scholarly journals Site-Directed Amino Acid Substitutions in the Hydroxylase α Subunit of Butane Monooxygenase from Pseudomonas butanovora: Implications for Substrates Knocking at the Gate

2006 ◽  
Vol 188 (13) ◽  
pp. 4962-4969 ◽  
Author(s):  
Kimberly H. Halsey ◽  
Luis A. Sayavedra-Soto ◽  
Peter J. Bottomley ◽  
Daniel J. Arp
Keyword(s):  
Amino Acid ◽  
Methane Oxidation ◽  
In Vivo Studies ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Butanol Production ◽  
Α Subunit ◽  
Wide Range ◽  
Pseudomonas Butanovora ◽  
Butane Monooxygenase

ABSTRACT Butane monooxygenase (BMO) from Pseudomonas butanovora has high homology to soluble methane monooxygenase (sMMO), and both oxidize a wide range of hydrocarbons; yet previous studies have not demonstrated methane oxidation by BMO. Studies to understand the basis for this difference were initiated by making single-amino-acid substitutions in the hydroxylase α subunit of butane monooxygenase (BMOH-α) in P. butanovora. Residues likely to be within hydrophobic cavities, adjacent to the diiron center, and on the surface of BMOH-α were altered to the corresponding residues from the α subunit of sMMO. In vivo studies of five site-directed mutants were carried out to initiate mechanistic investigations of BMO. Growth rates of mutant strains G113N and L279F on butane were dramatically slower than the rate seen with the control P. butanovora wild-type strain (Rev WT). The specific activities of BMO in these strains were sevenfold lower than those of Rev WT. Strains G113N and L279F also showed 277- and 5.5-fold increases in the ratio of the rates of 2-butanol production to 1-butanol production compared to Rev WT. Propane oxidation by strain G113N was exclusively subterminal and led to accumulation of acetone, which P. butanovora could not further metabolize. Methane oxidation was measurable for all strains, although accumulation of 23 μM methanol led to complete inhibition of methane oxidation in strain Rev WT. In contrast, methane oxidation by strain G113N was not completely inhibited until the methanol concentration reached 83 μM. The structural significance of the results obtained in this study is discussed using a three-dimensional model of BMOH-α.

scholarly journals Evidence for Modified Mechanisms of Chloroethene Oxidation in Pseudomonas butanovora Mutants Containing Single Amino Acid Substitutions in the Hydroxylase α-Subunit of Butane Monooxygenase

2007 ◽  
Vol 189 (14) ◽  
pp. 5068-5074 ◽  
Author(s):  
Kimberly H. Halsey ◽  
David M. Doughty ◽  
Luis A. Sayavedra-Soto ◽  
Peter J. Bottomley ◽  
Daniel J. Arp
Keyword(s):  
Amino Acid ◽  
Mutant Strain ◽  
Parent Compound ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Α Subunit ◽  
Mutant Strains ◽  
Pseudomonas Butanovora ◽  
Butane Monooxygenase ◽  
The Impact

ABSTRACT The properties of oxidation of dichloroethene (DCE) and trichloroethylene (TCE) by three mutant strains of Pseudomonas butanovora containing single amino acid substitutions in the α-subunit of butane monooxygenase hydroxylase (BMOH-α) were compared to the properties of the wild-type strain (Rev WT). The rates of oxidation of three chloroethenes (CEs) were reduced in mutant strain G113N and corresponded with a lower maximum rate of butane oxidation. The rate of TCE degradation was reduced by one-half in mutant strain L279F, whereas the rates of DCE oxidation were the same as those in Rev WT. Evidence was obtained that the composition of products of CE oxidation differed between Rev WT and some of the mutant strains. For example, while Rev WT released nearly all available chlorine stoichiometrically during CE oxidation, strain F321Y released about 40% of the chlorine during 1,2-cis-DCE and TCE oxidation, and strain G113N released between 14 and 25% of the available chlorine during oxidation of DCE and 56% of the available chlorine during oxidation of TCE. Whereas Rev WT, strain L279F, and strain F321Y formed stoichiometric amounts of 1,2-cis-DCE epoxide during oxidation of 1,2-cis-DCE, only about 50% of the 1,2-cis-DCE oxidized by strain G113N was detected as the epoxide. Evidence was obtained that 1,2-cis-DCE epoxide was a substrate for butane monooxygenase (BMO) that was oxidized after the parent compound was consumed. Yet all of the mutant strains released less than 40% of the available 1,2-cis-DCE chlorine, suggesting that they have altered activity towards the epoxide. In addition, strain G113N was unable to degrade the epoxide. TCE epoxide was detected during exposure of Rev WT and strain F321Y to TCE but was not detected with strains L279F and G113N. Lactate-dependent O2 uptake rates were differentially affected by DCE degradation in the mutant strains, providing evidence that some products released by the altered BMOs reduced the impact of CE on cellular toxicity. The use of CEs as substrates in combination with P. butanovora BMOH-α mutants might allow insights into the catalytic mechanism of BMO to be obtained.

