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Taifo Mahmud - One of the best experts on this subject based on the ideXlab platform.
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Structure of a Sedoheptulose 7‑Phosphate Cyclase: ValA from Streptomyces hygroscopicus
2016Co-Authors: Kelsey M Kean, Shumpei Asamizu, Taifo Mahmud, Sara J Codding, Andrew P KarplusAbstract:encompass three enzymes involved in producing the core cyclitol structures of pseudoglycosides and similar bioactive natural products. One such enzyme is ValA from Streptomyces hygroscopicus subsp. jinggangensis 5008, which makes 2-epi-5-epi-valiolone as part of the biosynthesis of the agricultural antifungal agent validamycin A. We present, as the first SH7PC structure, the 2.1 Å resolution crystal structure of ValA in complex with NAD+ and Zn2+ cofactors. ValA has a fold and active site organization resembling those of the sugar phosphate cyclase dehydroquinate synthase (DHQS) and contains two notable, previously unrecognized interactions between NAD+ and Asp side chains conserved in all sugar phosphate cyclases that may influence catalysis. Because the domains of ValA adopt a nearly closed conformation even though no sugar substrate is present, comparisons with a ligand-bound DHQS provide a model for aspects of substrate binding. One striking active site difference is a loop that adopts a distinct conformation as a result of an Asp → Asn change with respect to DHQS and alters the identity and orientation of a key Arg residue. This and other active site differences in ValA are mostly localized to areas where the ValA substrate differs from that of DHQS. Sequence comparison
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structure of a Sedoheptulose 7 phosphate cyclase vala from streptomyces hygroscopicus
Biochemistry, 2014Co-Authors: Kelsey M Kean, Shumpei Asamizu, Taifo Mahmud, Sara J Codding, Andrew P KarplusAbstract:Sedoheptulose 7-Phosphate cyclases (SH7PCs) encompass three enzymes involved in producing the core cyclitol structures of pseudoglycosides and similar bioactive natural products. One such enzyme is ValA from Streptomyces hygroscopicus subsp. jinggangensis 5008, which makes 2-epi-5-epi-valiolone as part of the biosynthesis of the agricultural antifungal agent validamycin A. We present, as the first SH7PC structure, the 2.1 A resolution crystal structure of ValA in complex with NAD+ and Zn2+ cofactors. ValA has a fold and active site organization resembling those of the sugar phosphate cyclase dehydroquinate synthase (DHQS) and contains two notable, previously unrecognized interactions between NAD+ and Asp side chains conserved in all sugar phosphate cyclases that may influence catalysis. Because the domains of ValA adopt a nearly closed conformation even though no sugar substrate is present, comparisons with a ligand-bound DHQS provide a model for aspects of substrate binding. One striking active site diff...
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Evolutionary Divergence of Sedoheptulose 7-Phosphate Cyclases Leads to Several Distinct Cyclic Products
2012Co-Authors: Shumpei Asamizu, Patricia M Flatt, Pengfei Xie, Corey J. Brumsted, Taifo MahmudAbstract:Sedoheptulose 7-Phosphate cyclases are enzymes that utilize the pentose phosphate pathway intermediate, Sedoheptulose 7-Phosphate, to generate cyclic precursors of many bioactive natural products, such as the antidiabetic drug acarbose, the crop protectant validamycin, and the natural sunscreens mycosporine-like amino acids. These proteins are phylogenetically related to the dehydroquinate (DHQ) synthases from the shikimate pathway and are part of the more recently recognized superfamily of sugar phosphate cyclases, which includes DHQ synthases, aminoDHQ synthases, and 2-deoxy-scyllo-inosose synthases. Through genome mining and biochemical studies, we identified yet another subset of DHQS-like proteins in the actinomycete Actinosynnema mirum and the myxobacterium Stigmatella aurantiaca DW4/3-1. These enzymes catalyze