Converting Escherichia coli to a Synthetic Methylotroph Growing Solely on Methanol.

Methanol, being electron rich and derivable from methane or CO2, is a potentially renewable one-carbon (C1) feedstock for microorganisms. Although the ribulose monophosphate (RuMP) cycle used by methylotrophs to assimilate methanol differs from the typical sugar metabolism by only three enzymes, turning a non-methylotrophic organism to a synthetic methylotroph that grows to a high cell density has been challenging. Here we reprogrammed E. coli using metabolic robustness criteria followed by laboratory evolution to establish a strain that can efficiently utilize methanol as the sole carbon source. This synthetic methylotroph alleviated a so far uncharacterized hurdle, DNA-protein crosslinking (DPC), by insertion sequence (IS)-mediated copy number variations (CNVs) and balanced the metabolic flux by mutations. Being capable of growing at a rate comparable with natural methylotrophs in a wide range of methanol concentrations, this synthetic methylotrophic strain illustrates genome editing and evolution for microbial tropism changes and expands the scope of biological C1 conversion.

The immune response to RNA suppresses nucleic acid synthesis by limiting ribose 5-phosphate.

During infection viruses hijack host cell metabolism to promote their replication. Here, analysis of metabolite alterations in macrophages exposed to poly I:C recognises that the antiviral effector Protein Kinase RNA-activated (PKR) suppresses glucose breakdown within the pentose phosphate pathway (PPP). This pathway runs parallel to central glycolysis and is critical to producing NADPH and pentose precursors for nucleotides. Changes in metabolite levels between wild-type and PKR-ablated macrophages show that PKR controls the generation of ribose 5-phosphate, in a manner distinct from its established function in gene expression but dependent on its kinase activity. PKR phosphorylates and inhibits the Ribose 5-Phosphate Isomerase A (RPIA), thereby preventing interconversion of ribulose- to ribose 5-phosphate. This activity preserves redox control but decreases production of ribose 5-phosphate for nucleotide biosynthesis. Accordingly, the PKR-mediated immune response to RNA suppresses nucleic acid production. In line, pharmacological targeting of the PPP during infection decreases the replication of the Herpes simplex virus. These results identify an immune response-mediated control of host cell metabolism and suggest targeting the RPIA as a potential innovative antiviral treatment.

Pyruvate kinase 2 from Synechocystis sp. PCC 6803 increased substrate affinity via glucose-6-phosphate and ribose-5-phosphate for phosphoenolpyruvate consumption.

Pyruvate kinase (Pyk, EC 2.7.1.40) is a glycolytic enzyme that generates pyruvate and adenosine triphosphate (ATP) from phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP), respectively. Pyk couples pyruvate and tricarboxylic acid metabolisms. Synechocystis sp. PCC 6803 possesses two pyk genes (encoded pyk1, sll0587 and pyk2, sll1275). A previous study suggested that pyk2 and not pyk1 is essential for cell viability; however, its biochemical analysis is yet to be performed. Herein, we biochemically analyzed Synechocystis Pyk2 (hereafter, SyPyk2). The optimum pH and temperature of SyPyk2 were 7.0 and 55 °C, respectively, and the Km values for PEP and ADP under optimal conditions were 1.5 and 0.053 mM, respectively. SyPyk2 is activated in the presence of glucose-6-phosphate (G6P) and ribose-5-phosphate (R5P); however, it remains unaltered in the presence of adenosine monophosphate (AMP) or fructose-1,6-bisphosphate. These results indicate that SyPyk2 is classified as PykA type rather than PykF, stimulated by sugar monophosphates, such as G6P and R5P, but not by AMP. SyPyk2, considering substrate affinity and effectors, can play pivotal roles in sugar catabolism under nonphotosynthetic conditions.

Cysteine and iron accelerate the formation of ribose-5-phosphate, providing insights into the evolutionary origins of the metabolic network structure.

