Conserved Amino Acid Moieties of Candidatus Desulforudis audaxviator MazF Determine Ribonuclease Activity and Specificity

The toxin-antitoxin (TA) system, inherent to various prokaryotes, plays a critical role in survival and adaptation to diverse environmental stresses. The toxin MazF, belonging to the type II TA system, functions as a sequence-specific ribonuclease that recognizes 3 to 7 bases. In recent studies, crystallographic analysis of MazFs from several species have suggested the presence of amino acid sites important for MazF substrate RNA binding and for its catalytic activity. Herein, we characterized MazF obtained from Candidatus Desulforudis audaxviator (MazF-Da) and identified the amino acid residues necessary for its catalytic function. MazF-Da, expressed using a cell-free protein synthesis system, is a six-base-recognition-specific ribonuclease that preferentially cleaves UACAAA sequences and weakly cleaves UACGAA and UACUAA sequences. We found that MazF-Da exhibited the highest activity at around 60°C. Analysis using mutants with a single mutation at an amino acid residue site that is well conserved across various MazF toxins showed that G18, E20, R25, and P26 were important for the ribonuclease activity of MazF-Da. The recognition sequence of the N36A mutant differed from that of the wild type. This mutant cleaved UACAAG sequences in addition to UACAAA sequences, but did not cleave UACGAA or UACUAA sequences, suggesting that Asn36 affects the loosening and narrowing of MazF-Da cleavage sequence recognition. Our study posits UACAAA as the recognition sequence of MazF-Da and provides insight into the amino acid sites that are key to its unique enzymatic properties.

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Insights into the Selective Pressures Restricting Pelargonium Flower Break Virus Genome Variability: Evidence for Host Adaptation

Journal of Virology ◽

10.1128/jvi.00603-06 ◽

2006 ◽

Vol 80 (16) ◽

pp. 8124-8132 ◽

Cited By ~ 43

Author(s):

Patricia Rico ◽

Pilar Ivars ◽

Santiago F. Elena ◽

Carmen Hernández

Keyword(s):

Amino Acid ◽

Leucine Zipper ◽

Critical Role ◽

Chenopodium Quinoa ◽

Virus Genome ◽

Acid Sites ◽

Genomic Region ◽

Amino Acid Sequences ◽

Nucleotide Substitutions ◽

Selective Pressures

ABSTRACT The molecular diversity of Pelargonium flower break virus (PFBV) was assessed using a collection of isolates from different geographical origins, hosts, and collecting times. The genomic region examined was 1,828 nucleotides (nt) long and comprised the coding sequences for the movement (p7 and p12) and the coat (CP) proteins, as well as flanking segments including the entire 3′ untranslated region (3′ UTR). Some constraints limiting viral heterogeneity could be inferred from sequence analyses, such as the conservation of the amino acid sequences of p7 and of the shell domain of the CP, the maintenance of a leucine zipper motif in p12, and the preservation of a particular folding in the 3′ UTR. A remarkable covariation, involving five specific amino acid sites, was found in the CP of isolates largely propagated in the local lesion host Chenopodium quinoa and in the progeny of a PFBV variant subjected to serial passages in this host. Concomitant with this covariation, up to 30 nucleotide substitutions in a 1,428-nt region of the viral RNA could be attributable to C. quinoa-specific adaptation, representing one of the most outstanding cases of host-driven genome variation for a plant virus. Globally, the results indicate that the selective pressures exerted by the host play a critical role in shaping PFBV populations and that these populations are likely being selected for at both protein and RNA levels.

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Poliovirus Polymerase Residue 5 Plays a Critical Role in Elongation Complex Stability

Journal of Virology ◽

10.1128/jvi.02147-09 ◽

2010 ◽

Vol 84 (16) ◽

pp. 8072-8084 ◽

Cited By ~ 20

Author(s):

Sarah E. Hobdey ◽

Brian J. Kempf ◽

Benjamin P. Steil ◽

David J. Barton ◽

Olve B. Peersen

Keyword(s):

