ISOLATION BY LYTIC COMPLEMENTATION OF A GENE ENCODING AN AMINO TRANSFERASE IN THE LYSINE BIOSYNTHETIC PATHWAY

Abstract Phosphonates are organophosphorus compounds possessing a characteristic C−P bond in which phosphorus is directly bonded to carbon. As phosphonates mimic the phosphates and carboxylates of biological molecules to potentially inhibit metabolic enzymes, they could be lead compounds for the development of a variety of drugs. Fosfomycin (FM) is a representative phosphonate natural product that is widely used as an antibacterial drug. Here, we review the biosynthesis of FM, which includes a recent breakthrough to find a missing link in the biosynthetic pathway that had been a mystery for a quarter-century. In addition, we describe the genome mining of phosphonate natural products using the biosynthetic gene encoding an enzyme that catalyzes C–P bond formation. We also introduce the chemoenzymatic synthesis of phosphonate derivatives. These studies expand the repertoires of phosphonates and the related biosynthetic machinery. This review mainly covers the years 2012-2020.

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Pathogen and Pest Responses Are Altered Due to RNAi-Mediated Knockdown of GLYCOALKALOID METABOLISM 4 in Solanum tuberosum

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-02-17-0033-r ◽

2017 ◽

Vol 30 (11) ◽

pp. 876-885 ◽

Cited By ~ 7

Author(s):

Jamuna Risal Paudel ◽

Charlotte Davidson ◽

Jun Song ◽

Itkin Maxim ◽

Asaph Aharoni ◽

...

Keyword(s):

Solanum Tuberosum ◽

Biosynthetic Pathway ◽

Plant Protection ◽

Insect Pest ◽

Metabolite Profile ◽

Gene Encoding ◽

Rnai Line ◽

Leaf Consumption ◽

Insect Mortality ◽

Potato Growth

Steroidal glycoalkaloids (SGAs) are major secondary metabolites constitutively produced in cultivated potato Solanum tuberosum, and α-solanine and α-chaconine are the most abundant SGAs. SGAs are toxic to humans at high levels but their role in plant protection against pests and pathogens is yet to be established. In this study, levels of SGAs in potato were reduced by RNA interference (RNAi)-mediated silencing of GLYCOALKALOID METABOLISM 4 (GAME4)—a gene encoding cytochrome P450, involved in an oxidation step in the conversion of cholesterol to SGA aglycones. Two GAME4 RNAi lines, T8 and T9, were used to investigate the effects of manipulation of the SGA biosynthetic pathway in potato. Growth and development of an insect pest, Colorado potato beetle (CPB), were affected in these lines. While no effect on CPB leaf consumption or weight gain was observed, early instar larval death and accelerated development of the insect was found while feeding on leaves of GAME4 RNAi lines. Modulation of SGA biosynthetic pathway in GAME4 RNAi plants was associated with a larger alteration to the metabolite profile, including increased levels of one or both the steroidal saponins or phytoecdysteroids, which could affect insect mortality as well as development time. Colonization by Verticillium dahliae on GAME4 RNAi plants was also tested. There were increased pathogen levels in the T8 GAME4 RNAi line but not in the T9. Metabolite differences between T8 and T9 were found and may have contributed to differences in V. dahliae infection. Drought responses created by osmotic stress were not affected by modulation of SGA biosynthetic pathway in potato.

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Isolation and functional expression of human COQ2, a gene encoding a polyprenyl transferase involved in the synthesis of CoQ

Biochemical Journal ◽

10.1042/bj20040261 ◽

2004 ◽

Vol 382 (2) ◽

pp. 519-526 ◽

Cited By ~ 81

Author(s):

Margareta FORSGREN ◽

Anneli ATTERSAND ◽

Staffan LAKE ◽

Jacob GRÜNLER ◽

Ewa SWIEZEWSKA ◽

...