The structure of the metabolic network is highly conserved, but we know little about its evolutionary origins. Key for explaining the early evolution of metabolism is solving a chicken-egg dilemma, which describes that enzymes are made from the very same molecules they produce. The recent discovery of several nonenzymatic reaction sequences that topologically resemble central metabolism has provided experimental support for a "metabolism first" theory, in which at least part of the extant metabolic network emerged on the basis of nonenzymatic reactions. But how could evolution kick-start on the basis of a metal catalyzed reaction sequence, and how could the structure of nonenzymatic reaction sequences be imprinted on the metabolic network to remain conserved for billions of years? We performed an in vitro screening where we add the simplest components of metabolic enzymes, proteinogenic amino acids, to a nonenzymatic, iron-driven reaction network that resembles glycolysis and the pentose phosphate pathway (PPP). We observe that the presence of the amino acids enhanced several of the nonenzymatic reactions. Particular attention was triggered by a reaction that resembles a rate-limiting step in the oxidative PPP. A prebiotically available, proteinogenic amino acid cysteine accelerated the formation of RNA nucleoside precursor ribose-5-phosphate from 6-phosphogluconate. We report that iron and cysteine interact and have additive effects on the reaction rate so that ribose-5-phosphate forms at high specificity under mild, metabolism typical temperature and environmental conditions. We speculate that accelerating effects of amino acids on rate-limiting nonenzymatic reactions could have facilitated a stepwise enzymatization of nonenzymatic reaction sequences, imprinting their structure on the evolving metabolic network.

Histones participate in base excision repair of 8-oxodGuo by transiently cross-linking with active repair intermediates in nucleosome core particles.

8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) is a biomarker of oxidative DNA damage and can be repaired by hOGG1 and APE1 via the base excision repair (BER) pathway. In this work, we studied coordinated BER of 8-oxodGuo by hOGG1 and APE1 in nucleosome core particles and found that histones transiently formed DNA-protein cross-links (DPCs) with active repair intermediates such as 3'-phospho-α,ß-unsaturated aldehyde (PUA) and 5'-deoxyribosephosphate (dRP). The effects of histone participation could be beneficial or deleterious to the BER process, depending on the circumstances. In the absence of APE1, histones enhanced the AP lyase activity of hOGG1 by cross-linking with 3'-PUA. However, the formed histone-PUA DPCs hampered the subsequent repair process. In the presence of APE1, both the AP lyase activity of hOGG1 and the formation of histone-PUA DPCs were suppressed. In this case, histones could catalyse removal of the 5'-dRP by transiently cross-linking with the active intermediate. That is, histones promoted the repair by acting as 5'-dRP lyases. Our findings demonstrate that histones participate in multiple steps of 8-oxodGuo repair in nucleosome core particles, highlighting the diverse roles that histones may play during DNA repair in eukaryotic cells.

A rare bacterial RNA motif is implicated in the regulation of the purF gene whose encoded enzyme synthesizes phosphoribosylamine.

The Fibro-purF motif is a putative structured noncoding RNA domain that was discovered previously in species of Fibrobacter by using comparative sequence analysis methods. An updated bioinformatics search yielded a total of only 30 unique-sequence representatives, exclusively found upstream of the purF gene that codes for the enzyme amidophosphoribosyltransferase. This enzyme synthesizes the compound 5-phospho-D-ribosylamine (PRA), which is the first committed step in purine biosynthesis. The consensus model for Fibro-purF motif RNAs includes a predicted three-stem junction that carries numerous conserved nucleotide positions within the regions joining the stems. This architecture appears to be of sufficient size and complexity for the formation of the ligand-binding aptamer portion of a riboswitch. In this study, we conducted biochemical analyses of a representative Fibro-purF motif RNA to confirm that the RNA generally folds according to the predicted consensus model. However, due to the instability of PRA, binding of this ligand candidate by the RNA could not be directly assessed. Genetic analyses were used to demonstrate that Fibro-purF motif RNAs regulate gene expression in accordance with predicted PRA concentrations. These findings indicate that Fibro-purF motif RNAs are genetic regulation elements that likely suppress PRA biosynthesis when sufficient levels of this purine precursor are present.

The plastidial pentose phosphate pathway is essential for postglobular embryo development in Arabidopsis.

