required for in vivo regulation were identified. The purR-independent regulation may be posttranscriptional. It is now established that all transcription units involved in de novo synthesis of purine nucleotides, nine pur operons, as well as purR itself and guaBA, are subject to purR control.
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PMCID: PMC205151  PMID: 8106311
12.  Genes of the Escherichia coli pur regulon are negatively controlled by a repressor-operator interaction. 
Journal of Bacteriology  1990;172(8):4555-4562.
Fusions of lacZ were constructed to genes in each of the loci involved in de novo synthesis of IMP. The expression of each pur-lacZ fusion was determined in isogenic purR and purR+ strains. These measurements indicated 5- to 17-fold coregulation of genes purF, purHD, purC, purMN, purL, and purEK and thus confirm the existence of a pur regulon. Gene purB, which encodes an enzyme involved in synthesis of IMP and in the AMP branch of the pathway, was not regulated by purR. Each locus of the pur regulon contains a 16-base-pair conserved operator sequence that overlaps with the promoter. The purR product, purine repressor, was shown to bind specifically to each operator. Thus, binding of repressor to each operator of pur regulon genes negatively coregulates expression.
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PMCID: PMC213288  PMID: 2198266
13.  Formylglycinamide Ribonucleotide Amidotransferase from Thermotoga maritima: Structural Insights into Complex Formation†‡ 
Biochemistry  2008;47(30):7816-7830.
In the fourth step of the purine biosynthetic pathway, formyl glycinamide ribonucleotide (FGAR) amidotransferase, also known as PurL, catalyzes the conversion of FGAR, ATP, and glutamine to formyl glycinamidine ribonucleotide (FGAM), ADP, Pi, and glutamate. Two forms of PurL have been characterized, large and small. Large PurL, present in most Gram-negative bacteria and eukaryotes, consists of a single polypeptide chain and contains three major domains: the N-terminal domain, the FGAM synthetase domain, and the glutaminase domain, with a putative ammonia channel located between the active sites of the latter two. Small PurL, present in Gram-positive bacteria and archaea, is structurally homologous to the FGAM synthetase domain of large PurL, and forms a complex with two additional gene products, PurQ and PurS. The structure of the PurS dimer is homologous with the N-terminal domain of large PurL, while PurQ, whose structure has not been reported, contains the glutaminase activity. In Bacillus subtilis, the formation of the PurLQS complex is dependent on glutamine and ADP and has been demonstrated by size-exclusion chromatography. In this work, a structure of the PurLQS complex from Thermotoga maritima is described revealing a 2:1:1 stoichiometry of PurS:Q:L, respectively. The conformational changes observed in TmPurL upon complex formation elucidate the mechanism of metabolite-mediated recruitment of PurQ and PurS. The flexibility of the PurS dimer is proposed to play a role in the activation of the complex and the formation of the ammonia channel. A potential path for the ammonia channel is identified.
doi:10.1021/bi800329p
PMCID: PMC2646663  PMID: 18597481
14.  Complexed Structures of Formylglycinamide Ribonucleotide Amidotransferase from Thermotoga maritima Describe a Novel ATP-binding Protein Superfamily†,‡ 
Biochemistry  2006;45(50):14880-14895.
Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) catalyzes the ATP-dependent synthesis of formylglycinamidine ribonucleotide (FGAM) from formylglycinamide ribonucleotide (FGAR) and glutamine in the fourth step of the purine biosynthetic pathway. FGAR-AT is encoded by the purL gene. Two types of PurL have been detected. The first type, found in eukaryotes and Gram-negative bacteria, consists of a single 140 kDa polypeptide chain and is designated large PurL (lgPurL). The second type, small PurL (smPurL), is found in archaea and Gram-positive bacteria and consists of an 80 kDa polypeptide chain. Small PurL requires two additional gene products, PurQ and PurS, for activity. PurL is a member of a protein superfamily that contains a novel ATP-binding domain. Structures of several members of this superfamily are available in the apo form. We determined five different structures of FGAR-AT from Thermotoga maritima in the presence of substrates, a substrate analog, and a product. These complexes have allowed a detailed description of the novel ATP-binding motif. Availability of a ternary complex enabled mapping of the active site thus identifying potential residues involved in catalysis. The complexes show a conformational change in the active site compared to the unliganded structure. A surprising discovery, an ATP molecule in an auxiliary site of the protein and the conformational changes associated with its binding, provoke speculations about the regulatory role of the auxiliary site in PurLSQ complex formation as well as the evolutionary relationship of PurL's from different organisms.