Keyword(s):

Biosynthetic Pathway ◽

Functional Expression ◽

Human Muscle ◽

Null Mutant ◽

Human Homologue ◽

Gene Encoding ◽

Reading Frame ◽

Human Enzyme ◽

Liver Cdna Library ◽

Mutant Cells

The COQ2 gene in Saccharomyces cerevisiae encodes a Coq2 (p-hydroxybenzoate:polyprenyl transferase), which is required in the biosynthetic pathway of CoQ (ubiquinone). This enzyme catalyses the prenylation of p-hydroxybenzoate with an all-trans polyprenyl group. We have isolated cDNA which we believe encodes the human homologue of COQ2 from a human muscle and liver cDNA library. The clone contained an open reading frame of length 1263 bp, which encodes a polypeptide that has sequence homology with the Coq2 homologues in yeast, bacteria and mammals. The human COQ2 gene, when expressed in yeast Coq2 null mutant cells, rescued the growth of this yeast strain in the absence of a non-fermentable carbon source and restored CoQ biosynthesis. However, the rate of CoQ biosynthesis in the rescued cells was lower when compared with that in cells rescued with the yeast COQ2 gene. CoQ formed when cells were incubated with labelled decaprenyl pyrophosphate and nonaprenyl pyrophosphate, showing that the human enzyme is active and that it participates in the biosynthesis of CoQ.

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Lipidomics of Candida albicans biofilms reveals phase-dependent production of phospholipid molecular classes and role for lipid rafts in biofilm formation

Microbiology ◽

10.1099/mic.0.051086-0 ◽

2011 ◽

Vol 157 (11) ◽

pp. 3232-3242 ◽

Cited By ~ 63

Author(s):

Ali Abdul Lattif ◽

Pranab K. Mukherjee ◽

Jyotsna Chandra ◽

Mary R. Roth ◽

Ruth Welti ◽

...

Keyword(s):

Candida Albicans ◽

Biosynthetic Pathway ◽

Bloodstream Infections ◽

Unsaturation Index ◽

Lipid Profiles ◽

Planktonic Cells ◽

Gene Encoding ◽

Aureobasidin A ◽

Developmental Phases ◽

Candida Albicans Biofilms

Candida albicans-associated bloodstream infections are linked to the ability of this yeast to form biofilms. In this study, we used lipidomics to compare the lipid profiles of C. albicans biofilms and planktonic cells, in early and mature developmental phases. Our results showed that significant differences exist in lipid composition in both developmental phases. Biofilms contained higher levels of phospholipid and sphingolipids than planktonic cells (nmol per g biomass, P<0.05 for all comparisons). In the early phase, levels of lipid in most classes were significantly higher in biofilms compared to planktonic cells (P≤0.05). The ratio of phosphatidylcholine to phosphatidylethanolamine was lower in biofilms compared to planktonic cells in both early (1.17 vs 2.52, P≤0.001) and late (2.34 vs 3.81, P≤0.001) developmental phases. The unsaturation index of phospholipids decreased with time, with this effect being particularly strong for biofilms. Inhibition of the biosynthetic pathway for sphingolipid [mannosyl diinositolphosphoryl ceramide, M(IP)2C] by myriocin or aureobasidin A, and disruption of the gene encoding inositolphosphotransferase (Ipt1p), abrogated the ability of C. albicans to form biofilms. The differences in lipid profiles between biofilms and planktonic Candida cells may have important implications for the biology and antifungal resistance of biofilms.

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Identification and characterization of the gene encoding the human phosphopantetheine adenylyltransferase and dephospho-CoA kinase bifunctional enzyme (CoA synthase)

Biochemical Journal ◽

10.1042/bj20020569 ◽

2002 ◽

Vol 365 (1) ◽

pp. 13-18 ◽

Cited By ~ 36

Author(s):

Suren AGHAJANIAN ◽

D.Margaret WORRALL

Keyword(s):

Biosynthetic Pathway ◽

Sequence Similarity ◽

Kinetic Properties ◽

Sequence Information ◽

Bifunctional Enzyme ◽

Gene Encoding ◽

Pig Liver ◽

Identification And Characterization ◽

Similar Kinetic

The final two enzymes in the CoA biosynthetic pathway, phosphopantetheine adenylyltransferase (PPAT; EC 2.7.7.3) and dephospho-CoA kinase (DPCK; EC 2.7.1.24), are separate proteins in prokaryotes, but exist as a bifunctional enzyme in pig liver. In the present study we have obtained sequence information from purified pig-liver enzyme, and identified the corresponding cDNA in a number of species. The human gene localizes to chromosome 17q12-21 and contains regions with sequence similarity to the monofunctional Escherichia coli DPCK and PPAT. The recombinant 564-amino-acid human protein confirmed the associated transferase and kinase activities, and gave similar kinetic properties to the wild-type pig enzyme.