Large numbers of genes essential for embryogenesis in Arabidopsis encode enzymes of plastidial metabolism. Disruption of many of these genes results in embryo arrest at the globular stage of development. However, the cause of lethality is obscure. We examined the role of the plastidial oxidative pentose phosphate pathway (OPPP) in embryo development. In nonphotosynthetic plastids the OPPP produces reductant and metabolic intermediates for central biosynthetic processes. Embryos with defects in various steps in the oxidative part of the OPPP had cell division defects and arrested at the globular stage, revealing an absolute requirement for the production via these steps of ribulose-5-phosphate. In the nonoxidative part of the OPPP, ribulose-5-phosphate is converted to ribose-5-phosphate (R5P)-required for purine nucleotide and histidine synthesis-and subsequently to erythrose-4-phosphate, which is required for synthesis of aromatic amino acids. We show that embryo development through the globular stage specifically requires synthesis of R5P rather than erythrose-4-phosphate. Either a failure to convert ribulose-5-phosphate to R5P or a block in purine nucleotide biosynthesis beyond R5P perturbs normal patterning of the embryo, disrupts endosperm development, and causes early developmental arrest. We suggest that seed abortion in mutants unable to synthesize R5P via the oxidative part of the OPPP stems from a lack of substrate for synthesis of purine nucleotides, and hence nucleic acids. Our results show that the plastidial OPPP is essential for normal developmental progression as well as for growth in the embryo.

Unlike the Escherichia coli counterpart, archaeal RNase HII cannot process ribose monophosphate abasic sites and oxidized ribonucleotides embedded in DNA.

The presence of ribonucleoside monophosphates (rNMPs) in nuclear DNA decreases genome stability. To ensure survival despite rNMP insertions, cells have evolved a complex network of DNA repair mechanisms, in which the ribonucleotide excision repair pathway, initiated by type 2 RNase H (RNase HII/2), plays a major role. We recently demonstrated that eukaryotic RNase H2 cannot repair damage, that is, ribose monophosphate abasic (both apurinic or apyrimidinic) site (rAP) or oxidized rNMP embedded in DNA. Currently, it remains unclear why RNase H2 is unable to repair these modified nucleic acids having either only a sugar moiety or an oxidized base. Here, we compared the endoribonuclease specificity of the RNase HII enzymes from the archaeon Pyrococcus abyssi and the bacterium Escherichia coli, examining their ability to process damaged rNMPs embedded in DNA in vitro We found that E. coli RNase HII cleaves both rAP and oxidized rNMP sites. In contrast, like the eukaryotic RNase H2, P. abyssi RNase HII did not display any rAP or oxidized rNMP incision activities, even though it recognized them. Notably, the archaeal enzyme was also inactive on a mismatched rNMP, whereas the E. coli enzyme displayed a strong preference for the mispaired rNMP over the paired rNMP in DNA. On the basis of our biochemical findings and also structural modeling analyses of RNase HII/2 proteins from organisms belonging to all three domains of life, we propose that RNases HII/2's dual roles in ribonucleotide excision repair and RNA/DNA hydrolysis result in limited acceptance of modified rNMPs embedded in DNA.

Ribose-5-phosphate isomerases: characteristics, structural features, and applications.

Ribose-5-phosphate isomerase (Rpi, EC 5.3.1.6) is widespread in microorganisms, animals, and plants. It has a pivotal role in the pentose phosphate pathway and responsible for catalyzing the isomerization between D-ribulose 5-phosphate and D-ribose 5-phosphate. In recent years, Rpi has received considerable attention as a multipurpose biocatalyst for production of rare sugars, including D-allose, L-rhamnulose, L-lyxose, and L-tagatose. Besides, it has been thought of as a potential drug target in the treatment of trypanosomatid-caused diseases such as Chagas' disease, leishmaniasis, and human African trypanosomiasis. Despite increased research activities, up to now, no systematic review of Rpi has been published. To fill this gap, this paper provides detailed information about the enzymatic properties of various Rpis. Furthermore, structural features, catalytic mechanism, and molecular modifications of Rpis are summarized based on extensive crystal structure research. Additionally, the applications of Rpi in rare sugar production and the role of Rpi in trypanocidal drug design are reviewed. Key points • Fundamental properties of various ribose-5-phosphate isomerases (Rpis). • Differences in crystal structure and catalytic mechanism between RpiA and RpiB. • Application of Rpi as a rare sugar producer and a potential drug target.

Structural insights into the catalytic mechanism of 5-methylthioribose 1-phosphate isomerase.

