Genetic complementation and enzyme correlates at the locus encoding the last two steps of de novo pyrimidine biosynthesis in Drosophila melanogaster

Summary Eight mutations of the rudimentary-like (r-l) locus were isolated following mutagenesis with ethylmethanesulfonate and inter se crosses revealed three basic complementation groups, using the wing phenotype as an index of complementation. One group consists of three entirely noncomplementing mutants that each specify severe reductions in levels of both r-l-encoded enzymes, orotate phosphoribosyltransferase (OPR-Tase) and orotidylate decarboxylase (ODCase). The other two groups consist of complementing mutants, such that any member of one group fully complements all members of the other group. One of these groups consists of two mutants that each specify severly reduced OPRTase, but normal ODCase. The other group consists of three mutants that specify severe OPRTase and OD-Case reductions in homoallelic flies, but that appear to contribute OPRTase in certain heteroallelic genotypes. It is concluded that the reciprocal and complementing enzymatic phenotypes of mutants in these two groups account for most instances of genetic trans complementation among r-l mutants. These findings are discussed relative to extant information on OPRTase and OD-Case in animals and an hypothesis is developed that the r-l locus encodes a single polypeptide product that contains both enzyme activities.     

The structure of adenylosuccinate lyase, an enzyme with dual activity in the de novo purine biosynthetic pathway

Background: Adenylosuccinate lyase is an enzyme that plays a critical role in both cellular replication and metabolism via its action in the de novo purine biosynthetic pathway. Adenylosuccinate lyase is the only enzyme in this pathway to catalyze two separate reactions, enabling it to participate in the addition of a nitrogen at two different positions in adenosine monophosphate. Both reactions catalyzed by adenylosuccinate lyase involve the beta-elimination of fumarate. Enzymes that catalyze this type of reaction belong to a superfamily, the members of which are homotetramers. Because adenylosuccinate lyase plays an integral part in maintaining proper cellular metabolism, mutations in the human enzyme can have severe clinical consequences, including mental retardation with autistic features. Results: The 1.8 A crystal structure of adenylosuccinate lyase from Thermotoga maritima has been determined by multiwavelength anomalous dispersion using the selenomethionine-substituted enzyme. The fold of the monomer is reminiscent of other members of the beta-elimination superfamily. However, its active tetrameric form exhibits striking differences in active-site architecture and cleft size. Conclusions: This first structure of an adenylosuccinate lyase reveals that, along with the catalytic base (His141) and the catalytic acid (His68), Gln212 and Asn270 might play a vital role in catalysis by properly orienting the succinyl moiety of the substrates. We propose a model for the dual activity of adenylosuccinate lyase: a single 180 degrees bond rotation must occur in the substrate between the first and second enzymatic reactions. Modeling of the pathogenic human S413P mutation indicates that the mutation destabilizes the enzyme by disrupting the C-terminal extension.     

The product of theade1 gene inSchizosaccharomyces pombe: A bifunctional enzyme catalysing two distinct steps in purine biosynthesis

Summary The assignment of the knownade genes to steps in purine biosynthesis inSchizosaccharomyces pombe has been completed with the demonstration that anade3 mutants lacks FGAR amidotransferase,ade1A mutants lack GAR synthetase andade1B mutants lack AIR synthetase. A comparison of enzyme activity with map position forade1 mutants shows that (1) complementingade1A mutants lack GAR synthetase but possess wild type amounts of AIR synthetase, (2) complementingade1B mutants lack AIR synthetase but posses variable amounts of GAR synthetase, (3) non-complementing mutants lack both activities. In wild type strains the two activities fractionate together throughout a hundred-fold purification. Hence theade1 gene appears to code for a bifunctional enzyme catalysing two distinct steps in purine biosynthesis. The two activities are catalysed by two different regions of the polypeptide chain which can be altered independendently by mutation. Gel filtration studies on partially purified enzymes from wild type and various complementing mutant strains, indicate that the bifunctional enzyme is a multimer consisting of between four and six sub-units of 40,000 daltons each. GAR synthetase activity is associated with both the monomeric and multimeric forms but AIR synthetase is only associated with the multimer. A comparison of enzyme levels between diploids and their original complementing haploid strains suggests that complementation is due to hybrid enzyme formation.     

