scholarly journals Bioinformatic and experimental evidence for suicidal and catalytic plant THI4s

2020 ◽  
Author(s):  
Jaya Joshi ◽  
Guillaume A.W. Beaudoin ◽  
Jenelle A. Patterson ◽  
Jorge D. García-García ◽  
Catherine E. Belisle ◽  
...  
Keyword(s):  
Active Site ◽  
Expressed Sequence Tag ◽  
De Novo ◽  
Indirect Evidence ◽  
Grain Filling ◽  
Prima Facie ◽  
Major Type ◽  
In Planta ◽  
Grain Filling Period ◽  
Expressed Sequence

ABSTRACTLike fungi and some prokaryotes, plants use a thiazole synthase (THI4) to make the thiazole precursor of thiamin. Fungal THI4s are suicide enzymes that destroy an essential active-site Cys residue to obtain the sulfur atom needed for thiazole formation. In contrast, certain prokaryotic THI4s have no active-site Cys, use sulfide as sulfur donor, and are truly catalytic. The presence of a conserved active-site Cys in plant THI4s and other indirect evidence implies that they are suicidal. To confirm this, we complemented the Arabidopsis tz-1 mutant, which lacks THI4 activity, with a His-tagged Arabidopsis THI4 construct. LC-MS analysis of tryptic peptides of the THI4 extracted from leaves showed that the active-site Cys was predominantly in desulfurated form, consistent with THI4 having a suicide mech-anism in planta. Unexpectedly, transcriptome datamining and deep proteome profiling showed that barley, wheat, and oat have both a widely expressed canonical THI4 with an active-site Cys, and a THI4-like paralog (non-Cys THI4) that has no active-site Cys and is the major type of THI4 in devel-oping grains. Transcriptomic evidence also indicated that barley, wheat, and oat grains synthesize thiamin de novo, implying that their non-Cys THI4s synthesize thiazole. Structure modeling supported this inference, as did demonstration that non-Cys THI4s have significant capacity to complement thia-zole auxotrophy in Escherichia coli. There is thus a prima facie case that non-Cys cereal THI4s, like their prokaryotic counterparts, are catalytic thiazole synthases. Bioenergetic calculations show that, relative to suicide THI4s, such enzymes could save substantial energy during the grain filling period.AbbreviationsDHAla, dehydroalanine; EST, expressed sequence tag; FDR, false discovery rate; IPTG, β-D-1-thiogalactopyranoside

scholarly journals Bioinformatic and experimental evidence for suicidal and catalytic plant THI4s

2020 ◽  
Vol 477 (11) ◽  
pp. 2055-2069 ◽  
Author(s):  
Jaya Joshi ◽  
Guillaume A.W. Beaudoin ◽  
Jenelle A. Patterson ◽  
Jorge D. García-García ◽  
Catherine E. Belisle ◽  
...  
Keyword(s):  
Active Site ◽  
De Novo ◽  
Indirect Evidence ◽  
Grain Filling ◽  
Prima Facie ◽  
Major Type ◽  
In Planta ◽  
Prima Facie Case ◽  
Grain Filling Period ◽  
Developing Grains

Like fungi and some prokaryotes, plants use a thiazole synthase (THI4) to make the thiazole precursor of thiamin. Fungal THI4s are suicide enzymes that destroy an essential active-site Cys residue to obtain the sulfur atom needed for thiazole formation. In contrast, certain prokaryotic THI4s have no active-site Cys, use sulfide as sulfur donor, and are truly catalytic. The presence of a conserved active-site Cys in plant THI4s and other indirect evidence implies that they are suicidal. To confirm this, we complemented the Arabidopsistz-1 mutant, which lacks THI4 activity, with a His-tagged Arabidopsis THI4 construct. LC–MS analysis of tryptic peptides of the THI4 extracted from leaves showed that the active-site Cys was predominantly in desulfurated form, consistent with THI4 having a suicide mechanism in planta. Unexpectedly, transcriptome data mining and deep proteome profiling showed that barley, wheat, and oat have both a widely expressed canonical THI4 with an active-site Cys, and a THI4-like paralog (non-Cys THI4) that has no active-site Cys and is the major type of THI4 in developing grains. Transcriptomic evidence also indicated that barley, wheat, and oat grains synthesize thiamin de novo, implying that their non-Cys THI4s synthesize thiazole. Structure modeling supported this inference, as did demonstration that non-Cys THI4s have significant capacity to complement thiazole auxotrophy in Escherichia coli. There is thus a prima facie case that non-Cys cereal THI4s, like their prokaryotic counterparts, are catalytic thiazole synthases. Bioenergetic calculations show that, relative to suicide THI4s, such enzymes could save substantial energy during the grain-filling period.