scholarly journals Identification of Escherichia coli dnaE(polC) Mutants with Altered Sensitivity to 2′,3′-Dideoxyadenosine

2000 ◽  
Vol 182 (14) ◽  
pp. 3942-3947 ◽  
Author(s):  
Koji Hiratsuka ◽  
Linda J. Reha-Krantz
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Dna Polymerase ◽  
Protein Product ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Α Subunit ◽  
Nucleotide Interactions ◽  
Increased Sensitivity ◽  
Mutant Phenotypes

ABSTRACT Bacteria with reduced DNA polymerase I activity have increased sensitivity to killing by chain-terminating nucleotides (S. A. Rashbaum and N. R. Cozzarelli, Nature 264:679–680, 1976). We have used this observation as the basis of a genetic strategy to identify mutations in the dnaE (polC) gene ofEscherichia coli that alter sensitivity to 2′,3′-dideoxyadenosine (ddA). Two dnaE (polC) mutant strains with increased sensitivity to ddA and one strain with increased resistance were isolated and characterized. The mutant phenotypes are due to single amino acid substitutions in the α subunit, the protein product of the dnaE (polC) gene. Increased sensitivity to ddA is produced by the L329F and H417Y substitutions, and increased resistance is produced by the G365S substitution. The L329F and H417Y substitutions also reduce the accuracy of DNA replication (the mutator phenotype), while the G365S substitution increases accuracy (the antimutator phenotype). All of the amino acid substitutions are in conserved regions near essential aspartate residues. These results prove the effectiveness of the genetic strategy in identifying informative dnaE(polC) mutations that can be used to elucidate the molecular basis of nucleotide interactions in the α subunit of the DNA polymerase III holoenzyme.

scholarly journals Mutational analysis of an autoantibody: differential binding and pathogenicity.

1994 ◽  
Vol 180 (3) ◽  
pp. 925-932 ◽  
Author(s):  
J B Katz ◽  
W Limpanasithikul ◽  
B Diamond
Keyword(s):  
Amino Acid ◽  
Mutational Analysis ◽  
Site Directed Mutagenesis ◽  
In Vivo Studies ◽  
Single Amino Acid ◽  
Antigen Binding ◽  
Amino Acid Substitutions ◽  
Renal Tubules ◽  
Dsdna Antibody ◽  
Dna Specificity

We have used site-directed mutagenesis to change amino acid residues in the heavy chain of the pathogenic R4A anti-double-stranded DNA (dsDNA) antibody and have looked for resultant alterations in DNA binding and in pathogenicity. The data demonstrate that single amino acid substitutions in both complementarity determining and framework regions alter antigen binding. Changes in only a few amino acids entirely ablate DNA specificity or cause a 10-fold increase in relative binding. In vivo studies in mice of the pathogenicity of the mutated antibodies show that a single amino acid substitution leading to a loss of dsDNA binding leads also to a loss of glomerular sequestration. Amino acid substitutions that increase relative affinity for dsDNA cause a change in localization of immunoglobulin deposition from glomeruli to renal tubules. These studies demonstrate that small numbers of amino acid substitutions can dramatically alter antigen binding and pathogenicity, and that the pathogenicity of anti-DNA antibodies does not strictly correlate with affinity for DNA.

scholarly journals A Single Point Mutation, Asn16→Lys, Dictates the Temperature-Sensitivity of the Reovirus tsG453 Mutant

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 289
Author(s):  
Kathleen K. M. Glover ◽  
Danica M. Sutherland ◽  
Terence S. Dermody ◽  
Kevin M. Coombs
Keyword(s):  
Amino Acid ◽  
Temperature Sensitivity ◽  
Reverse Genetics ◽  
Core Protein ◽  
Single Point ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Temperature Sensitive ◽  
Amino Acid Substitutions ◽  
Gene Encoding

Studies of conditionally lethal mutants can help delineate the structure-function relationships of biomolecules. Temperature-sensitive (ts) mammalian reovirus (MRV) mutants were isolated and characterized many years ago. Two of the most well-defined MRV ts mutants are tsC447, which contains mutations in the S2 gene encoding viral core protein σ2, and tsG453, which contains mutations in the S4 gene encoding major outer-capsid protein σ3. Because many MRV ts mutants, including both tsC447 and tsG453, encode multiple amino acid substitutions, the specific amino acid substitutions responsible for the ts phenotype are unknown. We used reverse genetics to recover recombinant reoviruses containing the single amino acid polymorphisms present in ts mutants tsC447 and tsG453 and assessed the recombinant viruses for temperature-sensitivity by efficiency-of-plating assays. Of the three amino acid substitutions in the tsG453 S4 gene, Asn16-Lys was solely responsible for the tsG453ts phenotype. Additionally, the mutant tsC447 Ala188-Val mutation did not induce a temperature-sensitive phenotype. This study is the first to employ reverse genetics to identify the dominant amino acid substitutions responsible for the tsC447 and tsG453 mutations and relate these substitutions to respective phenotypes. Further studies of other MRV ts mutants are warranted to define the sequence polymorphisms responsible for temperature sensitivity.