A mutant of CHO-K1 cells deficient in two nonsequential steps of de novo purine biosynthesis

An unusual new purine-requiring mutant, Ade^?P_AB, of Chinese hamster ovary cells (CHO-K1) is described. Ade^?P_AB will grow in medium supplemented with hypoxanthine, adenine, or aminoimidazole carboxamide. Ade^?P_AB fails to show genetic complementation with either Ade^?A, defective in amidophosphoribosyltransferase (E.C. 2.4.2.14), or Ade^?B, defective in phosphoribosylformylglycinamidine FGAM) synthetase (E.C. 6.3.5.3.), but will complement all five of our other hypoxanthine-requiring Ade^? complementation groups. Analysis of purine synthesis in wild-type, mutant, and revertant cells and analysis of relevant enzyme activities in cell-free extracts prepared from these cells demonstrates that Ade^?P_AB is similar to Ade^?B in that it has lost FGAM synthetase activity, and is similar to Ade^?A in that it has lost glutamine-dependent amidophosphoribosyltransferase activity. Unlike Ade^?A, however, Ade^?P_AB retains the ability to synthesize phosphoribosylamine (PRA), the product of the amidophosphoribosyltransferase reaction, if NH₄Cl is substituted for glutamine as the nitrogen donor. Moreover, partial revertants of Ade^?P_AB can apparently synthesize sufficient purines for growth using the NH₄Cl-dependent reaction. The available evidence indicates that neither a double mutation nor a deletion is probable in Ade^?P_AB. We discuss the relevance of these observations for our understanding of both the regulation of purine biosynthesis in mammalian cells and the structural organization of the enzymes defective in Ade^?P_AB and the genes coding for these enzymes.     

The arabidopsis DELAYED DEHISCENCE1 gene encodes an enzyme in the jasmonic acid synthesis pathway

delayed dehiscence1 is an Arabidopsis T-DNA mutant in which anthers release pollen grains too late for pollination to occur. The delayed dehiscence1 defect is caused by a delay in the stomium degeneration program. The gene disrupted in delayed dehiscence1 encodes 12-oxophytodienoate reductase, an enzyme in the jasmonic acid biosynthesis pathway. We rescued the mutant phenotype by exogenous application of jasmonic acid and obtained seed set from previously male-sterile plants. In situ hybridization studies showed that during the early stages of floral development, DELAYED DEHISCENCE1 mRNA accumulated within all floral organs. Later, DELAYED DEHISCENCE1 mRNA accumulated specifically within the pistil, petals, and stamen filaments. DELAYED DEHISCENCE1 mRNA was not detected in the stomium and septum cells of the anther that are involved in pollen release. The T-DNA insertion in delayed dehiscence1 eliminated both DELAYED DEHISCENCE1 mRNA accumulation and 12-oxophytodienoate reductase activity. These experiments suggest that jasmonic acid signaling plays a role in controlling the time of anther dehiscence within the flower.     

The Drosophila melanogaster retinophilin gene encodes the peripheral membrane protein in photoreceptor cells

The Drosophila melanogaster retinophilin (rtp) gene encoding the protein containing MORN (Membrane Occupation and Recognition Nexus) motifs that are known to interact with the plasma membrane has been identified to be expressed predominantly in adult eyes by several independent studies. Isolation and characterization of rtp mutant flies showed that the gene is involved in the phototransduction process by interacting with NINAC (neither inactivation nor afterpotential C) protein in adult photoreceptor cells. The gene was also reported to be involved in phagocytosis in embryos. We examined rtp gene expression during D. melanogaster development and in adult head tissues. The results showed that the gene is expressed at detectable levels only in adult photoreceptor cells but not in other developmental stages and other adult tissues, confirming its phototransduction functions. The RTP protein contains only 4 MORN motifs, whereas 8 MORN motifs are reported to be required for interactions with the plasma membrane. We found that RTP protein is present free in the cytosol and also is bound peripherally to the plasma membrane; this binding ability was found to be modulated by light. Our results suggest that the D. melanogaster RTP protein is a light-regulated peripheral membrane protein of photoreceptor cells.     