doi:10.1021/bi061591u
PMCID: PMC2527724  PMID: 17154526
15.  Structure of N 5-carboxyaminoimidazole ribonucleotide synthase (PurK) from Bacillus anthracis  
The crystal structure of N 5-carboxyaminoimidazole ribonucleotide synthase from Bacillus anthracis with only an Mg cation provides some insight into the catalytic mechanism of this enzyme and the role of a crucial loop during catalysis.
The apo structure of N 5-carboxyaminoimidazole ribonucleotide synthase (PurK) from Bacillus anthracis (baPurK) with Mg2+ in the active site is reported at 1.96 Å resolution. PurK is an enzyme in the purine-biosynthetic pathway, unique to prokaryotes, that converts 5-aminoimidazole ribonucleotide to N 5-carboxyaminoimidazole ribonucleotide and has been suggested as a potential antimicrobial drug target. Two interesting features of baPurK are a flexible B-loop (residues 149/150–157) that is in close contact with the active site and the binding of Mg2+ to the active site without additional ligands.
doi:10.1107/S0907444911029210
PMCID: PMC3270386  PMID: 21931218
N5-carboxy­aminoimidazole ribonucleotide synthase; PurK; Bacillus anthracis; purine biosynthesis
16.  Repression of Escherichia coli purB is by a transcriptional roadblock mechanism. 
Journal of Bacteriology  1992;174(22):7121-7127.
Escherichia coli purB is regulated by a repressor-operator interaction. The purB operator is 242 bp downstream from the transcription start site and overlaps condons 62 to 67 in the protein-coding sequence (B. He, J. M. Smith, and H. Zalkin, J. Bacteriol. 174:130-136, 1992). The mechanism by which the repressor-operator interaction functions to repress transcription was investigated by a combination of promoter replacement experiments and RNA analyses. By using a trp promoter replacement that deleted 5' flanking DNA to position -986, purB expression was increased sevenfold, yet normal two- to threefold regulation was maintained. This indicates that repressor-operator control is independent of the purB promoter and other 5' flanking sequences. Transcriptional regulation was likewise independent of coupled translation. An approximately 260-nucleotide truncated in vivo purB mRNA was identified which was dependent upon repressor-operator interaction. Thus, binding of purine repressor to the purB operator inhibits transcription elongation by a roadblock mechanism. The roadblock was not influenced by a sevenfold increase in promoter strength or by an operator mutation resulting in a 2.5-fold increase in repressor-operator affinity.
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PMCID: PMC207401  PMID: 1429435
17.  Molecular Characterization of Borrelia persica, the Agent of Tick Borne Relapsing Fever in Israel and the Palestinian Authority 
PLoS ONE  2010;5(11):e14105.
