scholarly journals Characterization of LytA-Like N-Acetylmuramoyl-l-Alanine Amidases from Two New Streptococcus mitis Bacteriophages Provides Insights into the Properties of the Major Pneumococcal Autolysin

2004 ◽  
Vol 186 (24) ◽  
pp. 8229-8239 ◽  
Author(s):  
Patricia Romero ◽  
Rubens López ◽  
Ernesto García
Keyword(s):  
Amino Acid ◽  
Biochemical Properties ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Lytic Phage ◽  
Streptococcus Mitis ◽  
Single Amino Acid Residue ◽  
Low Activity

ABSTRACT Two new temperate bacteriophages exhibiting a Myoviridae (φB6) and a Siphoviridae (φHER) morphology have been isolated from Streptococcus mitis strains B6 and HER 1055, respectively, and partially characterized. The lytic phage genes were overexpressed in Escherichia coli, and their encoded proteins were purified. The lytA HER and lytA B6 genes are very similar (87% identity) and appeared to belong to the group of the so-called typical LytA amidases (atypical LytA displays a characteristic two-amino-acid deletion signature). although they exhibited several differential biochemical properties with respect to the pneumococcal LytA, e.g., they were inhibited in vitro by sodium deoxycholate and showed a more acidic pH for optimal activity. However, and in sharp contrast with the pneumococcal LytA, a short dialysis of LytAHER or LytAB6 resulted in reversible deconversion to the low-activity state (E-form) of the fully active phage amidases (C-form). Comparison of the amino acid sequences of LytAHER and LytAB6 with that of the pneumococcal amidase suggested that Val317 might be responsible for at least some of the peculiar properties of S. mitis phage enzymes. Site-directed mutagenesis that changed Val317 in the pneumococcal LytA amidase to a Thr residue (characteristic of LytAB6 and LytAHER) produced a fully active pneumococcal enzyme that differs from the parental one only in that the mutant amidase can reversibly recover the low-activity E-form upon dialysis. This is the first report showing that a single amino acid residue is involved in the conversion process of the major S. pneumoniae autolysin. Our results also showed that some lysogenic S. mitis strains possess a lytA-like gene, something that was previously thought to be exclusive to Streptococcus pneumoniae. Moreover, the newly discovered phage lysins constitute a missing link between the typical and atypical pneumococcal amidases known previously.

scholarly journals Single amino acid changes in the mumps virus haemagglutinin–neuraminidase and polymerase proteins are associated with neuroattenuation

2009 ◽  
Vol 90 (7) ◽  
pp. 1741-1747 ◽  
Author(s):  
Tahir H. Malik ◽  
Candie Wolbert ◽  
Laura Nerret ◽  
Christian Sauder ◽  
Steven Rubin
Keyword(s):  
Amino Acid ◽  
Clinical Isolate ◽  
Amino Acid Change ◽  
Mumps Virus ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Supporting Evidence ◽  
L Protein ◽  
Single Amino Acid Change

It has previously been shown that three amino acid changes, one each in the fusion (F; Ala/Thr-91→Thr), haemagglutinin–neuraminidase (HN; Ser-466→Asn) and polymerase (L; Ile-736→Val) proteins, are associated with attenuation of a neurovirulent clinical isolate of mumps virus (88-1961) following serial passage in vitro. Here, using full-length cDNA plasmid clones and site-directed mutagenesis, it was shown that the single amino acid change in the HN protein and to a lesser extent, the change in the L protein, resulted in neuroattenuation, as assessed in rats. The combination of both amino acid changes caused neuroattenuation of the virus to levels previously reported for the clinical isolate following attenuation in vitro. The amino acid change in the F protein, despite having a dramatic effect on protein function in vitro, was previously shown to not be involved in the observed neuroattenuation, highlighting the importance of conducting confirmatory in vivo studies. This report provides additional supporting evidence for the role of the HN protein as a virulence factor and, as far as is known, is the first report to associate an amino acid change in the L protein with mumps virus neuroattenuation.

