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.     

Cloned mouse ribonucleotide reductase subunit M1 cDNA reveals amino acid sequence homology with Escherichia coli and herpesvirus ribonucleotide reductases

We have isolated and sequenced overlapping cDNA clones containing the entire coding region of mouse ribonucleotide reductase subunit M1. The coding region comprises 2.4 kilobases and predicts a polypeptide of 792 amino acids (Mr 90,234) which shows striking homology with ribonucleotide reductases from Escherichia coli and the herpesviruses, Epstein-Barr virus and herpes simplex virus. The homologies reveal three domains: an N-terminal domain common to the mammalian and bacterial enzymes, a C-terminal domain common to the mammalian and viral ribonucleotide reductases, and a central domain common to all three. We speculate on the functional basis of this conservation.     

Identification of inhibitors of N5-carboxyaminoimidazole ribonucleotide synthetase by high-throughput screening

The increasing risk of drug-resistant bacterial infections indicates that there is a growing need for new and effective antimicrobial agents. One promising, but unexplored area in antimicrobial drug design is de novo purine biosynthesis. Recent research has shown that de novo purine biosynthesis in microbes is different from that in humans. The differences in the pathways are centered around the synthesis of 4-carboxyaminoimidazole ribonucleotide (CAIR) which requires the enzyme N⁵-carboxyaminoimidazole ribonucleotide (N⁵-CAIR) synthetase. Humans do not require and have no homologs of this enzyme. Unfortunately, no studies aimed at identifying small-molecule inhibitors of N⁵-CAIR synthetase have been published. To remedy this problem, we have conducted high-throughput screening (HTS) against Escherichia coli N⁵-CAIR synthetase using a highly reproducible phosphate assay. HTS of 48,000 compounds identified 14 compounds that inhibited the enzyme. The hits identified could be classified into three classes based on chemical structure. Class I contains compounds with an indenedione core. Class II contains an indolinedione group, and Class III contains compounds that are structurally unrelated to other inhibitors in the group. We determined the Michaelis–Menten kinetics for five compounds representing each of the classes. Examination of compounds belonging to Class I indicates that these compounds do not follow normal Michaelis–Menten kinetics. Instead, these compounds inhibit N⁵-CAIR synthetase by reacting with the substrate AIR. Kinetic analysis indicates that the Class II family of compounds are non-competitive with both AIR and ATP. One compound in Class III is competitive with AIR but uncompetitive with ATP, whereas the other is non-competitive with both substrates. Finally, these compounds display no inhibition of human AIR carboxylase:SAICAR synthetase indicating that these agents are selective inhibitors of N⁵-CAIR synthetase.     

Response of ad-2 mutants of Saccharomyces cerevisiae to carbon dioxide

Summary Confirmation that the ad-2 locus of yeast controls the carboxylation of aminoimidazole ribonucleotide (AIR) to 5-amino-4-imidazole carboxylate ribonucleotide (CAIR) is provided by the observation that 21 out of a sample of 113 ad-2 mutants were affected by CO₂. 19 of the mutants were stimulated by CO₂ and 2 were inhibited. The majority of the CO₂-stimulated mutants were confined to one section of the complementation map of the ad-2 locus.     

Isolation of a multifunctional protein with aminoimidazole ribonucleotide synthetase, glycinamide ribonucleotide synthetase, and glycinamide ribonucleotide transformylase activities: characterization of aminoimidazole ribonucleotide synthetase

5-Aminoimidazole ribonucleotide (AIR) synthetase, glycinamide ribonucleotide (GAR) synthetase, and GAR transformylase activities from chicken liver exist on a single polypeptide of Mr 110,000 [Daubner, C. S., Schrimsher, J. L., Schendel, F. J., Young, M., Henikoff, S., Patterson, D., Stubbe, J., & Benkovic, S. J. (1985) Biochemistry 24, 7059-7062]. Details of copurification of these three activities through four chromatographic steps are reported. The ratios of these activities remain constant throughout the purification. AIR synthetase has an absolute requirement for K+ for activity and under these conditions has apparent molecular weights of 330,000, determined by Sephadex G-200 chromatography, and 133,000, determined by sucrose density gradient ultracentrifugation. Incubation of 18O-labeled formylglycinamidine ribonucleotide (FGAM) with AIR synthetase results in stoichiometric production of AIR, ADP, and [18O]Pi. NMR spectra of beta-FGAM and beta-AIR are reported.     