Amino Acid ◽

Rna Binding ◽

Critical Role ◽

Polymerase Activity ◽

Structural Basis ◽

Wild Type ◽

Elongation Complex ◽

Virus Growth ◽

The Stability

ABSTRACT The structures of polio-, coxsackie-, and rhinovirus polymerases have revealed a conserved yet unusual protein conformation surrounding their buried N termini where a β-strand distortion results in a solvent-exposed hydrophobic amino acid at residue 5. In a previous study, we found that coxsackievirus polymerase activity increased or decreased depending on the size of the amino acid at residue 5 and proposed that this residue becomes buried during the catalytic cycle. In this work, we extend our studies to show that poliovirus polymerase activity is also dependent on the nature of residue 5 and further elucidate which aspects of polymerase function are affected. Poliovirus polymerases with mutations of tryptophan 5 retain wild-type elongation rates, RNA binding affinities, and elongation complex formation rates but form unstable elongation complexes. A large hydrophobic residue is required to maintain the polymerase in an elongation-competent conformation, and smaller hydrophobic residues at position 5 progressively decrease the stability of elongation complexes and their processivity on genome-length templates. Consistent with this, the mutations also reduced viral RNA production in a cell-free replication system. In vivo, viruses containing residue 5 mutants produce viable virus, and an aromatic phenylalanine was maintained with only a slightly decreased virus growth rate. However, nonaromatic amino acids resulted in slow-growing viruses that reverted to wild type. The structural basis for this polymerase phenotype is yet to be determined, and we speculate that amino acid residue 5 interacts directly with template RNA or is involved in a protein structural interaction that stabilizes the elongation complex.

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Identification of the structural similarity in the functionally related amidohydrolases acting on the cyclic amide ring

Biochemical Journal ◽

10.1042/bj3300295 ◽

1998 ◽

Vol 330 (1) ◽

pp. 295-302 ◽

Cited By ~ 54

Author(s):

G.-J. Kim ◽

H.-S. Kim

Keyword(s):

Amino Acid ◽

Critical Role ◽

Evolutionary Relationship ◽

Site Directed Mutagenesis ◽

Amino Acid Residues ◽

Conserved Sequence ◽

Conserved Regions ◽

Catalytic Function ◽

Region I ◽

Cyclic Amide

The functionally related amidohydrolases, including D-hydantoinases, dihydropyrimidinases, allantoinases and dihydro-orotases, share a similar catalytic function of acting on the cyclic amide ring. We aligned 16 amidohydrolases by taking account of the conservative substitution and found a number of highly conserved regions and invariant amino acid residues. Analyses of the secondary structure and hydropathy profile of the enzymes revealed a significant degree of similarity in the conserved regions. Among the regions, the long stretched region I is of particular interest, because it is mainly composed of invariant amino acid residues, showing a similarity of 69% for the enzymes. A search of the protein data bank using the sequence of the conserved region I identified a number of proteins possessing a similar catalytic property, providing a clue that this region might be linked with the catalytic function. As a particular sequence, one aspartic acid and four histidine residues are found to be rigidly conserved in the functionally related amidohydrolases. In order to investigate the significance of the conserved residues, site-directed mutagenesis was carried out typically for the D-hydantoinase gene cloned from Bacillus stearothermophilus SD1. These residues were found to be essential for metal binding as well as catalysis, strongly implying that these invariant residues play a critical role in other enzymes as well as in D-hydantoinase. On the basis of the similar catalytic function and existence of the rigidly conserved sequence, we propose a close evolutionary relationship among the functionally related amidohydrolases, including D-hydantoinase, dihydropyrimidinase, allantoinase and dihydro-orotase.

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RNA-binding proteins La and HuR cooperatively modulate translation repression of PDCD4 mRNA

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014894 ◽

2020 ◽

pp. jbc.RA120.014894

Author(s):

Ravi Kumar ◽

Dipak Kumar Poria ◽

Partho Sarothi Ray

Keyword(s):