5-Methylthioribose 1-phosphate isomerase (M1Pi) is a crucial enzyme involved in the universally conserved methionine salvage pathway (MSP) where it is known to catalyze the conversion of 5-methylthioribose 1-phosphate (MTR-1-P) to 5-methylthioribulose 1-phosphate (MTRu-1-P) via a mechanism which remains unspecified till date. Furthermore, although M1Pi has a discrete function, it surprisingly shares high structural similarity with two functionally non-related proteins such as ribose-1,5-bisphosphate isomerase (R15Pi) and the regulatory subunits of eukaryotic translation initiation factor 2B (eIF2B). To identify the distinct structural features that lead to divergent functional obligations of M1Pi as well as to understand the mechanism of enzyme catalysis, the crystal structure of M1Pi from a hyperthermophilic archaeon Pyrococcus horikoshii OT3 was determined. A meticulous structural investigation of the dimeric M1Pi revealed the presence of an N-terminal extension and a hydrophobic patch absent in R15Pi and the regulatory α-subunit of eIF2B. Furthermore, unlike R15Pi in which a kink formation is observed in one of the helices, the domain movement of M1Pi is distinguished by a forward shift in a loop covering the active-site pocket. All these structural attributes contribute towards a hydrophobic microenvironment in the vicinity of the active site of the enzyme making it favorable for the reaction mechanism to commence. Thus, a hydrophobic active-site microenvironment in addition to the availability of optimal amino-acid residues surrounding the catalytic residues in M1Pi led us to propose its probable reaction mechanism via a cis-phosphoenolate intermediate formation.

Vestibular neurons with direct projections to the solitary nucleus in the rat.

Anterograde and retrograde tract tracing were combined with neurotransmitter and modulator immunolabeling to identify the chemical anatomy of vestibular nuclear neurons with direct projections to the solitary nucleus in rats. Direct, sparsely branched but highly varicose axonal projections from neurons in the caudal vestibular nuclei to the solitary nucleus were observed. The vestibular neurons giving rise to these projections were predominantly located in ipsilateral medial vestibular nucleus. The cell bodies were intensely glutamate immunofluorescent, and their axonal processes contained vesicular glutamate transporter 2, supporting the interpretation that the cells utilize glutamate for neurotransmission. The glutamate-immunofluorescent, retrogradely filled vestibular cells also contained the neuromodulator imidazoleacetic acid ribotide, which is an endogenous CNS ligand that participates in blood pressure regulation. The vestibulo-solitary neurons were encapsulated by axo-somatic GABAergic terminals, suggesting that they are under tight inhibitory control. The results establish a chemoanatomical basis for transient vestibular activation of the output pathways from the caudal and intermediate regions of the solitary nucleus. In this way, changes in static head position and movement of the head in space may directly influence heart rate, blood pressure, respiration, as well as gastrointestinal motility. This would provide one anatomical explanation for the synchronous heart rate and blood pressure responses observed after peripheral vestibular activation, as well as disorders ranging from neurogenic orthostatic hypotension, postural orthostatic tachycardia syndrome, and vasovagal syncope to the nausea and vomiting associated with motion sickness.NEW & NOTEWORTHY Vestibular neurons with direct projections to the solitary nucleus utilize glutamate for neurotransmission, modulated by imidazoleacetic acid ribotide. This is the first direct demonstration of the chemical neuroanatomy of the vestibulo-solitary pathway.

Unencumbered Pol ß lyase activity in nucleosome core particles.

Packaging of DNA into the nucleosome core particle (NCP) is considered to exert constraints to all DNA-templated processes, including base excision repair where Pol ß catalyzes two key enzymatic steps: 5'-dRP lyase gap trimming and template-directed DNA synthesis. Despite its biological significance, knowledge of Pol ß activities on NCPs is still limited. Here, we show that removal of the 5'-dRP block by Pol ß is unaffected by NCP constraints at all sites tested and is even enhanced near the DNA ends. In contrast, strong inhibition of DNA synthesis is observed. These results indicate 5'-dRP gap trimming proceeds unperturbed within the NCP; whereas, gap filling is strongly limited. In the absence of additional factors, base excision repair in NCPs will stall at the gap-filling step.

A Phosphofructokinase Homolog from Pyrobaculum calidifontis Displays Kinase Activity towards Pyrimidine Nucleosides and Ribose 1-Phosphate.

