scholarly journals It happened again: convergent evolution of acylglucose specialized metabolism in black nightshade and wild tomato

2021 ◽  
Author(s):  
Yann-Ru Lou ◽  
Thilani M. Anthony ◽  
Paul D. Fiesel ◽  
Rachel E. Arking ◽  
Elizabeth M. Christensen ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
2D Nmr ◽  
Solanum Nigrum ◽  
Metabolic Enzyme ◽  
Virus Induced Gene Silencing ◽  
Wild Tomato ◽  
Black Nightshade ◽  
In The Wild

Plants synthesize myriad phylogenetically-restricted specialized (aka secondary) metabolites with diverse structures. Metabolism of acylated sugar esters in epidermal glandular secreting trichomes across the Solanaceae (nightshade) family are ideal for investigating the mechanisms of evolutionary metabolic diversification. We developed methods to structurally analyze acylhexose mixtures by 2D NMR, which led to the insight that the Old World species black nightshade (Solanum nigrum) accumulates acylglucoses and acylinositols in the same tissue. Detailed in vitro biochemistry - cross validated by in vivo virus induced gene silencing - revealed two unique features of the four-step acylglucose biosynthetic pathway: a trichome-expressed, neofunctionalized invertase-like enzyme, SnASFF1, converts BAHD-produced acylsucroses to acylglucoses, which in turn are substrates for the first-reported acylglucose acyltransferase, SnAGAT1. This biosynthetic pathway evolved independently from that recently described in the wild tomato S. pennellii, reinforcing that acylsugar biosynthesis is evolutionarily dynamic with independent examples of primary metabolic enzyme cooption and additional variation in BAHD acyltransferases.

scholarly journals Evolution of metabolic novelty: A trichome-expressed invertase creates specialized metabolic diversity in wild tomato

2019 ◽  
Vol 5 (4) ◽  
pp. eaaw3754 ◽  
Author(s):  
Bryan J. Leong ◽  
Daniel B. Lybrand ◽  
Yann-Ru Lou ◽  
Pengxiang Fan ◽  
Anthony L. Schilmiller ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Gland Cell ◽  
Metabolic Enzyme ◽  
South American ◽  
Metabolic Diversity ◽  
Solanum Pennellii ◽  
Wild Tomato ◽  
Specialized Metabolites ◽  
New Class ◽  
In The Wild

Plants produce a myriad of taxonomically restricted specialized metabolites. This diversity—and our ability to correlate genotype with phenotype—makes the evolution of these ecologically and medicinally important compounds interesting and experimentally tractable. Trichomes of tomato and other nightshade family plants produce structurally diverse protective compounds termed acylsugars. While cultivated tomato (Solanum lycopersicum) strictly accumulates acylsucroses, the South American wild relative Solanum pennellii produces copious amounts of acylglucoses. Genetic, transgenic, and biochemical dissection of the S. pennellii acylglucose biosynthetic pathway identified a trichome gland cell–expressed invertase-like enzyme that hydrolyzes acylsucroses (Sopen03g040490). This enzyme acts on the pyranose ring–acylated acylsucroses found in the wild tomato but not on the furanose ring–decorated acylsucroses of cultivated tomato. These results show that modification of the core acylsucrose biosynthetic pathway leading to loss of furanose ring acylation set the stage for co-option of a general metabolic enzyme to produce a new class of protective compounds.

scholarly journals Evolution of metabolic novelty: a trichome-expressed invertase creates specialized metabolic diversity in wild tomato

2018 ◽  
Author(s):  
Bryan J. Leong ◽  
Daniel Lybrand ◽  
Yann-Ru Lou ◽  
Pengxiang Fan ◽  
Anthony L. Schilmiller ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Gland Cell ◽  
Metabolic Enzyme ◽  
South American ◽  
Metabolic Diversity ◽  
Solanum Pennellii ◽  
Wild Tomato ◽  
Specialized Metabolites ◽  
New Class ◽  
In The Wild