The identification of the Tick Borne Relapsing Fever (TBRF) agent in Israel and the Palestinian Authority relies on the morphology and the association of Borrelia persica with its vector Ornithodoros tholozani. Molecular based data on B. persica are very scarce as the organism is still non-cultivable. In this study, we were able to sequence three complete 16S rRNA genes, 12 partial flaB genes, 18 partial glpQ genes, 16 rrs-ileT intergenic spacers (IGS) from nine ticks and ten human blood samples originating from the West Bank and Israel. In one sample we sequenced 7231 contiguous base pairs that covered completely the region from the 5′end of the 16S rRNA gene to the 5′end of the 23S rRNA gene comprising the whole 16S rRNA (rrs), and the following genes: Ala tRNA (alaT), Ile tRNA (ileT), adenylosuccinate lyase (purB), adenylosuccinate synthetase (purA), methylpurine-DNA glycosylase (mag), hypoxanthine-guanine phosphoribosyltransferase (hpt), an hydrolase (HAD superfamily) and a 135 bp 5′ fragment of the 23S rRNA (rrlA) genes. Phylogenic sequence analysis defined all the Borrelia isolates from O. tholozani and from human TBRF cases in Israel and the West Bank as B. persica that clustered between the African and the New World TBRF species. Gene organization of the intergenic spacer between the 16S rRNA and the 23S rRNA was similar to that of other TBRF Borrelia species and different from the Lyme disease Borrelia species. Variants of B. persica were found among the different genes of the different isolates even in the same sampling area.
doi:10.1371/journal.pone.0014105
PMCID: PMC2991353  PMID: 21124792
18.  Structural and Biochemical Characterization of Human Adenylosuccinate Lyase (ADSL) and the R303C ADSL Deficiency Associated Mutation 
Biochemistry  2012;51(33):6701-6713.
Adenylosuccinate lyase (ADSL) deficiency is a rare autosomal recessive disorder, which causes a defect in purine metabolism resulting in neurological and physiological symptoms. ADSL executes two non-sequential steps in the de novo synthesis of AMP: the conversion of phosphoribosylsuccinyl-aminoimidazole carboxamide (SAICAR) to phosphoribosylaminoimidazole carboxamide (AICAR), which occurs in the de novo synthesis of IMP, and the conversion of adenylosuccinate (AMPS) to AMP, which occurs in the de novo synthesis of AMP and also in the purine nucleotide cycle, using the same active site. Mutation of ADSL’s arginine 303 to a cysteine is known to lead to ADSL deficiency. Interestingly, unlike other mutations leading to ADSL deficiency, the R303C mutation has been suggested to more significantly affect the enzyme’s ability to catalyze the conversion of SAMP than that of SAICAR to their respective products. To better understand the causation of disease due to the R303C mutation, as well as to gain insights as to why the R303C mutation potentially has a disproportional decrease in activity toward its substrates, the wild-type (WT) and the R303C mutation of ADSL were investigated enzymatically, and thermodynamically. Additionally, the X-ray structures of ADSL in its apo form as well as with the R303C mutation were elucidated, providing insight into ADSL’s cooperativity. By utilizing this information a model for the interaction between ADSL and SAICAR is proposed.
doi:10.1021/bi300796y
PMCID: PMC3424377  PMID: 22812634
19.  ATP Requirement for Acidic Resistance in Escherichia coli ▿ 
Journal of Bacteriology  2011;193(12):3072-3077.
ATP participates in many cellular metabolic processes as a major substrate to supply energy. Many systems for acidic resistance (AR) under extremely acidic conditions have been reported, but the role of ATP has not been examined. To clarify whether or not ATP is necessary for the AR in Escherichia coli, the AR of mutants deficient in genes for ATP biosynthesis was investigated in this study. The deletion of purA or purB, each of which encodes enzymes to produce AMP from inosinate (IMP), markedly decreased the AR. The content of ATP in these mutants decreased rapidly at pH 2.5 compared to that of the wild type. The AR was again decreased significantly by the mutation of adk, which encoded an enzyme to produce ADP from AMP. The DNA damage in the purA and purB mutants was higher than that in the wild type. These results demonstrated that metabolic processes that require ATP participate in survival under extremely acidic conditions, and that one such system is the ATP-dependent DNA repair system.
doi:10.1128/JB.00091-11
PMCID: PMC3133219  PMID: 21478347
20.  Identification of a chromosomal determinant of alpha-toxin production in Staphylococcus aureus. 
Infection and Immunity  1980;30(1):36-42.