The genome of the hyperthermophilic archaeon Pyrobaculum calidifontis contains an open reading frame, Pcal_0041, annotated as encoding a PfkB family ribokinase, consisting of phosphofructokinase and pyrimidine kinase domains. Among the biochemically characterized enzymes, the Pcal_0041 protein was 37% identical to the phosphofructokinase (Ape_0012) from Aeropyrum pernix, which displayed kinase activity toward a broad spectrum of substrates, including sugars, sugar phosphates, and nucleosides, and 36% identical to a phosphofructokinase from Desulfurococcus amylolyticus To examine the biochemical function of the Pcal_0041 protein, we cloned and expressed the gene and purified the recombinant protein. Although the Pcal_0041 protein contained a putative phosphofructokinase domain, it exhibited only low levels of phosphofructokinase activity. The recombinant enzyme catalyzed the phosphorylation of nucleosides and, to a lower extent, sugars and sugar phosphates. Surprisingly, among the substrates tested, the highest activity was detected with ribose 1-phosphate (R1P), followed by cytidine and uridine. The catalytic efficiency (kcat/Km ) toward R1P was 11.5 mM-1 · s-1 ATP was the most preferred phosphate donor, followed by GTP. Activity measurements with cell extracts of P. calidifontis indicated the presence of nucleoside phosphorylase activity, which would provide the means to generate R1P from nucleosides. The study suggests that, in addition to the recently identified ADP-dependent ribose 1-phosphate kinase (R1P kinase) in Thermococcus kodakarensis that functions in the pentose bisphosphate pathway, R1P kinase is also present in members of the Crenarchaeota.IMPORTANCE The discovery of the pentose bisphosphate pathway in Thermococcus kodakarensis has clarified how this archaeon can degrade nucleosides. Homologs of the enzymes of this pathway are present in many members of the Thermococcales, suggesting that this metabolism occurs in these organisms. However, this is not the case in other archaea, and degradation mechanisms for nucleosides or ribose 1-phosphate are still unknown. This study reveals an important first step in understanding nucleoside metabolism in Crenarchaeota and identifies an ATP-dependent ribose 1-phosphate kinase in Pyrobaculum calidifontis The enzyme is structurally distinct from previously characterized archaeal members of the ribokinase family and represents a group of proteins found in many crenarchaea.

Engineering Artificial Fusion Proteins for Enhanced Methanol Bioconversion.

Methanol is a low-cost and abundantly available feedstock derived from natural gas and syngas. Although bioconversion holds promise for producing desired chemicals from methanol under economically viable operating conditions, the efficiency is limited by unfavorable kinetics of methanol oxidation and assimilation. Herein, artificial fusion proteins were engineered to enhance methanol bioconversion. Nicotinamide adenine dinucleotide (NAD)-dependent methanol dehydrogenase (Mdh), 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloisomerase (Phi) from different sources were first screened for catalytic activity. Next, we designed six fusion proteins using the best enzyme candidates and flexible linkers. Fusing Mdh with Hps or Hps-Phi increased the Vmax of methanol oxidation up to 5.8-fold, and enhanced methanol conversion to fructose-6-phosphate up to 1.3-fold. Interestingly, fusion engineering changed the polymerization states of proteins and produced larger multimers, which may be responsible for the changed catalytic characteristics. This fusion engineering approach can be coupled with other metabolic engineering strategies for enhanced methanol bioconversion to valuable chemicals.

2-Deoxy-D-ribose-5-phosphate aldolase (DERA): applications and modifications.

2-Deoxy-D-ribose-5-phosphate aldolase (DERA) is a class I aldolase that offers access to several building blocks for organic synthesis. It catalyzes the stereoselective C-C bond formation between acetaldehyde and numerous other aldehydes. However, the practical application of DERA as a biocatalyst is limited by its poor tolerance towards industrially relevant concentrations of aldehydes, in particular acetaldehyde. Therefore, the development of proper experimental conditions, including protein engineering and/or immobilization on appropriate supports, is required. The present review is aimed to provide a brief overview of DERA, its history, and progress made in understanding the functioning of the enzyme. Furthermore, the current understanding regarding aldehyde resistance of DERA and the various optimizations carried out to modify this property are discussed.

Isotope-specific and amino acid-specific heavy atom substitutions alter barrier crossing in human purine nucleoside phosphorylase.