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Increased L-Ornithine Production in Corynebacterium glutamicum by Overexpression of a Gene Encoding a Putative Aminotransferase

Journal of Molecular Microbiology and Biotechnology ◽

10.1159/000375124 ◽

2015 ◽

Vol 25 (1) ◽

pp. 45-50 ◽

Cited By ~ 8

Author(s):

Dai-Joong Kim ◽

Gui-Hye Hwang ◽

Ji-Na Um ◽

Jae-Yong Cho

Keyword(s):

Biosynthetic Pathway ◽

Gene Promoter ◽

Open Reading Frame ◽

Aminotransferase Activity ◽

Wild Type ◽

Gene Encoding ◽

Reading Frame ◽

Dehydrogenase Gene ◽

Putative Aminotransferase ◽

Combined Overexpression

Overexpression of the NCgl0462 open reading frame, encoding a class II aminotransferase, was studied in conjunction with other enzymes in <smlcap>L</smlcap>-ornithine biosynthesis in an <smlcap>L</smlcap>-ornithine-producing strain. Expression of the wild-type NCgl0462 open reading frame, which displayed aminotransferase activity, was amplified by placing it under the control of the glyceraldehyde 3-phosphate dehydrogenase gene promoter in the pEK0 plasmid and in the genome. <smlcap>L</smlcap>-Ornithine production in Corynebacteriumglutamicum SJC8260 harboring plasmid and the genomic NCgl0462 open reading frame was increased by 8.8 and 21.6%, respectively. In addition, the combined overexpression of the NCgl0462 open reading frame within the genome along with the mutated <smlcap>L</smlcap>-ornithine biosynthesis genes (argCJBD) placed in the pEK0 plasmid in C. glutamicum SJC8260 resulted in significant improvement in <smlcap>L</smlcap>-ornithine production (12.48 g/l for combined overexpression compared with 8.42 g/l for the control). These results suggest that overexpression of the aminotransferase-encoding NCgl0462 open reading frame plays an unequivocal role in the <smlcap>L</smlcap>-ornithine biosynthetic pathway, with overlapping substrate specificity in C. glutamicum.

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Lysine Represses Transcription of the Escherichia coli dapB Gene by Preventing Its Activation by the ArgP Activator

Journal of Bacteriology ◽

10.1128/jb.01782-07 ◽

2008 ◽

Vol 190 (15) ◽

pp. 5224-5229 ◽

Cited By ~ 15

Author(s):

Jean Bouvier ◽

Patrick Stragier ◽

Violette Morales ◽

Elisabeth Rémy ◽

Claude Gutierrez

Keyword(s):

Escherichia Coli ◽

Transcription Start Site ◽

Biosynthetic Pathway ◽

Genomic Library ◽

Regulatory Region ◽

Null Mutation ◽

Start Site ◽

Transcription Start ◽

Gene Encoding ◽

Gel Retardation Assay

ABSTRACT The Escherichia coli dapB gene encodes one of the enzymes of the biosynthetic pathway leading to lysine and its immediate precursor, diaminopimelate. Expression of dapB is repressed by lysine, but no trans-acting regulator has been identified so far. Our analysis of the dapB regulatory region shows that sequences located in the −81/−118 interval upstream of the transcription start site are essential for full expression of dapB, as well as for lysine repression. Screening a genomic library for a gene that could alleviate lysine repression when present in multicopy led to the recovery of argP, a gene encoding an activating protein of the LysR-type family, known to use lysine as an effector. An argP null mutation strongly decreases dapB transcription that becomes insensitive to lysine. Purified His6-tagged ArgP protein binds with an apparent K d of 35 nM to the dapB promoter in a gel retardation assay, provided that sequences up to −103 are present. In the presence of l-lysine and l-arginine, the binding of ArgP to dapB is partly relieved. These results fit with a model in which ArgP contributes to enhanced transcription of dapB when lysine becomes limiting.