scholarly journals Natural variants in SARS-CoV-2 Spike protein pinpoint structural and functional hotspots with implications for prophylaxis and therapeutic strategies

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Suman Pokhrel ◽  
Benjamin R. Kraemer ◽  
Scott Burkholz ◽  
Daria Mochly-Rosen
Keyword(s):  
Amino Acid ◽  
Severe Disease ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Severe Respiratory Distress ◽  
S Protein ◽  
Individual Sequences ◽  
Natural Variants ◽  
Novel Coronavirus ◽  
The Individual

AbstractIn December 2019, a novel coronavirus, termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified as the cause of pneumonia with severe respiratory distress and outbreaks in Wuhan, China. The rapid and global spread of SARS-CoV-2 resulted in the coronavirus 2019 (COVID-19) pandemic. Earlier during the pandemic, there were limited genetic viral variations. As millions of people became infected, multiple single amino acid substitutions emerged. Many of these substitutions have no consequences. However, some of the new variants show a greater infection rate, more severe disease, and reduced sensitivity to current prophylaxes and treatments. Of particular importance in SARS-CoV-2 transmission are mutations that occur in the Spike (S) protein, the protein on the viral outer envelope that binds to the human angiotensin-converting enzyme receptor (hACE2). Here, we conducted a comprehensive analysis of 441,168 individual virus sequences isolated from humans throughout the world. From the individual sequences, we identified 3540 unique amino acid substitutions in the S protein. Analysis of these different variants in the S protein pinpointed important functional and structural sites in the protein. This information may guide the development of effective vaccines and therapeutics to help arrest the spread of the COVID-19 pandemic.

Single Amino Acid Substitutions Disrupt Tetramer Formation in the Dihydroneopterin Aldolase Enzyme ofPneumocystis carinii†

Biochemistry ◽  
1998 ◽  
Vol 37 (33) ◽  
pp. 11629-11636 ◽  
Author(s):  
M. Carmen Thomas ◽  
Stuart P. Ballantine ◽  
Susanne S. Bethell ◽  
Satty Bains ◽  
Paul Kellam ◽  
...  
Keyword(s):  
Amino Acid ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Tetramer Formation ◽  
Dihydroneopterin Aldolase

scholarly journals Pathogenic alleles in microtubule, secretory granule and extracellular matrix-related genes in familial keratoconus

2021 ◽  
Author(s):  
Vishal Shinde ◽  
Nara Sobreira ◽  
Elizabeth S Wohler ◽  
George Maiti ◽  
Nan Hu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Cultures ◽  
Cell Biology ◽  
Stromal Cell ◽  
Primary Cilia ◽  
Single Amino Acid ◽  
European Ancestry ◽  
Base Substitution ◽  
Amino Acid Substitutions ◽  
Functional Link

Abstract Keratoconus is a common corneal defect with a complex genetic basis. By whole exome sequencing of affected members from 11 multiplex families of European ancestry, we identified 23 rare, heterozygous, potentially pathogenic variants in 8 genes. These include nonsynonymous single amino acid substitutions in HSPG2, EML6 and CENPF in two families each, and in NBEAL2, LRP1B, PIK3CG and MRGPRD in three families each; ITGAX had nonsynonymous single amino acid substitutions in two families and an indel with a base substitution producing a nonsense allele in the third family. Only HSPG2, EML6 and CENPF have been associated with ocular phenotypes previously. With the exception of MRGPRD and ITGAX, we detected the transcript and encoded protein of the remaining genes in the cornea and corneal cell cultures. Cultured stromal cells showed cytoplasmic punctate staining of NBEAL2, staining of the fibrillar cytoskeletal network by EML6, while CENPF localized to the basal body of primary cilia. We inhibited the expression of HSPG2, EML6, NBEAL2 and CENPF in stromal cell cultures and assayed for the expression of COL1A1 as a readout of corneal matrix production. An upregulation in COL1A1 after siRNA inhibition indicated their functional link to stromal cell biology. For ITGAX, encoding a leukocyte integrin, we assayed its level in the sera of 3 affected families compared with 10 unrelated controls to detect an increase in all affecteds. Our study identified genes that regulate the cytoskeleton, protein trafficking and secretion, barrier tissue function and response to injury and inflammation, as being relevant to keratoconus.

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