Computational chemistry predicts that atomic motions on the femtosecond timescale are coupled to transition-state formation (barrier-crossing) in human purine nucleoside phosphorylase (PNP). The prediction is experimentally supported by slowed catalytic site chemistry in isotopically labeled PNP (13C, 15N, and 2H). However, other explanations are possible, including altered volume or bond polarization from carbon-deuterium bonds or propagation of the femtosecond bond motions into slower (nanoseconds to milliseconds) motions of the larger protein architecture to alter catalytic site chemistry. We address these possibilities by analysis of chemistry rates in isotope-specific labeled PNPs. Catalytic site chemistry was slowed for both [2H]PNP and [13C, 15N]PNP in proportion to their altered protein masses. Secondary effects emanating from carbon-deuterium bond properties can therefore be eliminated. Heavy-enzyme mass effects were probed for local or global contributions to catalytic site chemistry by generating [15N, 2H]His8-PNP. Of the eight His per subunit, three participate in contacts to the bound reactants and five are remote from the catalytic sites. [15N, 2H]His8-PNP had reduced catalytic site chemistry larger than proportional to the enzymatic mass difference. Altered barrier crossing when only His are heavy supports local catalytic site femtosecond perturbations coupled to transition-state formation. Isotope-specific and amino acid specific labels extend the use of heavy enzyme methods to distinguish global from local isotope effects.

Identification of a d-Arabinose-5-Phosphate Isomerase in the Gram-Positive Clostridium tetani.

d-Arabinose-5-phosphate (A5P) isomerases (APIs) catalyze the interconversion of d-ribulose-5-phosphate and d-arabinose-5-phosphate. Various Gram-negative bacteria, such as the uropathogenic Escherichia coli strain CFT073, contain multiple API paralogs (KdsD, GutQ, KpsF, and c3406) that have been assigned various cellular functions. The d-arabinose-5-phosphate formed by these enzymes seems to play important roles in the biosynthesis of lipopolysaccharide (LPS) and group 2 K-antigen capsules, as well as in the regulation of the cellular d-glucitol uptake and uropathogenic infectivity/virulence. The genome of a Gram-positive pathogenic bacterium, Clostridium tetani, contains a gene encoding a putative API, C. tetani API (CtAPI), even though C. tetani lacks both LPS and capsid biosynthetic genes. To better understand the physiological role of d-arabinose-5-phosphate in this Gram-positive organism, recombinant CtAPI was purified and characterized. CtAPI displays biochemical characteristics similar to those of APIs from Gram-negative organisms and complements the API deficiency of an E. coli API knockout strain. Thus, CtAPI represents the first d-arabinose-5-phosphate isomerase to be identified and characterized from a Gram-positive bacterium.IMPORTANCE The genome of Clostridium tetani, a pathogenic Gram-positive bacterium and the causative agent of tetanus, contains a gene (the CtAPI gene) that shares high sequence similarity with those of genes encoding d-arabinose-5-phosphate isomerases. APIs play an important role within Gram-negative bacteria in d-arabinose-5-phosphate production for lipopolysaccharide biosynthesis, capsule formation, and regulation of cellular d-glucitol uptake. The significance of our research is in identifying and characterizing CtAPI, the first Gram-positive API. Our findings show that CtAPI is specific to the interconversion of arabinose-5-phosphate and ribulose-5-phosphate while having no activity with the other sugars and sugar phosphates tested. We have speculated a regulatory role for this API in C. tetani, an organism that does not produce lipopolysaccharide.

Transketolase activity modulates glycerol-3-phosphate levels in Escherichia coli.