scholarly journals Functional effects of single amino acid substitutions in the region of Phe113 to Asp138 in the plasminogen activator inhibitor 1 molecule

1998 ◽  
Vol 331 (2) ◽  
pp. 409-415 ◽  
Author(s):  
Guang-Chao SUI ◽  
Björn WIMAN
Keyword(s):  
Amino Acid ◽  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Plasminogen Activator Inhibitor 1 ◽  
Low Activity ◽  
Activator Inhibitor ◽  
Pai 1

Thirteen amino acid substitutions have been introduced within the stretch Phe113 to Asp138 in the plasminogen activator inhibitor 1 (PAI-1) molecule by site-directed mutagenesis. The different proteins and wild-type (wt) PAI-1 have been overexpressed in Escherichia coliand purified by chromatography on heparin–Sepharose and on anhydrotrypsin–agarose. The PAI-1 variants have been characterized by their reactivity with tissue plasminogen activator (tPA), interactions with vitronectin or heparin, and stability. Most PAI-1 variants, except for Asp125 → Lys, Phe126 → Ser and Arg133 → Asp, displayed a high spontaneous inhibitory activity towards tPA, which did not change greatly on reactivation with 4 M guanidinium chloride, followed by dialysis at pH 5.5. The variants Asp125 → Lys and Arg133 → Asp became much more active after reactivation and they were also more rapidly transformed to inactive forms (t½ 22–31 min) at physiological pH and temperature than the other variants. However, in the presence of vitronectin they were both almost equally stable (t½ 2.3 h) as wtPAI-1 (t½ 3.0 h). The mutant Glu130 → Lys showed an increased stability, both in the absence and in the presence of vitronectin compared with wtPAI-1. Nevertheless a similar affinity between all the active PAI-1 variants and vitronectin was observed. Further, all mutants, including the three mutants with low activity, were to a large extent adsorbed on anhydrotrypsin–agarose and were eluted in a similar fashion. In accordance with these data, the three variants with a low activity were all to a large extent cleaved as a result of their reaction with tPA, suggesting that they occurred predominantly in the substrate conformation. Our results do not support the presence of a binding site for vitronectin in this part of the molecule, but rather that it might be involved in controlling the active PAI-1 to substrate transition. Partly, this region of the PAI-1 molecule (Arg115 to Arg118) seems also to be involved in the binding of heparin to PAI-1.

scholarly journals Three Drosophila beta-tubulin sequences: a developmentally regulated isoform (beta 3), the testis-specific isoform (beta 2), and an assembly-defective mutation of the testis-specific isoform (B2t8) reveal both an ancient divergence in metazoan isotypes and structural constraints for beta-tubulin function.

1987 ◽  
Vol 7 (6) ◽  
pp. 2231-2242 ◽  
Author(s):  
J E Rudolph ◽  
M Kimble ◽  
H D Hoyle ◽  
M A Subler ◽  
E C Raff
Keyword(s):  
Amino Acid ◽  
Sequence Divergence ◽  
Amino Acid Sequences ◽  
Single Amino Acid ◽  
Structural Constraints ◽  
Wild Type ◽  
Beta Tubulin ◽  
Developmentally Regulated ◽  
Single Amino Acid Residue ◽  
Beta 2