Production of alpha-toxin (the Hla+ phenotype, controlled by the Hla gene and scored as alpha-hemolytic activity) is a property of some isolates of Staphylococcus aureus NCTC 8325 and not of others. Genetic transformation between strains differing in the Hla phenotype revealed that the hla+ gene resides in the following sequence: purB110-bla+-hla+-ilv-129-pig-131; previously, the enterotoxin A (entA) gene of strain S-6 was shown to map very close to hla+. The hla+ mutations occurring naturally in strain Ps6 and after various mutagenic treatments in strains 8325 and 233 also mapped between bla+ and ilv-129. Among the isolates of strain 8325, the Hla+ phenotype was always associated with fibrinolytic activity, whereas Hla- isolates were non-fibrinolytic. This relationship was also observed among transformants selected for their Hla+ or Hla- phenotypes. The failure of Hla- strains and mutants to revert to hla+ at detectable frequencies, the instability of the Hla+ phenotype, and the previously observed pattern of recombination of the hla+ and entA+ determinants lend support to the view that hla+ may reside on a transposon; according to this view, Hla- mutants have lost the hla+-bearing transposon. It remains unclear whether hla+ is the structural gene for alpha-toxin.
PMCID: PMC551273  PMID: 6254884
21.  Structure and function of PA4872 from Pseudomonas aeruginosa, a novel class of oxaloacetate decarboxylase from the PEP mutase / isocitrate lyase superfamily†‡ 
Biochemistry  2007;47(1):167-182.
Pseudomonas aeruginosa PA4872 was identified by sequence analysis as a structurally and functionally novel member of the PEP mutase/isocitrate lyase superfamily and therefore targeted for investigation. Substrate screens ruled out overlap with known catalytic functions of superfamily members. The crystal structure of PA4872 in complex with oxalate (a stable analog of the shared family α-oxyanion carboxylate intermediate/transition state) and Mg2+ was determined at 1.9 Å resolution. As with other PEP mutase/isocitrate lyase superfamily members, the protein assembles into a dimer of dimers with each subunit adopting an α/β barrel fold and two subunits swapping their barrel's C-terminal α-helices. Mg2+ and oxalate bind in the same manner as observed with other superfamily members. The active site gating loop, known to play a catalytic role in the PEP mutase and lyase branches of the superfamily, adopts an open conformation. The Nε of His235, an invariant residue in the PA4872 sequence family, is oriented towards a C(2) oxygen of oxalate analogous to the C(3) of a pyruvyl moiety. Deuterium exchange into α-oxocarboxylate-containing compounds was confirmed by 1H-NMR spectroscopy. Having ruled out known activities, the involvement of a pyruvate enolate intermediate suggested a decarboxylase activity of an α-oxocarboxylate substrate. Enzymatic assays led to the discovery that PA4872 decarboxylates oxaloacetate (kcat = 7500 s−1 and Km = 2.2 mM) and 3-methyloxaloacetate (kcat = 250 s−1 and Km = 0.63 mM). Genome context of the fourteen sequence family members indicates that the enzyme is used by select group of Gram-negative bacteria to maintain cellular concentrations of bicarbonate and pyruvate; however the decarboxylation activity cannot be attributed to a pathway common to the various bacterial species.
doi:10.1021/bi701954p
PMCID: PMC2892964  PMID: 18081320
22.  Cloning, expression, purification, crystallization and preliminary X-ray diffraction studies of N-acetylneuraminate lyase from methicillin-resistant Staphylococcus aureus  
N-Acetylneuraminate lyase, an enzyme involved in the bacterial uptake and metabolism of sialic acid, is a promising target for antibiotic development against pathogenic bacteria. Here, the cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of N-acetylneuraminate lyase from methicillin-resistant S. aureus to 1.70 Å resolution are reported.