Ribulose-1, 5-bisphosphate carboxylase activity,chlorophyll and protein concentrations in differentPopulus clones

Ribulose-1,5-bisphosphate carboxylase activity (RuBPC), chlorophyll (chl) and protein (prot) concentrations and chlorophyll/protein (chl/prot) ratios were determined in five differentPopulus clones together with their maximal net CO₂ uptake rates (P_max). A classic reference clone (Populus ×euramericana “Robusta” (Dode) Guinier) was compared with four recently selected euramerican and interamerican crossings. Chl/prot ratio and RuBPC activity varied among the different clones, while chl a/chlb ratio showed only a very low coefficient of variation (1.7%) for the five clones. Poplar clone “Robusta” could be distinguished from the recent faster growing clones based on the different biochemical characteristics. A significant correlation was found between both total chl concentration and chl/prot ratio with P_max for the five clones.     

The presequence of the precursor to the nucleus-encoded 30 kDa protein of photosystem II in Euglena gracilis Z includes two hydrophobic domains

A cDNA clone for the extrinsic 30 kDa protein (OEC30) of photosystem II in Euglena gracilis Z was isolated and characterized. The open reading frame of the cDNA encoded a polypeptide of 338 amino acids, which consisted of a long presequence of 93 amino acids and a mature polypeptide of 245 amino acids. Two hydrophobic domains were identified in the presequence, in contrast to the presence of a single hydrophobic domain in the presequence of the corresponding proteins from higher plants. At the N- and C-terminal regions, respectively, of the presequence, a signal-peptide-like sequence and a thylakoid-transfer domain were identified. The presence of a long and unique presequence in the precursor to OEC30 is probably related to the complexity of the intracellular processes required for the synthesis and/or transport of the protein in Euglena.Abbreviations ER endoplasmic reticulum - cDNA complementary DNA - SSU small subunit; Rubisco, ribulose 1,5-bisphosphate carboxylase/oxygenase - Rubico, ribulose 1,5 bisphosphate carboxylase/oxygenase - LHC II light-harvesting chlorophyll protein of photosystem II - PS II photosystem II - OEC30 the extrinsic 30 kDa protein of photosystem II in Euglena - PCR polymerase chain reaction - SDS sodium dodecyl sulfate - TE a solution containing 10 mM Tris-HCl and 1 mM EDTA pH 8.0 - SSPE a solution containing 0.15 M NaCl, 10 mM NaH₂PO₄ and 1 mM EDTA pH 7.4 - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - PVDF poly(vinylidene difluoride)     

Crystal structure of the invertebrate bifunctional purine biosynthesis enzyme PAICS at 2.8 Å resolution

Two important steps of the de novo purine biosynthesis pathway are catalyzed by the 5‐aminoimidazole ribonucleotide carboxylase and the 4‐(N‐succinylcarboxamide)‐5‐aminoimidazole ribonucleotide synthetase enzymes. In most eukaryotic organisms, these two activities are present in the bifunctional enzyme complex known as PAICS. We have determined the 2.8‐Å resolution crystal structure of the 350‐kDa invertebrate PAICS from insect cells (Trichoplusia ni) using single‐wavelength anomalous dispersion methods. Comparison of insect PAICS to human and prokaryotic homologs provides insights into substrate binding and reveals a highly conserved enzymatic framework across divergent species. Proteins 2013; 81:1473–1478. © 2013 Wiley Periodicals, Inc.     

Octameric structure of the human bifunctional enzyme PAICS in purine biosynthesis

Phosphoribosylaminoimidazole carboxylase/phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) is an important bifunctional enzyme in de novo purine biosynthesis in vertebrate with both 5-aminoimidazole ribonucleotide carboxylase (AIRc) and 4-(N-succinylcarboxamide)-5-aminoimidazole ribonucleotide synthetase (SAICARs) activities. It becomes an attractive target for rational anticancer drug design, since rapidly dividing cancer cells rely heavily on the purine de novo pathway for synthesis of adenine and guanine, whereas normal cells favor the salvage pathway. Here, we report the crystal structure of human PAICS, the first in the entire PAICS family, at 2.8 Å resolution. It revealed that eight PAICS subunits, each composed of distinct AIRc and SAICARs domains, assemble a compact homo-octamer with an octameric-carboxylase core and four symmetric periphery dimers formed by synthetase domains. Based on structural comparison and functional complementation analyses, the active sites of SAICARs and AIRc were identified, including a putative substrate CO₂-binding site. Furthermore, four symmetry-related, separate tunnel systems in the PAICS octamer were found that connect the active sites of AIRc and SAICARs. This study illustrated the octameric nature of the bifunctional enzyme. Each carboxylase active site is formed by structural elements from three AIRc domains, demonstrating that the octamer structure is essential for the carboxylation activity. Furthermore, the existence of the tunnel system implies a mechanism of intermediate channeling and suggests that the quaternary structure arrangement is crucial for effectively executing the sequential reactions. In addition, this study provides essential structural information for designing PAICS-specific inhibitors for use in cancer chemotherapy.     