Inflammatory Response ◽

Chronic Inflammation ◽

Rna Binding ◽

Rna Binding Proteins ◽

Critical Role ◽

Regulation Of Gene Expression ◽

Mrna Translation ◽

Suppressor Gene ◽

Cooperative Action ◽

Translation Repression

Post-transcriptional regulation of gene expression plays a critical role in controlling the inflammatory response. An uncontrolled inflammatory response results in chronic inflammation, often leading to tumorigenesis. Programmed cell death 4 (PDCD4) is a pro-inflammatory tumor-suppressor gene which helps to prevent the transition from chronic inflammation to cancer. PDCD4 mRNA translation is regulated by an interplay between the oncogenic microRNA miR-21 and the RNA-binding protein (RBP) HuR in response to LPS stimulation, but the role of other regulatory factors remain unknown. Here we report that the RBP Lupus antigen (La) interacts with the 3’UTR of PDCD4 mRNA and prevents miR-21-mediated translation repression. While LPS causes nuclear-cytoplasmic translocation of HuR, it enhances cellular La expression. Remarkably, La and HuR were found to bind cooperatively to the PDCD4 mRNA and mitigate miR-21-mediated translation repression. The cooperative action of La and HuR reduced cell proliferation and enhanced apoptosis, reversing the pro-oncogenic function of miR-21. Together, these observations demonstrate a cooperative interplay between two RBPs, triggered differentially by the same stimulus, which exerts a synergistic effect on PDCD4 expression and thereby helps maintain a balance between inflammation and tumorigenesis.

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Insight into the mechanism of thermostabilization of GH10 xylanase from Bacillus sp. strain TAR-1 by the mutation of S92 to E

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbaa003 ◽

2021 ◽

Vol 85 (2) ◽

pp. 386-390

Author(s):

Manami Suzuki ◽

Teisuke Takita ◽

Kohei Kuwata ◽

Kota Nakatani ◽

Tongyang Li ◽

...

Keyword(s):

Amino Acid ◽

Thermodynamic Analysis ◽

Thermal Inactivation ◽

Entropy Change ◽

Crystallographic Analysis ◽

Bacillus Sp ◽

Amino Acid Residues ◽

Gh10 Xylanase ◽

Insight Into

ABSTRACT The mechanism of thermostabilization of GH10 xylanase, XynR, from Bacillus sp. strain TAR-1 by the mutation of S92 to E was investigated. Thermodynamic analysis revealed that thermostabilization was driven by the decrease in entropy change of activation for thermal inactivation. Crystallographic analysis suggested that this mutation suppressed the fluctuation of the amino acid residues at position 92-95.

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Alteration of high and low spin equilibrium by a single mutation of amino acid 209 in mouse cytochromes P450

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)67803-8 ◽

1991 ◽

Vol 266 (6) ◽

pp. 3380-3382

Author(s):

M Iwasaki ◽

R Juvonen ◽

R Lindberg ◽

M Negishi

Keyword(s):

Amino Acid ◽

Cytochromes P450 ◽

Single Mutation

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Codon-Substitution Models for Heterogeneous Selection Pressure at Amino Acid Sites

Genetics ◽

10.1093/genetics/155.1.431 ◽

2000 ◽

Vol 155 (1) ◽

pp. 431-449 ◽

Cited By ~ 41

Author(s):

Ziheng Yang ◽

Rasmus Nielsen ◽

Nick Goldman ◽

Anne-Mette Krabbe Pedersen

Keyword(s):

Amino Acid ◽

Positive Selection ◽

Selective Pressure ◽

Acid Sites ◽

Data Sets ◽

Protein Coding ◽

Important Indicator ◽

Diversifying Selection ◽

Codon Substitution ◽

Neutral Mutations

AbstractComparison of relative fixation rates of synonymous (silent) and nonsynonymous (amino acid-altering) mutations provides a means for understanding the mechanisms of molecular sequence evolution. The nonsynonymous/synonymous rate ratio (ω = dN/dS) is an important indicator of selective pressure at the protein level, with ω = 1 meaning neutral mutations, ω < 1 purifying selection, and ω > 1 diversifying positive selection. Amino acid sites in a protein are expected to be under different selective pressures and have different underlying ω ratios. We develop models that account for heterogeneous ω ratios among amino acid sites and apply them to phylogenetic analyses of protein-coding DNA sequences. These models are useful for testing for adaptive molecular evolution and identifying amino acid sites under diversifying selection. Ten data sets of genes from nuclear, mitochondrial, and viral genomes are analyzed to estimate the distributions of ω among sites. In all data sets analyzed, the selective pressure indicated by the ω ratio is found to be highly heterogeneous among sites. Previously unsuspected Darwinian selection is detected in several genes in which the average ω ratio across sites is <1, but in which some sites are clearly under diversifying selection with ω > 1. Genes undergoing positive selection include the β-globin gene from vertebrates, mitochondrial protein-coding genes from hominoids, the hemagglutinin (HA) gene from human influenza virus A, and HIV-1 env, vif, and pol genes. Tests for the presence of positively selected sites and their subsequent identification appear quite robust to the specific distributional form assumed for ω and can be achieved using any of several models we implement. However, we encountered difficulties in estimating the precise distribution of ω among sites from real data sets.