The Drosophila gene brainiac encodes a glycosyltransferase putatively involved in glycosphingolipid synthesis

The Drosophila genes fringe and brainiac exhibit sequence similarities to glycosyltransferases. Drosophila and mammalian fringe homologs encode UDP-N-acetylglucosamine:fucose-O-Ser beta1,3-N-acetylglucosaminyltransferases that modulate the function of Notch family receptors. The biological function of brainiac is less well understood. brainiac is a member of a large homologous mammalian beta3-glycosyltransferase family with diverse functions. Eleven distinct mammalian homologs have been demonstrated to encode functional enzymes forming beta1-3 glycosidic linkages with different UDP donor sugars and acceptor sugars. The putative mammalian homologs with highest sequence similarity to brainiac encode UDP-N-acetylglucosamine:beta1,3-N-acetylglucosaminyltransferases (beta3GlcNAc-transferases), and in the present study we show that brainiac also encodes a beta3GlcNAc-transferase that uses beta-linked mannose as well as beta-linked galactose as acceptor sugars. The inner disaccharide core structures of glycosphingolipids in mammals (Galbeta1-4Glcbeta1-Cer) and insects (Manbeta1-4Glcbeta1-Cer) are different. Both disaccharide glycolipids served as substrates for brainiac, but glycolipids of insect cells have so far only been found to be based on the GlcNAcbeta1-3Manbeta1-4Glcbeta1-Cer core structure. Infection of High Five(TM) cells with baculovirus containing full coding brainiac cDNA markedly increased the ratio of GlcNAcbeta1-3Manbeta1-4Glcbeta1-Cer glycolipids compared with Galbeta1-4Manbeta1-4Glcbeta1-Cer found in wild type cells. We suggest that brainiac exerts its biological functions by regulating biosynthesis of glycosphingolipids.     

The hprK gene of Enterococcus faecalis encodes a novel bifunctional enzyme: the HPr kinase/phosphatase

The HPr kinase of Gram-positive bacteria is an ATP-dependent serine protein kinase, which phosphorylates the HPr protein of the bacterial phosphotransferase system (PTS) and is involved in the regulation of carbohydrate metabolism. The hprK gene from Enterococcus faecalis was cloned via polymerase chain reaction (PCR) and sequenced. The deduced amino acid sequence was confirmed by microscale Edman degradation and mass spectrometry combined with collision-induced dissociation of tryptic peptides derived from the HPr kinase of E. faecalis . The gene was overexpressed in Escherichia coli , which does not contain any ATP-dependent HPr kinase or phosphatase activity. The homogeneous recombinant protein exhibits the expected HPr kinase activity as well as a P-Ser-HPr phosphatase activity, which was assumed to be a separate enzyme activity. The bifunctional HPr kinase/phosphatase acts preferentially as a kinase at high ATP levels of 2 mM occurring in glucose-metabolizing Streptococci . At low ATP levels, the enzyme hydrolyses P-Ser-HPr. In addition, high concentrations of phosphate present under starvation conditions inhibit the HPr kinase activity. Thus, a putative function of the enzyme may be to adjust the ratio of HPr and P-Ser-HPr according to the metabolic state of the cell; P-Ser-HPr is involved in carbon catabolite repression and regulates sugar uptake via the phosphotransferase system (PTS). Reinvestigation of the previously described Bacillus subtilis HPr kinase revealed that it also possesses P-Ser-HPr phosphatase activity. However, contrary to the E. faecalis enzyme, ATP alone was not sufficient to switch the phosphatase activity of the B. subtilis enzyme to the kinase activity. A change in activity of the B. subtilis HPr kinase was only observed when fructose-1,6-bisphosphate was also present.     

A Drosophila melanogaster gene encodes a protein homologous to the mouse t complex polypeptide 1

We have isolated and sequenced a cDNA from Drosophila melanogaster that is homologous to the mouse Tcp-1 gene encoding the t complex polypeptide 1, TCP-1. The Drosophila gene maps by in situ hybridization to bands 94B1-2 of the polytene chromosomes. It shares 66% nucleotide sequence identity with the mouse gene. The predicted Drosophila protein consists of 557 amino acids and shares 72% identity with the mouse polypeptide. The TCP-1 polypeptide appears to be highly conserved in evolution from mammals to simple eukaryotes because the Drosophila gene probe also detects related sequences in DNA from the yeast, Saccharomyces cerevisiae. The presence of TCP-1-related polypeptides in organisms such as Drosophila and yeast should facilitate biochemical and genetic analysis of its function.     