Purification and characterization of aminoimidazole ribonucleotide synthetase from Escherichia coli

Aminoimidazole ribonucleotide (AIR) synthetase has been purified 15-fold to apparent homogeneity from Escherichia coli which contains a multicopy plasmid containing the purM, AIR synthetase, gene. The protein is a dimer composed of two identical subunits of Mr 38,500. The N-terminal sequence, amino acid composition, and steady-state kinetics of the protein have been determined. AIR synthetase has been shown to catalyze the transfer of the formyl oxygen of [18O]formylglycinamide ribonucleotide to Pi.     

Isolation of cDNA clones of genes induced upon transfer of Chlamydomonas reinhardtii cells to low CO2

Unicellular algae grow well under limiting CO₂ conditions, aided by a carbon concentrating mechanism (CCM). In C. reinhardtii, this mechanism is inducible and is present only in cells grown under low CO₂ conditions. We constructed a cDNA library from cells adapting to low CO₂, and screened the library for cDNAs specific to low CO₂-adapting cells. Six classes of low CO₂-inducible clones were identified. One class of clone, reported here, represents a novel gene associated with adaptation of cells to air. A second class of clones corresponds to the air-inducible periplasmic carbonic anhydrase I (CAH1). These clones represent genes that respond to the level of CO₂ in the environment.     

Majority of random cDNA clones correspond to single loci in the tomato genome

Summary The number of loci corresponding to each of 34 unique, random cDNA clones has been determined for the diploid plant species Lycopersicon esculentum (tomato) (2n=24). Fifty-three percent of the clones are homologous to loci represented only once in the tomato chromosomes. Thirty-two percent of the clones correspond to two genetically independent loci. The remaining clones belong to gene families represented by 3–5 loci. To determine the number of gene copies per locus, reconstruction experiments were performed on six of the single locus clones. The majority of these loci were estimated to contain only 1 or 2 copies of the gene. These results support the concept that the majority of structural genes in this diploid plant species are arranged in single loci and present in low copy number. Multigene families, such as those for the small subunit of ribulose bisphosphate carboxylase, chlorophyll a/b binding polypeptide and actin are exceptions to this rule.     

Isolation and characterization of a cDNA encoding a 3-hydroxy-3-methylglutaryl coenzyme A reductase from Nicotiana sylvestris

A cDNA library from freshly isolated mesophyll protoplasts of Nicotiana sylvestris was differentially screened using cDNAs from leaves, leaf strips submitted to the same stress as protoplasts during the isolation procedure, and cell suspension cultures. One of the selected clones (6P2) was found to encode a putative polypeptide highly homologous to previously characterized 3-hydroxy-3-methylglutaryl coenzyme A reductases. The C-terminal region of the polypeptide was highly conserved whereas its N-terminal region including the trans-membrane domains and the linker was more variable. Apart from protoplasts, the 6P2 gene was found to be expressed in apexes, anthers, roots, and in stressed leaf strips after 24h of culture, during the hypersensitive reaction to viral infection and after HgCl₂ treatment. This pattern of expression is consistent with a role in plant defence mechanisms.     