The enzyme N-acetylneuraminate lyase (EC 4.1.3.3) is involved in the metabolism of sialic acids. Specifically, the enzyme catalyzes the retro-aldol cleavage of N-acetylneuraminic acid to form N-acetyl-d-mannosamine and pyruvate. Sialic acids comprise a large family of nine-carbon amino sugars, all of which are derived from the parent compound N-acetylneuraminic acid. In recent years, N-acetylneuraminate lyase has received considerable attention from both mechanistic and structural viewpoints and has been recognized as a potential antimicrobial drug target. The N-acetylneuraminate lyase gene was cloned from methicillin-resistant Staphylococcus aureus genomic DNA, and recombinant protein was expressed and purified from Escherichia coli BL21 (DE3). The enzyme crystallized in a number of crystal forms, predominantly from PEG precipitants, with the best crystal diffracting to beyond 1.70 Å resolution in space group P21. Molecular replacement indicates the presence of eight monomers per asymmetric unit. Understanding the structural biology of N-acetylneuraminate lyase in pathogenic bacteria, such as methicillin-resistant S. aureus, will provide insights for the development of future antimicrobials.
doi:10.1107/S1744309113003060
PMCID: PMC3606580  PMID: 23519810
antibiotic resistance; N-acetylneuraminate lyase; NAL; sialic acid metabolism; Staphylococcus aureus; MRSA
23.  Fragment-based drug discovery using a multi-domain, parallel MD-MM/PBSA screening protocol 
We have developed a rigorous computational screening protocol to identify novel fragment-like inhibitors of N5-CAIR mutase (PurE), a key enzyme involved in de novo purine synthesis that represents a novel target for the design of antibacterial agents. This computational screening protocol utilizes molecular docking, graphics processing unit (GPU)-accelerated molecular dynamics and Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) free energy estimations to investigate the binding modes and energies of fragments in the active sites of PurE. PurE is a functional octamer comprised of identical subunits. The octameric structure, with its eight active sites, provided a distinct advantage in these studies because, for a given simulation length, we were able to place eight separate fragment compounds in the active sites to increase the throughput of the MM/PBSA analysis. To validate this protocol, we have screened an in-house fragment library consisting of 352 compounds. The theoretical results were then compared with the results of two experimental fragment screens, Nuclear Magnetic Resonance (NMR) and Surface Plasmon Resonance (SPR) binding analyses. In these validation studies, the protocol was able to effectively identify the competitive binders that had been independently identified by experimental testing, suggesting the potential utility of this method for the identification of novel fragments for future development as PurE inhibitors.
doi:10.1021/ci300502h
PMCID: PMC3752004  PMID: 23432621
Fragment-based drug discovery; Molecular dynamics; MM/PBSA; N5-CAIR mutase; PurE
24.  Structural basis of nucleic-acid recognition and double-strand unwinding by the essential neuronal protein Pur-alpha 
eLife  null;5:e11297.
The neuronal DNA-/RNA-binding protein Pur-alpha is a transcription regulator and core factor for mRNA localization. Pur-alpha-deficient mice die after birth with pleiotropic neuronal defects. Here, we report the crystal structure of the DNA-/RNA-binding domain of Pur-alpha in complex with ssDNA. It reveals base-specific recognition and offers a molecular explanation for the effect of point mutations in the 5q31.3 microdeletion syndrome. Consistent with the crystal structure, biochemical and NMR data indicate that Pur-alpha binds DNA and RNA in the same way, suggesting binding modes for tri- and hexanucleotide-repeat RNAs in two neurodegenerative RNAopathies. Additionally, structure-based in vitro experiments resolved the molecular mechanism of Pur-alpha's unwindase activity. Complementing in vivo analyses in Drosophila demonstrated the importance of a highly conserved phenylalanine for Pur-alpha's unwinding and neuroprotective function. By uncovering the molecular mechanisms of nucleic-acid binding, this study contributes to understanding the cellular role of Pur-alpha and its implications in neurodegenerative diseases.