AbstractPlants produce myriad taxonomically restricted specialized metabolites. This diversity – and our ability to correlate genotype with phenotype – makes the evolution of these ecologically and medicinally important compounds interesting and experimentally tractable. Trichomes of tomato and other nightshade family plants produce structurally diverse protective compounds termed acylsugars. While cultivated tomato (Solanum lycopersicum) accumulates strictly acylsucroses, the South American wild relative Solanum pennellii produces copious amounts of acylglucoses. Genetic, transgenic and biochemical dissection of the S. pennellii acylglucose biosynthetic pathway identified a trichome gland cell expressed invertase-like enzyme that hydrolyzes acylsucroses (Sopen03g040490). This enzyme acts on the pyranose ring-acylated acylsucroses found in the wild tomato but not the furanose ring-decorated acylsucroses of cultivated tomato. These results show that modification of the core acylsucrose biosynthetic pathway leading to loss of furanose ring acylation set the stage for co-option of a general metabolic enzyme to produce a new class of protective compounds.

Sirtuin-dependent reversible lysine acetylation controls the activity of acetyl-Coenzyme A synthetase in Campylobacter jejuni

2021 ◽  
Author(s):  
Victoria L. Jeter ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Campylobacter Jejuni ◽  
Posttranslational Modifications ◽  
Active Site ◽  
Protein Function ◽  
Metabolic Enzyme ◽  
Lysine Acetylation ◽  
Class Iii ◽  
Acetyl Coa

Posttranslational modifications are mechanisms for rapid control of protein function used by cells from all domains of life. Acetylation of the epsilon amino group ( N ε ) of an active-site lysine of the AMP-forming acetyl-CoA synthetase (Acs) enzyme is the paradigm for the posttranslational control of the activity of metabolic enzymes. In bacteria, the alluded active-site lysine of Acs enzymes can be modified by a number of different GCN5-type N -acetyltransferases (GNATs). Acs activity is lost as a result of acetylation, and restored by deacetylation. Using a heterologous host, we show that Campylobacter jejuni NCTC11168 synthesizes enzymes that control Acs function by reversible lysine acetylation (RLA). This work validates the function of gene products encoded by the cj1537c , cj1715, and cj1050c loci, namely the AMP-forming acetate:CoA ligase ( Cj Acs), a type IV GCN5-type lysine acetyltransferase (GNAT, hereafter Cj LatA), and a NAD + -dependent (class III) sirtuin deacylase ( Cj CobB), respectively. To our knowledge, these are the first in vivo and in vitro data on C. jejuni enzymes that control the activity of Cj Acs. IMPORTANCE This work is important because it provides the experimental evidence needed to support the assignment of function to three key enzymes, two of which control the reversible posttranslational modification of an active-site lysyl residue of the central metabolic enzyme acetyl-CoA synthetase ( Cj Acs). We can now generate Campylobacter jejuni mutant strains defective in these functions, so we can establish the conditions in which this mode of regulation of Cj Acs is triggered in this bacterium. Such knowledge may provide new therapeutic strategies for the control of this pathogen.

scholarly journals Characterization of gossypol biosynthetic pathway

2018 ◽  
Vol 115 (23) ◽  
pp. E5410-E5418 ◽  
Author(s):  
Xiu Tian ◽  
Ju-Xin Ruan ◽  
Jin-Quan Huang ◽  
Chang-Qing Yang ◽  
Xin Fang ◽  
...  
Keyword(s):  
Plant Disease Resistance ◽  
Biosynthetic Pathway ◽  
Virus Induced Gene Silencing ◽  
Defense Compounds ◽  
Oxidative Reactions ◽  
Substrate Conversion ◽  
Transcriptome Comparison ◽  
Tight Correlation