scholarly journals Application of EST-SSR markers developed from the transcriptome of Torreya grandis (Taxaceae), a threatened nut-yielding conifer tree

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5606 ◽  
Author(s):  
Jun Zeng ◽  
Jie Chen ◽  
Yixuan Kou ◽  
Yujin Wang
Keyword(s):  
Ssr Markers ◽  
Expressed Sequence Tag ◽  
De Novo ◽  
Population Genetic Analysis ◽  
Protein Databases ◽  
Genetic Groups ◽  
Conifer Tree ◽  
Torreya Grandis ◽  
Expressed Sequence ◽  
Distribution Frequency

Torreya grandis (Taxaceae) is an ancient conifer species endemic to southeast China. Because of its nutrient-rich and delicious seeds, this species has been utilized for centuries by the Chinese. However, transcriptome data and transcriptome-derived microsatellite markers for population genetics studies are still insufficient for understanding of this species’ genetic basis. In this study, a transcriptome from T. grandis leaves was generated using Illumina sequencing. A total of 69,920 unigenes were generated after de novo assembly, and annotated by searching against seven protein databases. In addition, 2,065 expressed sequence tag–simple sequence repeats (EST-SSRs) were detected, with the distribution frequency of 2.75% of total unigenes and average number of 0.03 SSRs per unigene. Among these EST-SSRs, 1,339 primer pairs were successfully designed, and 106 primer pairs were randomly selected for the development of potential molecular markers. Among them, 11 EST-SSR markers revealed a moderate level of genetic diversity, and were used to investigate the population structure of T. grandis. Two different genetic groups within this species were revealed using these EST-SSR markers, indicating that these markers developed in this study can be effectively applied to the population genetic analysis of T. grandis.

scholarly journals Identification of sugarcane genes involved in the purine synthesis pathway

2001 ◽  
Vol 24 (1-4) ◽  
pp. 251-255 ◽  
Author(s):  
Mario A. Jancso ◽  
Susana A. Sculaccio ◽  
Otavio H. Thiemann
Keyword(s):  
Expressed Sequence Tag ◽  
De Novo ◽  
Statistical Significance ◽  
Local Alignment ◽  
Central Importance ◽  
Purine Nucleotides ◽  
Nucleotide Synthesis ◽  
Purine Synthesis ◽  
Search Tool ◽  
Expressed Sequence

Nucleotide synthesis is of central importance to all cells. In most organisms, the purine nucleotides are synthesized de novo from non-nucleotide precursors such as amino acids, ammonia and carbon dioxide. An understanding of the enzymes involved in sugarcane purine synthesis opens the possibility of using these enzymes as targets for chemicals which may be effective in combating phytopathogen. Such an approach has already been applied to several parasites and types of cancer. The strategy described in this paper was applied to identify sugarcane clusters for each step of the de novo purine synthesis pathway. Representative sequences of this pathway were chosen from the National Center for Biotechnology Information (NCBI) database and used to search the translated sugarcane expressed sequence tag (SUCEST) database using the available basic local alignment search tool (BLAST) facility. Retrieved clusters were further tested for the statistical significance of the alignment by an implementation (PRSS3) of the Monte Carlo shuffling algorithm calibrated using known protein sequences of divergent taxa along the phylogenetic tree. The sequences were compared to each other and to the sugarcane clusters selected using BLAST analysis, with the resulting table of p-values indicating the degree of divergence of each enzyme within different taxa and in relation to the sugarcane clusters. The results obtained by this strategy allowed us to identify the sugarcane proteins participating in the purine synthesis pathway.