Copurification of glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridyltransferase activities of Escherichia coli: characterization of the glmU gene product as a bifunctional enzyme catalyzing two subsequent steps in the pathway for UDP-N-acetylglucosamine synthesis.

The glmU gene product of Escherichia coli was recently identified as the N-acetylglucosamine-1-phosphate uridyltransferase activity which catalyzes the formation of UDP-N-acetylglucosamine, an essential precursor for cell wall peptidoglycan and lipopolysaccharide biosyntheses (D. Mengin-Lecreulx and J. van Heijenoort, J. Bacteriol. 175:6150-6157, 1993). Evidence that the purified GlmU protein is in fact a bifunctional enzyme which also catalyzes acetylation of glucosamine-1-phosphate, the preceding step in the same pathway, is now provided. Kinetic parameters of both reactions were investigated, indicating in particular that the acetyltransferase activity of the enzyme is fivefold higher than its uridyltransferase activity. In contrast to the uridyltransferase activity, which is quite stable and insensitive to thiol reagents, the acetyltransferase activity was rapidly lost when the enzyme was stored in the absence of reducing thiols or acetyl coenzyme A or was treated with thiol-alkylating agents, suggesting the presence of at least one essential cysteine residue in or near the active site. The acetyltransferase activity is greatly inhibited by its reaction product N-acetylglucosamine-1-phosphate and, interestingly, also by UDP-N-acetylmuramic acid, which is one of the first precursors specific for the peptidoglycan pathway. The detection in crude cell extracts of a phosphoglucosamine mutase activity finally confirms that the route from glucosamine-6-phosphate to UDP-N-acetylglucosamine occurs via glucosamine-1-phosphate in bacteria.     

A Methanocaldococcus jannaschii archaeal signature gene encodes for a 5-formaminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5'-monophosphate synthetase. A new enzyme in purine biosynthesis

We have identified and characterized a new member of the ATP-grasp enzyme family that catalyzes the ATP- and formate-dependent formylation of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5'-monophosphate (AICAR) to 5-formaminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5'-monophosphate (FAICAR) in the absence of folates. The enzyme, which we designate as PurP, is the product of the Methanocaldococcus jannaschii purP gene (MJ0136), which is a signature gene for Archaea. As is characteristic of reactions catalyzed by this family of enzymes, the other products of the reaction, ADP and P(i), were produced stoichiometrically with the amount of ATP, formate, and AICAR used. Formyl phosphate was found to substitute for ATP and formate in the reaction, yet the methylene analog, phosphonoacetaldehyde, was not an inhibitor or substrate for the reaction. The enzyme, along with PurO, which catalyzes the cyclization of FAICAR to inosine 5'-monophosphate, catalyzes the same overall transformation in purine biosynthesis as is accomplished by PurH in bacteria and eukaryotes. No homology exists between PurH and either PurO or PurP. 1H NMR and gas chromatography-mass spectrometry analysis of an M. jannaschii cell extract showed the presence of free formate that can be used by the enzyme for purine biosynthesis. This formate arises by the reduction of CO2 with hydrogen; this was demonstrated by incorporating 13C into the formate when M. jannaschii cell extracts were incubated with H13CO3- and hydrogen gas. The presence of this signature gene in all of the Archaea indicates the presence of a purine biosynthetic pathway proceeding in the absence of folate coenzymes.     

Metabolic anomalies in cri du chat syndrome (5p-) lymphocytes and de novo purine synthesis.

Having previously demonstrated that patients with cri du chat, 5p- syndrome, have a highly significant excess of the plasmatic and urinary relative amount of asparagine and aspartate, the authors tested the hypothesis according to which this excess could be in relation with a defect of purine metabolism. Using a previously reported in vitro assay, they found a paradoxal increase in the mitotic index in the presence of L-alanosine in lymphocyte cultures of patients with 5p- who were on no medication. They also observed particularly severe toxicity to HAT medium. This response, apparently characteristic for 5p- syndrome, was highly significant when compared to the one observed in samples of normal controls, of patients with mental retardation of various etiologies, patients with Down syndrome or with Xqfra syndrome. When patients with cri du chat syndrome received inosine with folinic acid, an inversion of their response to alanosine was observed as well as the normalization of their response to HAT medium. These findings suggest that deletion of 5p14-5p15 leads to some impairment of de novo purine synthesis, the implications of these findings are discussed.     