Gossypol and related sesquiterpene aldehydes in cotton function as defense compounds but are antinutritional in cottonseed products. By transcriptome comparison and coexpression analyses, we identified 146 candidates linked to gossypol biosynthesis. Analysis of metabolites accumulated in plants subjected to virus-induced gene silencing (VIGS) led to the identification of four enzymes and their supposed substrates. In vitro enzymatic assay and reconstitution in tobacco leaves elucidated a series of oxidative reactions of the gossypol biosynthesis pathway. The four functionally characterized enzymes, together with (+)-δ-cadinene synthase and the P450 involved in 7-hydroxy-(+)-δ-cadinene formation, convert farnesyl diphosphate (FPP) to hemigossypol, with two gaps left that each involves aromatization. Of six intermediates identified from the VIGS-treated leaves, 8-hydroxy-7-keto-δ-cadinene exerted a deleterious effect in dampening plant disease resistance if accumulated. Notably, CYP71BE79, the enzyme responsible for converting this phytotoxic intermediate, exhibited the highest catalytic activity among the five enzymes of the pathway assayed. In addition, despite their dispersed distribution in the cotton genome, all of the enzyme genes identified show a tight correlation of expression. Our data suggest that the enzymatic steps in the gossypol pathway are highly coordinated to ensure efficient substrate conversion.

Yeast killer plasmid mutations affecting toxin secretion and activity and toxin immunity function

1982 ◽  
Vol 2 (4) ◽  
pp. 346-354
Author(s):  
H Bussey ◽  
W Sacks ◽  
D Galley ◽  
D Saville
Keyword(s):  
Molecular Weight ◽  
In Vitro Translation ◽  
Heat Curing ◽  
In The Wild ◽  
Toxin Secretion ◽  
Killer Plasmid ◽  
Yeast Killer ◽  
Immunity Function

M double-stranded RNA (MdsRNA) plasmid mutants were obtained by mutagenesis and screening of a diploid killer culture partially heat cured of the plasmid, so that a high proportion of the cells could be expected to have only on M plasmid. Mutants with neutral (nonkiller [K-], immune [R+]) or suicide (killer [K+], sensitive [R-] phenotypes were examined. All mutants became K- R- sensitives on heat curing of the MdsRNA plasmid, and showed cytoplasmic inheritance by random spore analysis. In some cases, M plasmid mutations were indicated by altered mobility of the MdsRNA by agarose gel electrophoresis or by altered size of in vitro translation products from denatured dsRNA. Neutral mutants were of two types: nonsecretors of the toxin protein or secretors of an inactive toxin. Of three neutral nonsecretors examined, one (NLP-1), probably a nonsense mutation, made a smaller protoxin precursor in vitro and in vivo, and two made full-size protoxin molecules. The in vivo protoxin of 43,000 molecular weight was unstable in the wild type and kinetically showed a precursor-product relationship to the processed, secreted 11,000-molecular-weight toxin. In one nonsecretor (N1), the protoxin appeared more stable in a pulse-chase experiment, and could be altered in a recognition site required for protein processing.

scholarly journals The Utilization of Protoporphyrin 9 in Heme Synthesis

Blood ◽  
1959 ◽  
Vol 14 (4) ◽  
pp. 476-485 ◽  
Author(s):  
MOISES GRINSTEIN ◽  
ROBIN M. BANNERMAN ◽  
CARL V. MOORE
Keyword(s):  
Cell Membrane ◽  
Human Blood ◽  
Biosynthetic Pathway ◽  
Aminolevulinic Acid ◽  
Thalassemia Major ◽  
Heme Synthesis ◽  
Equivalent Quantity ◽  
Chicken Erythrocytes

