Identification of the genes encoding enzymes involved in the early biosynthetic pathway of pteridines in<i>Synechocystis</i>sp. PCC 6803

Lee, Soo Woong; Lee, Hee Woo; Chung, Hyewon; Kim, Young-A; Kim, Yeon Jeong; Hahn, Yoon-Soo; Chung, Jae Hoon; Park, Young Shik

doi:10.1111/j.1574-6968.1999.tb13658.x

Cited by 24 publications

(4 citation statements)

References 20 publications

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“…PTPS activity was enhanced (75 %) by Mg 2+ , completely inhibited by Co 2+ , Mn 2+ and Cu 2+ , slightly inhibited by Fe 2+ , but unaffected by DTT. A positive effect of Mg 2+ on PTPS activity has been reported before (Lee et al , 1999; Park et al , 1990). SR activity was completely inhibited by Cu 2+ , inhibited to different degrees by Co 2+ , Fe 2+ and Mn 2+ , but unaffected by K + , Mg 2+ and DTT ().…”

Section: Resultssupporting

confidence: 72%

Biochemical characterization of the tetrahydrobiopterin synthesis pathway in the oleaginous fungus Mortierella alpina

et al. 2011

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We characterized the de novo biosynthetic pathway of tetrahydrobiopterin (BH 4 ) in the lipidproducing fungus Mortierella alpina. The BH 4 cofactor is essential for various cell processes, and is probably present in every cell or tissue of higher organisms. Genes encoding two copies of GTP cyclohydrolase I (GTPCH-1 and GTPCH-2) for the conversion of GTP to dihydroneopterin triphosphate (H 2 -NTP), 6-pyruvoyltetrahydropterin synthase (PTPS) for the conversion of H 2 -NTP to 6-pyruvoyltetrahydropterin (PPH 4 ), and sepiapterin reductase (SR) for the conversion of PPH 4 to BH 4 , were expressed heterologously in Escherichia coli. The recombinant enzymes were produced as His-tagged fusion proteins and were purified to homogeneity to investigate their enzymic activities. Enzyme products were analysed by HPLC and electrospray ionization-MS. Kinetic parameters and other properties of GTPCH, PTPS and SR were investigated. Physiological roles of BH 4 in M. alpina are discussed, and comparative analyses between GTPCH, PTPS and SR proteins and other homologous proteins were performed. The presence of two functional GTPCH enzymes has, as far as we are aware, not been reported previously, reflecting the unique ability of this fungus to synthesize both BH 4 and folate, using the GTPCH product as a common substrate. To our knowledge, this study is the first to report the comprehensive characterization of a BH 4 biosynthesis pathway in a fungus.

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Section: Resultssupporting

confidence: 72%

Biochemical characterization of the tetrahydrobiopterin synthesis pathway in the oleaginous fungus Mortierella alpina

et al. 2011

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“…Finally, sll0330 accumulated to approximately 10-times higher levels and was among the three most strongly induced mRNAs. The corresponding protein is annotated as sepiapterin reductase or 3-ketoacyl-ACP reductase; however, sepiapterin reductase activity was not confirmed in vitro [ 40 ].…”

Section: Resultsmentioning

confidence: 99%

Insights into isoprene production using the cyanobacterium Synechocystis sp. PCC 6803

Pade

Erdmann

Enke³

et al. 2016

Biotechnol Biofuels

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BackgroundCyanobacteria are phototrophic prokaryotes that convert inorganic carbon as CO2 into organic compounds at the expense of light energy. They need only inorganic nutrients and can be cultivated to high densities using non-arable land and seawater. This has made cyanobacteria attractive organisms for the production of biofuels and chemical feedstock. Synechocystis sp. PCC 6803 is one of the most widely used cyanobacterial model strains. Based on its available genome sequence and genetic tools, Synechocystis has been genetically modified to produce different biotechnological products. Efficient isoprene production is an attractive goal because this compound is widely used as chemical feedstock.ResultsHere, we report on our attempts to generate isoprene-producing strains of Synechocystis using a plasmid-based strategy. As previously reported, a codon-optimized plant isoprene synthase (IspS) was expressed under the control of different Synechocystis promoters that ensure strong constitutive or light-regulated ispS expression. The expression of the ispS gene was quantified by qPCR and Western blotting, while the amount of isoprene was quantified using GC—MS. In addition to isoprene measurements in the headspace of closed culture vessels, single photon ionization time-of-flight mass spectrometry (SPI-MS) was applied, which allowed online measurements of isoprene production in open-cultivation systems under various conditions. Under standard conditions, a good correlation existed between ispS expression and isoprene production rate. The cultivation of isoprene production strains under NaCl-supplemented conditions decreased isoprene production despite enhanced ispS mRNA levels. The characterization of the metabolome of isoprene-producing strains indicated that isoprene production might be limited by insufficient precursor levels. Transcriptomic analysis revealed the upregulation of mRNA and regulatory RNAs characteristic of acclimation to metabolic stress.ConclusionsOur best production strains produced twofold higher isoprene amounts in the presence of low NaCl concentrations than previously reported strains. These results will guide future attempts to establish isoprene production in cyanobacterial hosts.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0503-4) contains supplementary material, which is available to authorized users.