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Sequence and structure of mtr, an amino acid transport gene of Neurospora crassa

Genome ◽

10.1139/g91-098 ◽

1991 ◽

Vol 34 (4) ◽

pp. 644-651 ◽

Cited By ~ 16

Author(s):

Kenneth Koo ◽

W. Dorsey Stuart

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Neurospora Crassa ◽

Rna Binding ◽

Signal Sequence ◽

Average Length ◽

Open Reading Frame ◽

Mrna Transcript ◽

S1 Nuclease ◽

Reading Frame

The gene product of the mtr locus of Neurospora crassa is required for the transport of neutral aliphatic and aromatic amino acids via the N system. We have previously cloned three cosmids containing Neurospora DNA that complement the mtr-6(r) mutant allele. The cloned DNAs were tightly linked to restriction fragment length polymorphisms that flank the mtr locus. A 2.9-kbp fragment from one cosmid was subcloned and found to complement the mtr-6(r) allele. Here we report the sequence of the fragment that hybridized to a poly(A)+ mRNA transcript of about 2300 nucleotides. We have identified an 845-bp open reading frame (ORF) having a 59-bp intron as the potential mtr ORF. S1 nuclease analysis of the transcript confirmed the transcript size and the presence of the intron. A second open reading frame was found upstream in the same reading frame as the mtr ORF and appears to be present in the mRNA transcript. The mtr ORF is predicted to encode a 261 amino acid polypeptide with a molecular mass of 28 613 Da. The proposed polypeptide exhibits six potential α-helical transmembrane domains with an average length of 23 amino acids, does not have a signal sequence, and contains amino acid sequence homologous to an RNA binding motif.Key words: sequence, membranes, ribonucleoprotein.

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Human eosinophil cationic protein. Molecular cloning of a cytotoxin and helminthotoxin with ribonuclease activity.

Journal of Experimental Medicine ◽

10.1084/jem.170.1.163 ◽

1989 ◽

Vol 170 (1) ◽

pp. 163-176 ◽

Cited By ~ 86

Author(s):

H F Rosenberg ◽

S J Ackerman ◽

D G Tenen

Keyword(s):

Amino Acid ◽

Cellular Localization ◽

Basic Protein ◽

Eosinophil Cationic Protein ◽

Ribonuclease Activity ◽

Monocytic Differentiation ◽

Cationic Protein ◽

Human Eosinophil ◽

Reading Frame ◽

The Matrix

We have isolated a 725-bp full-length cDNA clone for the human eosinophil cationic protein (ECP). ECP is a small, basic protein found in the matrix of the eosinophil's large specific granule that has cytotoxic, helminthotoxic, and ribonuclease activity, and is a member of the ribonuclease multigene family. The cDNA sequence shows 89% sequence identity with that reported for the related granule protein, eosinophil-derived neurotoxin (EDN). The open reading frame encodes a previously unidentified 27-amino acid leader sequence preceding a 133-residue mature ECP polypeptide with a molecular mass of 15.6 kD. The encoded amino acid sequence of ECP shows 66% identity to that of EDN and 31% identity to that of human pancreatic ribonuclease, including conservation of the essential structural cysteine and cataytic lysine and histidine residues. mRNA for ECP was detected in eosinophil-enriched peripheral granulocytes and in a subclone of the promyelocytic leukemia line, HL-60, induced toward eosinophilic differentiation with IL-5. No ECP mRNA was detected in uninduced HL-60 cells, or in HL-60 cells induced toward monocytic differentiation with vitamin D3 or toward neutrophilic differentiation with DMSO. In contrast, mRNA for EDN was detected in uninduced HL-60 cells and was upregulated in HL-60 cells induced with DMSO. Despite similarities in sequence and cellular localization, these results suggest that ECP and EDN are subject to different regulatory mechanisms.

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