Abstract The experiments described in this communication demonstrate that C14-tagged protoporphyrin 9 can be incorporated into the heme during the biosynthesis of hemoglobin. 1. In vitro observations: (a) C14 protoporphyrin 9 was found to be incorporated into heme by hemolysates of chicken and human blood incubated at 37 C. The degree of incorporation by washed chicken erythrocytes was less, presumably because the protoporphyrin was not readily transferred across the cell membrane. Incorporation by hemolysates was inhibited completely at 1 x 10-2 M KCN at 4 C., markedly by 1 x 10-2 M KCN at 37 C. and partially by 1 x 10-3 M Pb at 37 C. (b) The degree of incorporation was reduced by the addition of an equivalent quantity of delta-aminolevulinic acid. Furthermore, the incorporation of glycine-2-C14 into heme was reduced by the addition of an equivalent quantity of protoporphyrin 9. 2. In vivo observations: Intravenously administered C14 protoporphyrin was incorporated into the circulating hemoglobin of two rabbits with a phenylhydrazine-induced hemolytic anemia. These observations provide support for the view that protoporphyrin 9 itself is a true direct precursor of hemoglobin, in the biosynthetic pathway between porphobilinogen and heme. Comparative studies of rates of incorporation of C14 protoporphyrin 9 and its precursors into heme in vitro may provide a useful tool for the study of heme synthesis in normal and pathologic conditions. For instance, it was shown that hemolysates from the blood of patients with thalassemia major, with poor iron and glycine utilization, rapidly incorporated the tagged protoporphyrin into heme.

Polystachyasaponin with Adjuvant Activity from Entada polystachya

2010 ◽  
Vol 65 (5) ◽  
pp. 628-634 ◽  
Author(s):  
Bernadete P. da Silva ◽  
José P. Parente
Keyword(s):  
2D Nmr ◽  
In Vitro Assays ◽  
Triterpenoid Saponin ◽  
Spectroscopic Techniques ◽  
Adjuvant Activity ◽  
In Vivo Assays ◽  
Cellular Immune ◽  
C Nmr

A new complex triterpenoid saponin, polystachyasaponin, was isolated from leaves of Entada polystachya (L.) DC. (Leguminosae) by using chromatographic methods. Its structure was established as 15,16-dihydroxy-3-[[O-β -D-xylopyranosyl-(1→2)-O-α-L-arabinopyranosyl-(1→6)-2- (acetylamino)-2-deoxy-β -D-glucopyranosyl]oxy]-(3β ,15α,16α)-olean-12-en-28-oic acid O-D-apio- β -D-furanosyl-(1→3)-O-β -D-xylopyranosyl-(1→2)-O-[β -D-glucopyranosyl-(1→4)]-6-O-[(2E,6R)- 6-hydroxy-2,6-dimethyl-1-oxo-2,7-octadienyl]-β -D-glucopyranosyl ester. Structural elucidation was performed using detailed analyses of 1H and 13C NMR spectra including 2D NMR spectroscopic techniques and chemical conversions. The hemolytic activity of the saponin was evaluated using in vitro assays, and its adjuvant potential on the cellular immune response against ovalbumin antigen was investigated using in vivo assays.

scholarly journals The synthesis of murine ferrochelatase in vitro and in vivo

1988 ◽  
Vol 254 (3) ◽  
pp. 799-803 ◽  
Author(s):  
S R Karr ◽  
H A Dailey
Keyword(s):  
Membrane Protein ◽  
Biosynthetic Pathway ◽  
Integral Membrane Protein ◽  
Mitochondrial Proteins ◽  
Isolated Mitochondria ◽  
Mitochondrial Inner Membrane ◽  
Carbonyl Cyanide ◽  
A Cell

Ferrochelatase (protohaem ferro-lyase, EC 4.99.1.1), the terminal enzyme of the haem-biosynthetic pathway, is an integral membrane protein of the mitochondrial inner membrane. When murine erythroleukaemia cells are labelled in vivo with [35S]methionine, lysed, and the extract is immunoprecipitated with rabbit anti-(mouse ferrochelatase) antibody, a protein of Mr 40,000 is isolated. However, when isolated mouse RNA is translated in a cell-free reticulocyte extract, a protein of Mr 43,000 is isolated. Incubation of this Mr 43,000 protein with isolated mitochondria resulted in processing of the Mr 43,000 precursor to the Mr 40,000 mature-sized protein. Addition of carbonyl cyanide m-chlorophenylhydrazone and/or phenanthroline inhibits this processing. These data indicate that ferrochelatase, like most mitochondrial proteins, is synthesized in the cytoplasm as a larger precursor and is then translocated and processed to a mature-sized protein in an energy-required step.