Interrogating the mechanism of a tight binding inhibitor of AIR carboxylase

The enzyme aminoimidazole ribonucleotide (AIR) carboxylase catalyzes the synthesis of the purine intermediate, 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). Previously, we have shown that the compound 4-nitro-5-aminoimidazole ribonucleotide (NAIR) is a slow, tight binding inhibitor of the enzyme with a Ki of 0.34 nM. The structural attributes and the slow, tight binding characteristics of NAIR implicated this compound as a transition state or reactive intermediate analog. However, it is unclear what molecular features of NAIR contribute to the mimetic properties for either of the two proposed mechanisms of AIR carboxylase. In order to gain additional information regarding the mechanism for the potent inhibition of AIR carboxylase by NAIR, a series of heterocyclic analogs were prepared and evaluated. We find that all compounds are weaker inhibitors than NAIR and that CAIR analogs are not alternative substrates for the enzyme. Surprisingly, rather subtle changes in the structure of NAIR can lead to profound changes in binding affinity. Computational investigations of enzyme intermediates and these inhibitors reveal that NAIR displays an electrostatic potential surface similar to a proposed reaction intermediate. The result indicates that AIR carboxylase is likely sensitive to the electrostatic surface of reaction intermediates and thus compounds which mimic these surfaces should possess tight binding characteristics. Given the evolutionary relationship between AIR carboxylase and N⁵-CAIR mutase, we believe that this concept extends to the mutase enzyme as well. The implications of this hypothesis for the design of selective inhibitors of the N⁵-CAIR mutase are discussed.     

Regulation of the low-CO2-inducible polypeptides in Chlamydomonas reinhardtii

Polypeptides of 21, 36 and 37 kDa are induced in the unicellular green alga Chlamydomonas reinhardtii Dang. when cells are transferred from high (2%) to low (0.03%) CO₂ concentrations. The synthesis of these polypeptides is correlated with the induction of the CO₂-concentrating mechanism. In this work we studied the effect of the growth conditions on the synthesis of these polypeptides with the aim of clarifying whether the induction of all three of these low-CO₂-inducible polypeptides requires the same environmental factor. Our results showed that induction of the 21- and 36-kDa polypeptides under low-CO₂ conditions occurred only in the light, while the 37-kDa periplasmic carbonic anhydrase (EC 4.2.1.1) was induced in light, darkness, and in both synchronous and asynchronous cultures. In addition, induction of these polypeptides appeared to be determined more by the O₂/CO₂ ratio than by the CO₂ concentrations. None of these polypeptides could be induced in either of two different mutants of C. reinhardtii, one lacking ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) and the other with inactive enzyme. Our results indicate that the 21- and 36-kDa polypeptides are regulated by a mechanism different from that controlling the 37-kDa polypeptide.Abbreviations pCA (periplasmic) carbonic anhydrase - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - TAP Trisacetate phosphate medium The authors thank Prof. M. Spalding (Iowa State University, USA) for providing antisera to LIP-21 and LIP-36. We thank Prof. S. Bartlett and Dr. J. Moroney (Louisiana State University, USA) for providing antibodies to C. reinhardtii, Rubisco and 37-kDa pCA, respectively. This work was supported by the Instituto Tecnologico de Canarias.     

Regulation of Synthesis of Chlorophyll, Carotene, Ribulosel,5-diP Carboxylase and Phosphoribulokinase in a Temperature-sensitive Chlorophyll Mutant of Medicago sativa

The activities of several enzymes were studied in a temperature-sensitive chlorophyll mutant of alfalfa (Medicago saliva). In leaves grown at 10°C photosynthetic capacity was essentially nil with ribulose-1,5-diP carboxylase, chlorophyll, and carolene present in greatly limiting concentrations. The activity of phosphoribulokinase was 3.5 times lower at 10°C than at 27°C, but was still sufficiently high at 10°C to not limit the rate of CO₂ fixation. Activities of phosphoriboisomerase, phosphoenolpyruvate carboxylase, glucose-6-P dehydrogenase and malate dehydrogenase were not different at 10°C and 27°C. The low fraction I protein content (which also accounts for the ribulose-1,5-dip carboxylase activity in alfalfa) indicated that synthesis of the carboxylase was effectively blocked at 10°C. A large, comparable increase in carboxylase activity and in concentration of fraction I protein in alfalfa leaves grown at 27°C indicated that the carboxylase was synthesized de novo. The initial induction of the carboxylase, chlorophyll, and carotene may be related, but after induction the carboxylase was not linearly correlated with the other two and had a different temperature optima. Nevertheless, the synthesis of each appeared to be regulated by the temperature-sensitive gene of this mutant.     