the conversion of Sedoheptulose 7-Phosphate to 2-epi-valiolone, which is predicted to be an alternative precursor for aminocyclitol biosynthesis. Comparative bioinformatics and biochemical analyses of these proteins with 2-epi-5-epi-valiolone synthases (EEVS) and desmethyl-4-deoxygadusol synthases (DDGS) provided further insights into their genetic diversity, conserved amino acid sequences, and plausible catalytic mechanisms. The results further highlight the uniquely diverse DHQS-like sugar phosphate cyclases, which may provide new tools for chemoenzymatic, stereospecific synthesis of various cyclic molecules
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biosynthetic gene cluster of cetoniacytone a an unusual aminocyclitol from the endosymbiotic bacterium actinomyces sp lu 9419
ChemBioChem, 2009Co-Authors: Patricia M Flatt, Taifo MahmudAbstract:A gene cluster responsible for the biosynthesis of the antitumor agent cetoniacytone A was identified in Actinomyces sp. strain Lu 9419, an endosymbiotic bacterium isolated from the intestines of the rose chafer beetle (Cetonia aurata). The nucleotide sequence analysis of the 46 kb DNA region revealed the presence of 31 complete ORFs, including genes predicted to encode a 2-epi-5-epi-valiolone synthase (CetA), a glyoxalase/bleomycin resistance protein (CetB), an acyltransferase (CetD), an FAD-dependent dehydrogenase (CetF2), two oxidoreductases (CetF1 and CetG), two aminotransferases (CetH and CetM), and a pyranose oxidase (CetL). CetA has previously been demonstrated to catalyze the cyclization of Sedoheptulose 7-Phosphate to the cyclic intermediate, 2-epi-5-epi-valiolone. In this report, the glyoxalase/bleomycin resistance protein homolog CetB was identified as a 2-epi-5-epi-valiolone epimerase (EVE), a new member of the vicinal oxygen chelate (VOC) superfamily. The 24 kDa recombinant histidine-tagged CetB was found to form a homodimer; each monomer contains two betaalphabetabetabeta scaffolds that form a metal binding site with two histidine and two glutamic acid residues. A BLAST search using the newly isolated cet biosynthetic genes revealed an analogous suite of genes in the genome of Frankia alni ACN14a, suggesting that this plant symbiotic nitrogen-fixing bacterium is capable of producing a secondary metabolite related to the cetoniacytones.
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Genetic organization of the putative salbostatin biosynthetic gene cluster including the 2-epi-5-epi-valiolone synthase gene in Streptomyces albus ATCC 21838
Applied Microbiology and Biotechnology, 2008Co-Authors: Woo Sik Choi, Taifo Mahmud, Yong-hoon Choeng, Byeong Chul Jeong, Sang Hee Lee, Yong Keun Chang, Chang-joon Kim, Soon-kwang HongAbstract:The cyclization of Sedoheptulose 7-Phosphate to 2- epi -5- epi -valiolone, catalyzed by the 2- epi -5- epi -valiolone synthases, is the first committed step in the biosynthesis of C_ 7 N-aminocyclitol-containing natural products, such as validamycin and acarbose. These natural products contain in their structures a valienamine unit, which is important for their biological activity. The same core unit is also found in salbostatin, a related pseudodisaccharide that has strong trehalase inhibitory activity. In silico analysis of the putative biosynthetic gene cluster of salbostatin from Streptomyces albus ATCC 21838 revealed 20 open reading frames, including an acbC homolog gene ( salQ ), which is believed to be involved in the biosynthesis of salbostatin. The salQ gene was overexpressed in Escherichia coli and the catalytic function of the recombinant protein was confirmed to be a 2- epi -5- epi -valiolone synthase. In addition, SalF, SalL, SalM, SalN, SalO, and SalR were found to be homologous to AcbR, AcbM, AcbL, AcbN, AcbO, and AcbP from the acarbose pathway, respectively, which suggests that the biosynthesis of C_ 7 N-aminocyclitol moiety of salbostatin may be very similar to that of acarbose.
Hirotada Mori - One of the best experts on this subject based on the ideXlab platform.