The PROS-35 gene encodes the 35 kd protein subunit of Drosophila melanogaster proteasome.

The proteasome is a multicatalytic proteinase complex composed of nonidentical protein subunits. We have isolated a cDNA clone encoding the 35 kd proteasome subunit of Drosophila melanogaster and propose the designation PROS-35 for the corresponding gene. The deduced amino acid sequence reveals a region of striking homology to a tyrosine phosphorylation site of viral and cellular proteins suggesting a potential regulatory function for the 35 kd subunit within the proteinase complex. Immunocytochemical experiments reveal a tissue-dependent differential distribution of the proteasome between the nucleus and cytoplasm. In addition developmental analysis shows that the proteasome is highly expressed in the CNS of stage-16 embryos and in cardia, ventriculus and ovaries of adult flies. These data suggest a tissue- and development-dependent distribution of the proteasome in D. melanogaster.     

Functional analysis of the pyrimidine de novo synthesis pathway in solanaceous species

Pyrimidines are particularly important in dividing tissues as building blocks for nucleic acids, but they are equally important for many biochemical processes, including sucrose and cell wall polysaccharide metabolism. In recent years, the molecular organization of nucleotide biosynthesis in plants has been analyzed. Here, we present a functional analysis of the pyrimidine de novo synthesis pathway. Each step in the pathway was investigated using transgenic plants with reduced expression of the corresponding gene to identify controlling steps and gain insights into the phenotypic and metabolic consequences. Inhibition of expression of 80% based on steady-state mRNA level did not lead to visible phenotypes. Stepwise reduction of protein abundance of Asp transcarbamoylase or dihydro orotase resulted in a corresponding inhibition of growth. This was not accompanied by pleiotropic effects or by changes in the developmental program. A more detailed metabolite analysis revealed slightly different responses in roots and shoots of plants with decreased abundance of proteins involved in pyrimidine de novo synthesis. Whereas in leaves the nucleotide and amino acid levels were changed only in the very strong inhibited plants, the roots show a transient increase of these metabolites in intermediate plants followed by a decrease in the strong inhibited plants. Growth analysis revealed that elongation rates and number of organs per plant were reduced, without large changes in the average cell size. It is concluded that reduced pyrimidine de novo synthesis is compensated for by reduction in growth rates, and the remaining nucleotide pools are sufficient for running basic metabolic processes.     

A gene, cobA + hemD, from Selenomonas ruminantium encodes a bifunctional enzyme involved in the synthesis of vitamin B12.

Coenzymes derived from vitamin B12 (cyanocobalamin) are particularly important for core metabolism in ruminant animals. Selenomonas ruminantium, a Gram-positive obligate anaerobe isolated from cattle, is the main contributor of vitamin B12 to such ruminant animals. In nature, there are both aerobic and anaerobic pathways for B12 synthesis - the latter is only partly elucidated. Until now, there has been no investigation of B12 synthesis in S. ruminantium, which must use an anaerobic pathway. This paper reports the cloning of the chromosomal operon from S. ruminantium which is responsible for the first committed steps in corrinoid synthesis. Five open reading frames were found in the cloned fragment. All deduced amino acid sequences had similarity to defined proteins in the databases that are involved in porphyrin and corrin synthesis. Of particular interest is the gene designated cobA + hemD, which encodes a single polypeptide possessing two catalytic functions - uroporphyrinogen III synthase and uroporphyrinogen III 2,7-methyltransferase. This enzyme converts hydroxymethylbilane to precorrin-2. The functions of the protein coded by cobA + hemD were established by heterologous expression in Escherichia coli. The CobA activity has been demonstrated for three distinct types of proteins - monofunctional, bifunctional with siroheme formation and, this report, bifunctional with uroporphyrinogen III synthesis. The type found in S. ruminantium (cobA + hemD) is probably restricted to obligately anaerobic fermentative bacteria.