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“…The pfshmt gene from P. falciparum (PFL1720w) [41] encodes a product that has been functionally characterized as a conventional cytoplasmic SHMT [21-23]. However, a predicted SHMT-like gene product (PfSHMTm, encoded on PF14_ 0534) was also identified that carries a putative mitochondrial signal sequence [24] with 18% amino acid identity and 44% similarity to PfSHMTc, but lacks almost all (16 of 21) of the known, very highly conserved residues [42] contributing to the active site in SHMT orthologues from other organisms, whether cytoplasmic or organellar (See Additional file 2 Sequence alignments of the PfSHMT isoforms).…”

Section: Resultsmentioning

confidence: 99%

Dynamic subcellular localization of isoforms of the folate pathway enzyme serine hydroxymethyltransferase (SHMT) through the erythrocytic cycle of Plasmodium falciparum

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Müller

Mitchell

et al. 2010

Malar J

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BackgroundThe folate pathway enzyme serine hydroxymethyltransferase (SHMT) converts serine to glycine and 5,10-methylenetetrahydrofolate and is essential for the acquisition of one-carbon units for subsequent transfer reactions. 5,10-methylenetetrahydrofolate is used by thymidylate synthase to convert dUMP to dTMP for DNA synthesis. In Plasmodium falciparum an enzymatically functional SHMT (PfSHMTc) and a related, apparently inactive isoform (PfSHMTm) are found, encoded by different genes. Here, patterns of localization of the two isoforms during the parasite erythrocytic cycle are investigated.MethodsPolyclonal antibodies were raised to PfSHMTc and PfSHMTm, and, together with specific markers for the mitochondrion and apicoplast, were employed in quantitative confocal fluorescence microscopy of blood-stage parasites.ResultsAs well as the expected cytoplasmic occupancy of PfSHMTc during all stages, localization into the mitochondrion and apicoplast occurred in a stage-specific manner. Although early trophozoites lacked visible organellar PfSHMTc, a significant percentage of parasites showed such fluorescence during the mid-to-late trophozoite and schizont stages. In the case of the mitochondrion, the majority of parasites in these stages at any given time showed no marked PfSHMTc fluorescence, suggesting that its occupancy of this organelle is of limited duration. PfSHMTm showed a distinctly more pronounced mitochondrial location through most of the erythrocytic cycle and GFP-tagging of its N-terminal region confirmed the predicted presence of a mitochondrial signal sequence. Within the apicoplast, a majority of mitotic schizonts showed a marked concentration of PfSHMTc, whose localization in this organelle was less restricted than for the mitochondrion and persisted from the late trophozoite to the post-mitotic stages. PfSHMTm showed a broadly similar distribution across the cycle, but with a distinctive punctate accumulation towards the ends of elongating apicoplasts. In very late post-mitotic schizonts, both PfSHMTc and PfSHMTm were concentrated in the central region of the parasite that becomes the residual body on erythrocyte lysis and merozoite release.ConclusionsBoth PfSHMTc and PfSHMTm show dynamic, stage-dependent localization among the different compartments of the parasite and sequence analysis suggests they may also reversibly associate with each other, a factor that may be critical to folate cofactor function, given the apparent lack of enzymic activity of PfSHMTm.

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Identification of the genes encoding enzymes involved in the early biosynthetic pathway of pteridines inSynechocystissp. PCC 6803

Cited by 24 publications

References 20 publications

Biochemical characterization of the tetrahydrobiopterin synthesis pathway in the oleaginous fungus Mortierella alpina

Biochemical characterization of the tetrahydrobiopterin synthesis pathway in the oleaginous fungus Mortierella alpina

Insights into isoprene production using the cyanobacterium Synechocystis sp. PCC 6803

Dynamic subcellular localization of isoforms of the folate pathway enzyme serine hydroxymethyltransferase (SHMT) through the erythrocytic cycle of Plasmodium falciparum

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