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Systematic phenome analysis of Escherichia coli multiple‐knockout mutants reveals hidden reactions in central carbon metabolism
Molecular systems biology, 2009Co-Authors: Kenji Nakahigashi, Yoshihiro Toya, Nobuyoshi Ishii, Tomoyoshi Soga, Miki Hasegawa, Hisami Watanabe, Yuki Takai, Masayuki Honma, Hirotada Mori, Masaru TomitaAbstract:Central carbon metabolism is a basic and exhaustively analyzed pathway. However, the intrinsic robustness of the pathway might still conceal uncharacterized reactions. To test this hypothesis, we constructed systematic multiple-knockout mutants involved in central carbon catabolism in Escherichia coli and tested their growth under 12 different nutrient conditions. Differences between in silico predictions and experimental growth indicated that unreported reactions existed within this extensively analyzed metabolic network. These putative reactions were then confirmed by metabolome analysis and in vitro enzymatic assays. Novel reactions regarding the breakdown of Sedoheptulose-7-Phosphate to erythrose-4-phosphate and dihydroxyacetone phosphate were observed in transaldolase-deficient mutants, without any noticeable changes in gene expression. These reactions, triggered by an accumulation of Sedoheptulose-7-Phosphate, were catalyzed by the universally conserved glycolytic enzymes ATP-dependent phosphofructokinase and aldolase. The emergence of an alternative pathway not requiring any changes in gene expression, but rather relying on the accumulation of an intermediate metabolite may be a novel mechanism mediating the robustness of these metabolic networks.
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systematic phenome analysis of escherichia coli multiple knockout mutants reveals hidden reactions in central carbon metabolism
Molecular Systems Biology, 2009Co-Authors: Kenji Nakahigashi, Yoshihiro Toya, Nobuyoshi Ishii, Tomoyoshi Soga, Miki Hasegawa, Hisami Watanabe, Yuki Takai, Masayuki Honma, Hirotada MoriAbstract:Central carbon metabolism is a basic and exhaustively analyzed pathway. However, the intrinsic robustness of the pathway might still conceal uncharacterized reactions. To test this hypothesis, we constructed systematic multiple-knockout mutants involved in central carbon catabolism in Escherichia coli and tested their growth under 12 different nutrient conditions. Differences between in silico predictions and experimental growth indicated that unreported reactions existed within this extensively analyzed metabolic network. These putative reactions were then confirmed by metabolome analysis and in vitro enzymatic assays. Novel reactions regarding the breakdown of Sedoheptulose-7-Phosphate to erythrose-4-phosphate and dihydroxyacetone phosphate were observed in transaldolase-deficient mutants, without any noticeable changes in gene expression. These reactions, triggered by an accumulation of Sedoheptulose-7-Phosphate, were catalyzed by the universally conserved glycolytic enzymes ATP-dependent phosphofructokinase and aldolase. The emergence of an alternative pathway not requiring any changes in gene expression, but rather relying on the accumulation of an intermediate metabolite may be a novel mechanism mediating the robustness of these metabolic networks.
Yuri A Trotsenko - One of the best experts on this subject based on the ideXlab platform.
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characterization of recombinant ppi dependent 6 phosphofructokinases from methylosinus trichosporium ob3b and methylobacterium nodulans ors 2060
Biochemistry, 2012Co-Authors: Olga N Rozova, V N Khmelenina, Yuri A TrotsenkoAbstract:The properties of the purified recombinant PPi-dependent 6-phosphofructokinases (PPi-PFKs) from the methanotroph Methylosinus trichosporium OB3b and rhizospheric phytosymbiont Methylobacterium nodulans ORS 2060 were determined. The dependence of activities of PPi-PFK-His6-tag from Ms. trichosporium OB3b (6 × 45 kDa) and PPi-PFK from Mb. nodulans ORS 2060 (4 × 43 kDa) on the concentrations of substrates of forward and reverse reactions conformed to Michaelis-Menten kinetics. Besides fructose-6-phosphate, the enzymes also phosphorylated Sedoheptulose-7-Phosphate. ADP or AMP (1 mM each) inhibited activity of the Ms. trichosporium PPi-PFK but did not affect the activity of the Mb. nodulans enzyme. Preference of PPi-PFKs to fructose-1,6-bisphosphate implied a predominant function of the enzymes in hexose phosphate synthesis in these bacteria. PPi-PFKs from the methylotrophs have low similarity of translated amino acid sequences (17% identity) and belong to different phylogenetic subgroups of type II 6-phosphofructokinases. The relationship of PPi-PFKs with microaerophilic character of Ms. trichosporium OB3b and adaptation of Mb. nodulans ORS 2060 to anaerobic phase of phytosymbiosis are discussed.