Ndfip1-deficient mice have impaired DMT1 regulation and iron homeostasis

Blood ◽  
2011 ◽  
Vol 117 (2) ◽  
pp. 638-646 ◽  
Author(s):  
Natalie J. Foot ◽  
Yew Ann Leong ◽  
Loretta E. Dorstyn ◽  
Hazel E. Dalton ◽  
Kristen Ho ◽  
...  
Keyword(s):  
Metal Ion ◽  
Iron Homeostasis ◽  
Transferrin Saturation ◽  
Hypochromic Anemia ◽  
Adaptor Proteins ◽  
Normal Diet ◽  
Immunodeficient Mice ◽  
In The Wild

Abstract The divalent metal ion transporter DMT1 is critical for nonheme iron import. We have previously shown that DMT1 is regulated in vitro by ubiquitination that is facilitated by the adaptor proteins Ndfip1 and Ndfip2. Here we report that in Ndfip1−/− mice fed a low- iron diet, DMT1 expression and activity in duodenal enterocytes are significant higher than in the wild-type animals. This correlates with an increase in serum iron levels and transferrin saturation. Liver and spleen iron stores were also increased in Ndfip1−/− mice fed a normal diet. Counterintuitive to the increase in iron uptake, Ndfip1−/− mice fed a low iron diet develop severe microcytic, hypochromic anemia. We demonstrate that this is due to a combination of iron deficiency and inflammatory disease in Ndfip1−/− mice, because Ndfip1−/−/Rag1−/− immunodeficient mice fed a low iron diet did not develop anemia and showed an iron overload phenotype. These data demonstrate that Ndfip1 is a critical mediator of DMT1 regulation in vivo, particularly under iron restricted conditions.

scholarly journals Regulation of ppk Expression and In Vivo Function of Ppk in Streptomyces lividans TK24

2006 ◽  
Vol 188 (17) ◽  
pp. 6269-6276 ◽  
Author(s):  
Sofiane Ghorbel ◽  
Aleksey Smirnov ◽  
Hichem Chouayekh ◽  
Brice Sperandio ◽  
Catherine Esnault ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Sufficient Conditions ◽  
Streptomyces Lividans ◽  
Antibiotic Biosynthesis ◽  
Wild Type ◽  
In The Wild

ABSTRACT The ppk gene of Streptomyces lividans encodes an enzyme catalyzing, in vitro, the reversible polymerization of the γ phosphate of ATP into polyphosphate and was previously shown to play a negative role in the control of antibiotic biosynthesis (H. Chouayekh and M. J. Virolle, Mol. Microbiol. 43:919-930, 2002). In the present work, some regulatory features of the expression of ppk were established and the polyphosphate content of S. lividans TK24 and the ppk mutant was determined. In Pi sufficiency, the expression of ppk was shown to be low but detectable. DNA gel shift experiments suggested that ppk expression might be controlled by a repressor using ATP as a corepressor. Under these conditions, short acid-soluble polyphosphates accumulated upon entry into the stationary phase in the wild-type strain but not in the ppk mutant strain. The expression of ppk under Pi-limiting conditions was shown to be much higher than that under Pi-sufficient conditions and was under positive control of the two-component system PhoR/PhoP. Under these conditions, the polyphosphate content of the cell was low and polyphosphates were reproducibly found to be longer and more abundant in the ppk mutant strain than in the wild-type strain, suggesting that Ppk might act as a nucleoside diphosphate kinase. In light of our results, a novel view of the role of this enzyme in the regulation of antibiotic biosynthesis in S. lividans TK24 is proposed.

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