scholarly journals A new member of the chalcone synthase (CHS) family in sugarcane

2001 ◽  
Vol 24 (1-4) ◽  
pp. 257-261 ◽  
Author(s):  
Miriam G.G. Contessotto ◽  
Claudia B. Monteiro-Vitorello ◽  
Pilar D.S.C. Mariani ◽  
Luiz L. Coutinho
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Chalcone Synthase ◽  
Expressed Sequence Tag ◽  
Consensus Sequence ◽  
Stilbene Synthase ◽  
Amino Acid Sequences ◽  
Group 2 ◽  
Expressed Sequence ◽  
Group 1

Sequences from the sugarcane expressed sequence tag (SUCEST) database were analyzed based on their identities to genes encoding chalcone-synthase-like enzymes. The sorghum (Sorghum bicolor) chalcone-synthase (CHS, EC 2.3.1.74) protein sequence (gi|12229613) was used to search the SUCEST database for clusters of sequencing reads that were most similar to chalcone synthase. We found 121 reads with homology to sorghum chalcone synthase, which we were then able to sort into 14 clusters which themselves were divided into two groups (group 1 and group 2) based on the similarity of their deduced amino acid sequences. Clusters in group 1 were more similar to the sorghum enzyme than those in group 2, having the consensus sequence of the active site of chalcone and stilbene synthase. Analysis of gene expression (based on the number of reads from a specific library present in each group) indicated that most of the group 1 reads were from sugarcane flower and root libraries. Group 2 clusters were more similar to the amino acid sequence of an uncharacterized pathogen-induced protein (PI1, gi|9855801) from the S. bicolor expressed sequence tag (EST) database. The group 2 clusters sequences and PI1 proteins are 90% identical, having two amino acid changes at the chalcone and stilbene synthase consensi but conserving the cysteine residue at the active site. The PI1 EST has not been previously associated with chalcone synthase and has a different consensus sequence from the previously described chalcone synthase of sorghum. Most of the group 2 reads were from libraries prepared from sugarcane roots and plants infected with Herbaspirillum rubrisubalbicans and Gluconacetobacter diazotroficans. Our results indicate that we have identified a sugarcane chalcone synthase similar to the pathogen-induced PI1 protein found in the sorghum cDNA libraries, and it appears that both proteins represent new members of the chalcone and stilbene synthase super-family.

scholarly journals Genome Update of Botrytis cinerea Strains B05.10 and T4

2012 ◽  
Vol 11 (11) ◽  
pp. 1413-1414 ◽  
Author(s):  
Martijn Staats ◽  
Jan A. L. van Kan
Keyword(s):  
Botrytis Cinerea ◽  
Expressed Sequence Tag ◽  
De Novo ◽  
Rna Seq ◽  
High Coverage ◽  
Protein Coding ◽  
Content Type ◽  
Gene Predictor ◽  
Structure Annotation ◽  
Expressed Sequence

ABSTRACT We report here an update of the Botrytis cinerea strains B05.10 and T4 genomes, as well as an automated preliminary gene structure annotation. High-coverage de novo assemblies and reference-based alignments led to a correction of wrong base calls, elimination of sequence gaps, and improved joining of contigs. The new assemblies have substantially lower numbers of scaffolds and a concomitant increase in the N 50 .The list of protein-coding genes was generated using the evidence-driven gene predictor Augustus, with expressed sequence tag evidence and RNA-Seq data as input.

scholarly journals Homology-Driven Proteomics of Dinoflagellates with Unsequenced Genomes Using MALDI-TOF/TOF and AutomatedDe NovoSequencing

2011 ◽  
Vol 2011 ◽  
pp. 1-16 ◽  
Author(s):  
Da-Zhi Wang ◽  
Cheng Li ◽  
Zhang-Xian Xie ◽  
Hong-Po Dong ◽  
Lin Lin ◽  
...  
Keyword(s):  
Expressed Sequence Tag ◽  
De Novo ◽  
Alexandrium Tamarense ◽  
Database Searching ◽  
Protein Sequence Analysis ◽  
Protein Database ◽  
Blast Search ◽  
Maldi Tof ◽  
Ncbinr Database ◽  
Expressed Sequence