Three-dimensional structure of N5-carboxyaminoimidazole ribonucleotide synthetase: a member of the ATP grasp protein superfamily

Escherichia coli PurK, a dimeric N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) synthetase, catalyzes the conversion of 5-aminoimidazole ribonucleotide (AIR), ATP, and bicarbonate to N5-CAIR, ADP, and Pi. Crystallization of both a sulfate-liganded and the MgADP-liganded E. coli PurK has resulted in structures at 2.1 and 2.5 A resolution, respectively. PurK belongs to the ATP grasp superfamily of C-N ligase enzymes. Each subunit of PurK is composed of three domains (A, B, and C). The B domain contains a flexible, glycine-rich loop (B loop, T123-G130) that is disordered in the sulfate-PurK structure and becomes ordered in the MgADP-PurK structure. MgADP is wedged between the B and C domains, as with all members of the ATP grasp superfamily. Other enzymes in this superfamily contain a conserved Omega loop proposed to interact with the B loop, define the specificity of their nonnucleotide substrate, and protect the acyl phosphate intermediate formed from this substrate. PurK contains a minimal Omega loop without conserved residues. In the reaction catalyzed by PurK, carboxyphosphate is the putative acyl phosphate intermediate. The sulfate of the sulfate ion-liganded PurK interacts electrostatically with Arg 242 and the backbone amide group of Asn 245, components of the J loop of the C domain. This sulfate may reveal the location of the carboxyphosphate binding site. Conserved residues within the C-terminus of the C domain define a pocket that is proposed to bind AIR in collaboration with an N-terminal strand loop helix motif in the A domain (P loop, G8-L1). The P loop is proposed to bind the phosphate of AIR on the basis of similar binding sites observed in PurN and PurE and proposed in PurD and PurT, four other enzymes in the purine pathway.     

Sequence analysis of the large and small subunits of human ribonucleotide reductase.

We have isolated and sequenced overlapping cDNA clones from a breast carcinoma cDNA library containing the entire coding region of both the R1 and R2 subunits of the human ribonucleotide reductase gene. The coding region of the human R1 subunit comprises 2376 nucleotides and predicts a polypeptide of 792 amino acids (calculated molecular mass 90,081). The sequence of this subunit is almost identical to the equivalent mouse ribonucleotide reductase subunit with 97.7% homology between the mouse and human R1 subunit amino acid sequences. The coding region of the human R2 subunit of ribonucleotide reductase comprises 1170 nucleotides and predicts a polypeptide of 389 amino acids (calculated molecular mass 44,883), which is one amino acid shorter than the equivalent mouse subunit. The human and mouse R2 subunits display considerable homology in their carboxy-terminal amino acid sequences, with 96.3% homology downstream of amino acid 68 of the human and mouse R2 proteins. However, the amino-terminal portions of these two proteins are more divergent in sequence, with only 69.2% homology in the first 68 amino acids.     

Molecular analysis of barley mutants deficient in chloroplast glutamine synthetase

A barley leaf cDNA library has been screened with two oligonucleotide probes designed to hybridize to conserved sequences in glutamine synthetase (GS) genes from higher plants. Two GS cDNA clones were identified as hybridizing strongly to one or both probes. The larger clone (pcHvGS6) contained a 1.6 kb insert which was shown by primer extension analysis to be an almost full-length cDNA. Both clones were more closely related to cDNAs for the chloroplast form of GS (GS₂) from pea and Phaseolus vulgaris than to cDNAs for the cytosolic form (GS₁). A sequence identicalto an N-terminal sequence determined from a purified preparation of the mature GS₂ polypeptide (NH₂-XLGPETTGVIQRMQQ) was found in the pcHvGS6-encoded polypeptide at residues 46–61, indicating a pre-sequence of at least 45 amino acids. The pre-sequence has only limited sequence homology to the pre-sequences of pea and P. vulgaris GS₂ subunits, but is similarly rich in basic residues and possesses some of the structural features common to the targeting sequences of other chloroplast proteins. The molecular lesions responsible for the GS₂-deficient phenotypes of eight photorespiratory mutants of barley were investigated using a gene-specific probe from pcHvGS6 to assay for GS₂ mRNA, and an anti-GS antiserum to assay for GS₂ protein. Three classes of mutants were identified: class I, in which absence of cross-reacting material was correlated with low or undetectable levels of GS₂ mRNA; class II, which had normal or increased levels of GS₂ mRNA but very little GS₂ protein; and class III, which had significant amounts of GS₂ protein but little or no GS₂ activity.