DOI: http://dx.doi.org/10.7554/eLife.11297.001
eLife digest
Some proteins perform several different tasks inside cells. This is the case for a protein called Pur-alpha, which is essential for neurons to work correctly. For example, Pur-alpha can bind to DNA to regulate gene activity. It also binds to RNA molecules, which are copies of a gene, and helps to distribute them within the neuron. In humans, there are several neurodegenerative diseases in which Pur-alpha is involved. One example is the Fragile X-associated Tremor/Ataxia Syndrome (FXTAS), which causes memory and movement problems.
Experiments with isolated proteins and double-stranded DNA show that Pur-alpha is able to separate the two DNA strands. But it was not clear how this DNA unwinding occurs, and the biological significance of this activity was unknown. Other questions also remained unanswered: how does Pur-alpha recognize DNA and RNA? Does the loss of Pur-alpha’s binding to DNA and RNA contribute to neurodegenerative diseases?
To address these questions, Weber et al. obtained Pur-alpha from the fruit fly and crystallized the protein bound to DNA. A technique called X-ray crystallography was then used to determine the three-dimensional structure of the Pur-alpha/DNA complex in fine enough detail to work out the position of individual atoms.
Based on this structure, Weber et al. could introduce mutations that alter the DNA- and RNA-binding region of the protein to investigate the binding mechanism. The crystal structure and experiments with normal and mutant Pur-alpha protein revealed how it unwinds double-stranded DNA: binding of Pur-alpha to DNA causes a strong twist of the DNA molecule, which contributes to separating the strands. Further experiments in fruit flies revealed that both the DNA-unwinding activity and the ability of Pur-alpha to bind DNA/RNA are needed for the protein to work correctly in neurons.
Because Pur-alpha is involved in a range of different processes inside cells, a future goal is to identify the DNA and RNA sequences it specifically binds to. This information, together with the insights gained from Weber et al.’s study, should advance our understanding of why Pur-alpha is essential for maintaining neurons.
DOI: http://dx.doi.org/10.7554/eLife.11297.002
doi:10.7554/eLife.11297
PMCID: PMC4764581  PMID: 26744780
DNA-/RNA-protein interaction; DNA unwinding; FXTAS; ALS; 5q31.3 microdeletion syndrome; X-ray crystallography; D. melanogasterE. coli
25.  Biochemical and Biophysical Analysis of Five Disease-Associated Human Adenylosuccinate Lyase Mutants† 
Biochemistry  2009;48(23):5291-5302.
Adenylosuccinate lyase (ASL), a catalyst of key reactions in purine biosynthesis, is normally a homotetramer in which three subunits contribute to each of four active sites. Human ASL deficiency is an inherited metabolic disease associated with autism and mental retardation. We have characterized five disease-associated ASL mutants: R194C and K246E are located at subunit interfaces, L311V is in the central helical region away from the active site, and R396C and R396H are at the entrance to the active site. The Vmax (at 25 °C) for R194C is comparable to that of WT; while those of L311V, R396C, R396H and K246E are considerably reduced and affinity for adenylosuccinate is retained. The mutant enzymes have decreased positive cooperativity as compared to WT. K246E exists mainly as dimer or monomer, accounting for its negligible activity; whereas the other mutant enzymes are similar to WT in the predominance of tetramer. At 37 °C, the specific activity of WT and these mutant enzymes slowly decreases 30-40% with time and reaches a limiting specific activity without changing significantly the amount of tetramer. Mutant R194C is unique in being rapidly inactivated at the harsher temperature of 60°C, indicating that it is the least stable enzyme in vitro. Conformational changes in the mutant enzymes are evident from protein fluorescence intensity at 25 °C and after incubation at 37 °C, which correlates with the loss of enzymatic activity. Thus, these disease-associated single mutations can yield enzyme with reduced activity either by affecting the active site or by perturbing the enzyme’s structure and/or native conformation which are required for catalytic function.
doi:10.1021/bi802321m
PMCID: PMC2745324  PMID: 19405474

Results 1-25 (1407412)