This study developed a multilayered, gel-based, and underivatized strategy forde novoprotein sequence analysis of unsequenced dinoflagellates using a MALDI-TOF/TOF mass spectrometer with the assistance of DeNovo Explorer software. MASCOT was applied as the first layer screen to identify either known or unknown proteins sharing identical peptides presented in a database. Once the confident identifications were removed after searching against the NCBInr database, the remainder was searched against the dinoflagellate expressed sequence tag database. In the last layer, those borderline and nonconfident hits were further subjected tode novointerpretation using DeNovo Explorer software. Thede novosequences passing a reliability filter were subsequently submitted to nonredundant MS-BLAST search. Using this layer identification method, 216 protein spots representing 158 unique proteins out of 220 selected protein spots fromAlexandrium tamarense, a dinoflagellate with unsequenced genome, were confidently or tentatively identified by database searching. These proteins were involved in various intracellular physiological activities. This study is the first effort to develop a completely automated approach to identify proteins from unsequenced dinoflagellate databases and establishes a preliminary protein database for various physiological studies of dinoflagellates in the future.

Influence of Canopy Temperature (CT) During Grain-Filling Period on Yield and Effects of Several CT-Associated SSR Loci

2015 ◽  
Vol 41 (4) ◽  
pp. 548 ◽  
Author(s):  
Dong-Ling ZHANG ◽  
Hong-Na ZHANG ◽  
Chen-Yang HAO ◽  
Lan-Fen WANG ◽  
Tian LI ◽  
...  
Keyword(s):  
Canopy Temperature ◽  
Grain Filling ◽  
Ssr Loci ◽  
Grain Filling Period

Effect of PASP-KT-NAA on Leaf Senescence and Grain Filling Rate during the Grain-Filling Period in Different Temperature Zones

2013 ◽  
Vol 38 (9) ◽  
pp. 1698-1709
Author(s):  
Tian-Jun XU ◽  
Zhi-Qiang DONG ◽  
Jiao GAO ◽  
Chuan-Xiao CHEN ◽  
Liu JIAO ◽  
...  
Keyword(s):  
Leaf Senescence ◽  
Grain Filling ◽  
Filling Rate ◽  
Grain Filling Rate ◽  
Grain Filling Period

scholarly journals An enzymatic activation of formaldehyde for nucleotide methylation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Charles Bou-Nader ◽  
Frederick W. Stull ◽  
Ludovic Pecqueur ◽  
Philippe Simon ◽  
Vincent Guérineau ◽  
...  
Keyword(s):  
Amino Acids ◽  
Thymidylate Synthase ◽  
Active Site ◽  
De Novo ◽  
Crystallographic Structure ◽  
De Novo Synthesis ◽  
Active Site Residues ◽  
Enzymatic Activation ◽  
Enzyme Cofactors ◽  
Unique Ability

AbstractFolate enzyme cofactors and their derivatives have the unique ability to provide a single carbon unit at different oxidation levels for the de novo synthesis of amino-acids, purines, or thymidylate, an essential DNA nucleotide. How these cofactors mediate methylene transfer is not fully settled yet, particularly with regard to how the methylene is transferred to the methylene acceptor. Here, we uncovered that the bacterial thymidylate synthase ThyX, which relies on both folate and flavin for activity, can also use a formaldehyde-shunt to directly synthesize thymidylate. Combining biochemical, spectroscopic and anaerobic crystallographic analyses, we showed that formaldehyde reacts with the reduced flavin coenzyme to form a carbinolamine intermediate used by ThyX for dUMP methylation. The crystallographic structure of this intermediate reveals how ThyX activates formaldehyde and uses it, with the assistance of active site residues, to methylate dUMP. Our results reveal that carbinolamine species promote methylene transfer and suggest that the use of a CH2O-shunt may be relevant in several other important folate-dependent reactions.

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