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characterization of the recombinant pyrophosphate dependent 6 phosphofructokinases from methylomicrobium alcaliphilum 20z and methylococcus capsulatus bath
Methods in Enzymology, 2011Co-Authors: V N Khmelenina, Olga N Rozova, Yuri A TrotsenkoAbstract:Abstract The Embden–Meyerhof–Parnas (EMP) glycolysis is the starting point of the core carbon metabolism. Aerobic methanotrophs possessing activity of the pyrophosphate-dependent 6-phosphofructokinase (PPi-PFK) instead of the classical glycolytic enzyme ATP-dependent 6-phosphofructokinase (ATP-PFK) are promising model bacteria for elucidation of the role of inorganic pyrophosphate (PPi) and PPi-dependent glycolysis in microorganisms. Characterization of the His 6 -tagged PPi-PFKs from two methanotrophs, halotolerant alkaliphilic Methylomicrobium alcaliphilum 20Z and thermotolerant Methylococcus capsulatus Bath, showed differential capabilities of PPi-PFKs to phosphorylate Sedoheptulose-7-Phosphate and this property correlated well with the metabolic patterns of these bacteria assimilating C 1 substrate either via the ribulosemonophosphate (RuMP) pathway ( Mm. alcaliphilum 20Z) or simultaneously via the RuMP and serine pathways and the Calvin cycle ( Mc. capsulatus Bath). Analysis of the genomic draft of Mm. alcaliphilum 20Z ( https://www.genoscope.cns.fr/agc/mage ) has provided in silico evidence for the existence of a PPi-dependent pyruvate-phosphate dikinase (PPDK). Expression of the ppdk gene at oxygen limitation along with the presence of PPi-PFK in Mm. alcaliphilum 20Z implied functioning of PPi-dependent glycolysis and PPi recycling under conditions when oxidative phosphorylation is hampered.
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Characterization of recombinant pyrophosphate-dependent 6-phosphofructokinase from halotolerant methanotroph Methylomicrobium alcaliphilum 20Z.
Research in Microbiology, 2010Co-Authors: Olga N Rozova, V N Khmelenina, Stéphane Vuilleumier, Yuri A TrotsenkoAbstract:Pyrophosphate-dependent 6-phosphofructokinase (PPi-PFK) was obtained as His(6)-tagged protein by cloning of the pfp gene from the aerobic obligate methanotroph Methylomicrobium alcaliphilum 20Z and characterized. The recombinant PPi-PFK (4×45kDa) was highly active, non-allosteric and stringently specific to pyrophosphate as the phosphoryl donor. The enzyme was more specific for the reverse reaction substrate fructose-1,6-bisphosphate (K(m) 0.095mM, V(max) 805U/mg of protein) than for the forward reaction substrate fructose-6-phosphate (K(m) 0.64mM, V(max) 577U/mg of protein). It also phosphorylated Sedoheptulose-7-Phosphate with much lower efficiency (K(m) 1.01mM, V(max) 0.118U/mg of protein). The kinetic properties of the M. alcaliphilum PP(i)-PFK were analyzed and compared with those of PP(i)-PFKs from other methanotrophs. The PP(i)-PFK from M. alcaliphilum shows highest sequence identity to PPi-PFK from obligate mesophilic methanotroph Methylomonas methanica (89%), and only low identity to the enzyme from thermotolerant Methylococcus capsulatus Bath (16%). This extensive sequence divergence of PPi-PFKs correlated with differential ability to phosphorylate Sedoheptulose-7-Phosphate and with the metabolic patterns of these bacteria assimilating C(1) substrate either via the ribulose monophoshate (RuMP) cycle or simultaneously via the RuMP and the Calvin cycles. Based on enzymic and genomic data, the involvement of PPi-PFK in pyrophosphate-dependent glycolysis in M. alcaliphilum 20Z was fist proposed.