Transketolase activity provides an important link between the metabolic pathways of glycolysis and pentose phosphate shunt and catalyzes inter-conversions between pentose phosphates and glycolytic intermediates. It is widely conserved in life forms. A genetic screen for suppression of the growth defect of Escherichia coli tktA tktB mutant in LB medium revealed two mutations, one that rendered the glpK expression constitutive and another that inactivated deoB. Characterizing these mutations aided in uncovering the role of ribose-5-P (a transketolase substrate) as an inhibitor of glycerol assimilation and de novo glycerol-3-P synthesis. Using lacZ fusions, we show that ribose-5-P enhances GlpR-mediated repression of the glpFKX operon and inhibits glycerol assimilation. Electrophoretic Mobility Shift Assay (EMSA) showed ribose-5-P made the DNA-GlpR complex less sensitive to the inducer glycerol-3-P. In addition to inhibition of glycerol assimilation, obstruction of ribose-5-P metabolism retards growth from glycerol-3-P limitation. Glucose helps to overcome this limitation through a mechanism involving catabolite repression. To our knowledge, this report is the first to show ribose-5-P can modulate glycerol-3-P concentration in the cell by regulation of glycerol assimilation as well as its de novo synthesis. This regulation could be prevalent in other organisms.

Cell growth and homeostasis are disrupted in arabidopsis rns2-2 mutants missing the main vacuolar RNase activity.

BACKGROUND AND AIMS: Enzymes belonging to the RNase T2 family are essential for normal rRNA turnover in eukaryotes. In Arabidopsis thaliana, this function is performed by RNS2. The null mutant rns2-2 has increased rRNA half-life and constitutive autophagy. The aim of this work was to determine the molecular changes that take place in the rns2-2 mutant that may lead to altered cellular homeostasis, manifested by the observed cellular phenotype. METHODS: To determine the effect of defective rRNA turnover on cellular homeostasis, comparative transcriptome and metabolome analyses of 10-day-old wild-type and rns2-2 seedlings were used to identify molecular processes affected in the mutant. Bioinformatics analyses suggested additional phenotypes that were confirmed through direct plant size measurements and microscopy. KEY RESULTS: Few genes were differentially expressed in the rns2-2 mutant, indicating that control of autophagy in this genotype is mainly achieved at the post-transcriptional level. Among differentially expressed genes, transcripts related to carbon flux processes, particularly the pentose phosphate pathway (PPP), were identified. Metabolite analyses confirmed changes in the levels of PPP intermediates. Genes related to cell wall loosening were also differentially expressed in the mutant, and a decrease in monosaccharide components of cell wall hemicellulose were found. As a potential effect of weaker cell walls, rns2-2 plants are larger than wild-type controls, due to larger cells and increased water content. Elevated levels of reactive oxygen species (ROS) were also measured in rns2-2, and the constitutive autophagy phenotype was blocked by preventing ROS production via NADPH oxidase. CONCLUSIONS: Lack of rRNA recycling in rns2-2 cells triggers a change in carbon flux, which is redirected through the PPP to produce ribose-5-phosphate for de novo nucleoside synthesis. rRNA or ribosome turnover is thus essential for cellular homeostasis, probably through maintenance of nucleoside levels as part of the salvage pathway.

Crystal structures capture three states in the catalytic cycle of a pyridoxal phosphate (PLP) synthase.

PLP synthase (PLPS) is a remarkable single-enzyme biosynthetic pathway that produces pyridoxal 5'-phosphate (PLP) from glutamine, ribose 5-phosphate, and glyceraldehyde 3-phosphate. The intact enzyme includes 12 synthase and 12 glutaminase subunits. PLP synthesis occurs in the synthase active site by a complicated mechanism involving at least two covalent intermediates at a catalytic lysine. The first intermediate forms with ribose 5-phosphate. The glutaminase subunit is a glutamine amidotransferase that hydrolyzes glutamine and channels ammonia to the synthase active site. Ammonia attack on the first covalent intermediate forms the second intermediate. Glyceraldehyde 3-phosphate reacts with the second intermediate to form PLP. To investigate the mechanism of the synthase subunit, crystal structures were obtained for three intermediate states of the Geobacillus stearothermophilus intact PLPS or its synthase subunit. The structures capture the synthase active site at three distinct steps in its complicated catalytic cycle, provide insights into the elusive mechanism, and illustrate the coordinated motions within the synthase subunit that separate the catalytic states. In the intact PLPS with a Michaelis-like intermediate in the glutaminase active site, the first covalent intermediate of the synthase is fully sequestered within the enzyme by the ordering of a generally disordered 20-residue C-terminal tail. Following addition of ammonia, the synthase active site opens and admits the Lys-149 side chain, which participates in formation of the second intermediate and PLP. Roles are identified for conserved Asp-24 in the formation of the first intermediate and for conserved Arg-147 in the conversion of the first to the second intermediate.