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New Insight into the Ochratoxin A Biosynthetic Pathway through Deletion of a Nonribosomal Peptide Synthetase Gene in Aspergillus carbonarius

Applied and Environmental Microbiology ◽

10.1128/aem.02508-12 ◽

2012 ◽

Vol 78 (23) ◽

pp. 8208-8218 ◽

Cited By ~ 69

Author(s):

Antonia Gallo ◽

Kenneth S. Bruno ◽

Michele Solfrizzo ◽

Giancarlo Perrone ◽

Giuseppina Mulè ◽

...

Keyword(s):

Ochratoxin A ◽

Biosynthetic Pathway ◽

Nonribosomal Peptide Synthetase ◽

Gene Encoding ◽

Nonribosomal Peptide ◽

Content Type ◽

Peptide Synthetase ◽

Ochratoxin Α ◽

Hydrolysis Of ◽

Insight Into

ABSTRACTOchratoxin A (OTA), a mycotoxin produced byAspergillusandPenicilliumspecies, is composed of a dihydroisocoumarin ring linked to phenylalanine, and its biosynthetic pathway has not yet been completely elucidated. Most of the knowledge regarding the genetic and enzymatic aspects of OTA biosynthesis has been elucidated inPenicilliumspecies. InAspergillusspecies, onlypksgenes involved in the initial steps of the pathway have been partially characterized. In our study, the inactivation of a gene encoding a nonribosomal peptide synthetase (NRPS) in OTA-producingA. carbonariusITEM 5010 has eliminated the ability of this fungus to produce OTA. This is the first report on the involvement of annrpsgene product in OTA biosynthetic pathway in anAspergillusspecies. The absence of OTA and ochratoxin α, the isocoumaric derivative of OTA, and the concomitant increase of ochratoxin β, the dechloro analog of ochratoxin α, were observed in the liquid culture of transformed strain. The data provide the first evidence that the enzymatic step adding phenylalanine to polyketide dihydroisocoumarin precedes the chlorination step to form OTA inA. carbonariusand that ochratoxin α is a product of hydrolysis of OTA, giving an interesting new insight into the biosynthetic pathway of the toxin.

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Plasmodium falciparum hydroxymethylbilane synthase does not house any cosynthase activity within the haem biosynthetic pathway

10.1101/2021.02.09.429727 ◽

2021 ◽

Author(s):

Alan F. Scott ◽

Evelyne Deery ◽

Andrew D. Lawrence ◽

Martin J. Warren

Keyword(s):

Plasmodium Falciparum ◽

Hplc Analysis ◽

Biosynthetic Pathway ◽

Gene Encoding ◽

E Coli ◽

Aminolaevulinic Acid ◽

Porphobilinogen Synthase

AbstractThe production of uroporphyrinogen III, the universal progenitor of macrocyclic, modified tetrapyrroles, is produced from aminolaevulinic acid (ALA) by a conserved pathway involving three enzymes: porphobilinogen synthase (PBGS), hydroxymethylbilane synthase (HmbS) and uroporphyrinogen III synthase (UroS). The gene encoding uroporphyrinogen III synthase has not yet been identified in Plasmodium falciparum but it has been suggested that this activity is housed inside a bifunctional hybroxymethylbilane synthase (HmbS). In this present study it is demonstrated that P. falciparum HmbS does not have uroporphyrinogen III synthase activity. This was demonstrated by the failure of a codon optimised P. falciparum hemC gene, encoding HmbS, to compliment a defined E. coli hemD- mutant (SASZ31) deficient in uroporphyrinogen III synthase activity. Furthermore, HPLC analysis of the oxidsed reaction product from recombinant, purified HmbS showed that only uroporphyrin I could be detected (corresponding to hydroxymethylbilane production). No uroporphyrin III was detected, thus showing that P. falciparum HmbS does not have UroS activity and can only catalyse the formation of hydroxymethylbilane from porphobilinogen.

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