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characterization of the pyrophosphate dependent 6 phosphofructokinase from methylococcus capsulatus bath
Fems Microbiology Letters, 2008Co-Authors: A S Reshetnikov, Olga N Rozova, V N Khmelenina, Ildar I Mustakhimov, Alexander P Beschastny, Yuri A TrotsenkoAbstract:An active pyrophosphate-dependent 6-phosphofructokinase (PPi-PFK) from the thermotolerant methanotroph Methylococcus capsulatus Bath, containing a six-residue polyhistidine tag, was characterized. The enzyme was homodimeric (2 x 45 kDa), nonallosteric and most active at pH 7.0. PPi-PFK catalyzed reactions of PPi-dependent phosphorylation of fructose-6-phosphate (F-6-P) (K-m 2.27 mM and V-max 7.6 U mg(-1) of protein), Sedoheptulose-7-Phosphate (K-m 0.027 mM and V-max 31 U mg(-1)) and ribulose-5-phosphate. In the reaction with F-6-P, the apparent K-m for PPi was 0.027 mM, while in the reverse reaction, K-m for orthophosphate was 8.69 mM and that for fructose-1,6-bisphosphate 0.328 mM (V-max 9.0 U mg(-1)). Phylogenetically, M. capsulatus PPi-PFK was most similar to PPi-PFKs from the lithoautotrophic ammonia oxidizers Nitrosomonas europaea (74.0%), Nitrosospira multiformis (73.6%) and Betaproteobacterial methylotroph Methylibium petroleiphilum PM1 (71.6% identity). Genes coding PPi-PFK and a putative V-type H+-translocating pyrophosphatase (H+-PPi-ase) were cotranscribed as an operon. The potential significance of the PPi-PFK for regulation of carbon and energy fluxes in M. capsulatus Bath is discussed.
Paul F Long - One of the best experts on this subject based on the ideXlab platform.
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o methyltransferase is shared between the pentose phosphate and shikimate pathways and is essential for mycosporine like amino acid biosynthesis in anabaena variabilis atcc 29413
ChemBioChem, 2015Co-Authors: Matthew A Pope, Edward Spence, Valentina Seralvo, Ranko Gacesa, Sibylle Heidelberger, Andrew J Weston, Walter C Dunlap, Malcolm J Shick, Paul F LongAbstract:The parent core structure of mycosporine-like amino acids (MAAs) is 4-deoxygadusol, which, in cyanobacteria, is derived from conversion of the pentose phosphate pathway intermediate Sedoheptulose 7-Phosphate by the enzymes 2-epi-5-epivaliolone synthase (EVS) and O-methyltransferase (OMT). Yet, deletion of the EVS gene from Anabaena variabilis ATCC 29413 was shown to have little effect on MAA production, thus suggesting that its biosynthesis is not exclusive to the pentose phosphate pathway. Herein, we report how, using pathway-specific inhibitors, we demonstrated unequivocally that MAA biosynthesis occurs also via the shikimate pathway. In addition, complete in-frame gene deletion of the OMT gene from A. variabilis ATCC 29413 reveals that, although biochemically distinct, the pentose phosphate and shikimate pathways are inextricably linked to MAA biosynthesis in this cyanobacterium. Furthermore, proteomic data reveal that the shikimate pathway is the predominate route for UV-induced MAA biosynthesis.
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2-epi-5-epi-Valiolone synthase activity is essential for maintaining phycobilisome composition in the cyanobacterium Anabaena variabilis ATCC 29413 when grown in the presence of a carbon source
Photosynthesis Research, 2013Co-Authors: Edward Spence, Walter C Dunlap, Samantha J. Bryan, Mohamed Lisfi, John Cullum, J. Malcolm Shick, Conrad W. Mullineaux, Paul F LongAbstract:The cyclase 2- epi -5- epi -valiolone synthase (EVS) is reported to be a key enzyme for biosynthesis of the mycosporine-like amino acid shinorine in the cyanobacterium Anabaena variabilis ATCC 29413. Subsequently, we demonstrated that an in-frame complete deletion of the EVS gene had little effect on in vivo production of shinorine. Complete segregation of the EVS gene deletion mutant proved difficult and was achieved only when the mutant was grown in the dark and in a medium supplemented with fructose. The segregated mutant showed a striking colour change from native blue-green to pale yellow-green, corresponding to substantial loss of the photosynthetic pigment phycocyanin, as evinced by combinations of absorbance and emission spectra. Transcriptional analysis of the mutant grown in the presence of fructose under dark or light conditions revealed downregulation of the cpcA gene that encodes the alpha subunit of phycocyanin, whereas the gene encoding nblA, a protease chaperone essential for phycobilisome degradation, was not expressed. We propose that the substrate of EVS (Sedoheptulose 7-Phosphate) or possibly lack of its EVS-downstream products, represses transcription of cpcA to exert a hitherto unknown control over photosynthesis in this cyanobacterium. The significance of this finding is enhanced by phylogenetic analyses revealing horizontal gene transfer of the EVS gene of cyanobacteria to fungi and dinoflagellates. It is also conceivable that the EVS gene has been transferred from dinoflagellates, as evident in the host genome of symbiotic corals. A role of EVS in regulating Sedoheptulose 7-Phosphate concentrations in the photophysiology of coral symbiosis is yet to be determined.
Kenji Nakahigashi - One of the best experts on this subject based on the ideXlab platform.
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Systematic phenome analysis of Escherichia coli multiple‐knockout mutants reveals hidden reactions in central carbon metabolism
Molecular systems biology, 2009Co-Authors: Kenji Nakahigashi, Yoshihiro Toya, Nobuyoshi Ishii, Tomoyoshi Soga, Miki Hasegawa, Hisami Watanabe, Yuki Takai, Masayuki Honma, Hirotada Mori, Masaru TomitaAbstract:Central carbon metabolism is a basic and exhaustively analyzed pathway. However, the intrinsic robustness of the pathway might still conceal uncharacterized reactions. To test this hypothesis, we constructed systematic multiple-knockout mutants involved in central carbon catabolism in Escherichia coli and tested their growth under 12 different nutrient conditions. Differences between in silico predictions and experimental growth indicated that unreported reactions existed within this extensively analyzed metabolic network. These putative reactions were then confirmed by metabolome analysis and in vitro enzymatic assays. Novel reactions regarding the breakdown of Sedoheptulose-7-Phosphate to erythrose-4-phosphate and dihydroxyacetone phosphate were observed in transaldolase-deficient mutants, without any noticeable changes in gene expression. These reactions, triggered by an accumulation of Sedoheptulose-7-Phosphate, were catalyzed by the universally conserved glycolytic enzymes ATP-dependent phosphofructokinase and aldolase. The emergence of an alternative pathway not requiring any changes in gene expression, but rather relying on the accumulation of an intermediate metabolite may be a novel mechanism mediating the robustness of these metabolic networks.
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systematic phenome analysis of escherichia coli multiple knockout mutants reveals hidden reactions in central carbon metabolism
Molecular Systems Biology, 2009Co-Authors: Kenji Nakahigashi, Yoshihiro Toya, Nobuyoshi Ishii, Tomoyoshi Soga, Miki Hasegawa, Hisami Watanabe, Yuki Takai, Masayuki Honma, Hirotada MoriAbstract:Central carbon metabolism is a basic and exhaustively analyzed pathway. However, the intrinsic robustness of the pathway might still conceal uncharacterized reactions. To test this hypothesis, we constructed systematic multiple-knockout mutants involved in central carbon catabolism in Escherichia coli and tested their growth under 12 different nutrient conditions. Differences between in silico predictions and experimental growth indicated that unreported reactions existed within this extensively analyzed metabolic network. These putative reactions were then confirmed by metabolome analysis and in vitro enzymatic assays. Novel reactions regarding the breakdown of Sedoheptulose-7-Phosphate to erythrose-4-phosphate and dihydroxyacetone phosphate were observed in transaldolase-deficient mutants, without any noticeable changes in gene expression. These reactions, triggered by an accumulation of Sedoheptulose-7-Phosphate, were catalyzed by the universally conserved glycolytic enzymes ATP-dependent phosphofructokinase and aldolase. The emergence of an alternative pathway not requiring any changes in gene expression, but rather relying on the accumulation of an intermediate metabolite may be a novel mechanism mediating the robustness of these metabolic networks.