The genomic DNA sequence and deduced amino acid sequence are presented for three Drosophila melanogaster beta-tubulins: a developmentally regulated isoform beta 3-tubulin, the wild-type testis-specific isoform beta 2-tubulin, and an ethyl methanesulfonate-induced assembly-defective mutation of the testis isoform, B2t8. The testis-specific beta 2-tubulin is highly homologous to the major vertebrate beta-tubulins, but beta 3-tubulin is considerably diverged. Comparison of the amino acid sequences of the two Drosophila isoforms to those of other beta-tubulins indicates that these two proteins are representative of an ancient sequence divergence event which at least preceded the split between lines leading to vertebrates and invertebrates. The intron/exon structures of the genes for beta 2- and beta 3-tubulin are not the same. The structure of the gene for the variant beta 3-tubulin isoform, but not that of the testis-specific beta 2-tubulin gene, is similar to that of vertebrate beta-tubulins. The mutation B2t8 in the gene for the testis-specific beta 2-tubulin defines a single amino acid residue required for normal assembly function of beta-tubulin. The sequence of the B2t8 gene is identical to that of the wild-type gene except for a single nucleotide change resulting in the substitution of lysine for glutamic acid at residue 288. This position falls at the junction between two major structural domains of the beta-tubulin molecule. Although this hinge region is relatively variable in sequence among different beta-tubulins, the residue corresponding to glu 288 of Drosophila beta 2-tubulin is highly conserved as an acidic amino acid not only in all other beta-tubulins but in alpha-tubulins as well.

scholarly journals A single amino acid change converts Aurora-A into Aurora-B-like kinase in terms of partner specificity and cellular function

2009 ◽  
Vol 106 (17) ◽  
pp. 6939-6944 ◽  
Author(s):  
Jingyan Fu ◽  
Minglei Bian ◽  
Junjun Liu ◽  
Qing Jiang ◽  
Chuanmao Zhang
Keyword(s):  
Amino Acid ◽  
Aurora Kinase ◽  
Single Amino Acid ◽  
Aurora B ◽  
Aurora A ◽  
Single Amino Acid Residue ◽  
Single Amino Acid Change ◽  
Equivalent Residue ◽  
And Function

Aurora kinase-A and -B are key regulators of the cell cycle and tumorigenesis. It has remained a mystery why these 2 Aurora kinases, although highly similar in protein sequence and structure, are distinct in subcellular localization and function. Here, we report the striking finding that a single amino acid residue is responsible for these differences. We replaced the Gly-198 of Aurora-A with the equivalent residue Asn-142 of Aurora-B and found that in HeLa cells, Aurora-AG198N was recruited to the inner centromere in metaphase and the midzone in anaphase, reminiscent of the Aurora-B localization. Moreover, Aurora-AG198N compensated for the loss of Aurora-B in chromosome misalignment and cell premature exit from mitosis. Furthermore, Aurora-AG198N formed a complex with the Aurora-B partners, INCENP and Survivin, and its localization depended on this interaction. Aurora-AG198N phosphorylated the Aurora-B substrates INCENP and Survivin in vitro. Therefore, we propose that the presence of Gly or Asn at a single site assigns Aurora-A and -B to their respective partners and thus to their distinctive subcellular localizations and functions.

A single amino acid residue replacement in the β subunit of human chorionic gonadotrophin results in the loss of biological activity

1992 ◽  
Vol 8 (1) ◽  
pp. 87-89 ◽  
Author(s):  
F. Chen ◽  
D. Puett
Keyword(s):  
Amino Acid ◽  
Amino Acid Residue ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Amino Acid Sequences ◽  
Single Amino Acid ◽  
Β Subunit ◽  
Single Amino Acid Residue

ABSTRACT The heterodimer, human chorionic gonadotrophin (hCG), contains an a subunit that is common to the glycoprotein hormones and a hormone-specific β subunit. A comparison of all known β amino acid sequences shows that an aspartic acid at position 99 (with the numbering scheme for hCG-β) is one of the seven non-Cys invariant residues. Using site-directed mutagenesis we have replaced hCG-β Asp99 with Arg. Chinese hamster ovary cells, containing a stably integrated gene for bovine a subunit, were transiently transfected with plasmids containing wild-type and mutant hCG-β cDNAs. The Arg99 β mutant associated with the a subunit, but the resulting heterodimer failed to enhance intracellular cyclic AMP production in a gonadotrophin-responsive transformed murine Leydig cell line. Thus, a single amino acid residue replacement in this glycosylated heterodimer containing 237 amino acid residues is sufficient to abolish activity.

Three Drosophila beta-tubulin sequences: a developmentally regulated isoform (beta 3), the testis-specific isoform (beta 2), and an assembly-defective mutation of the testis-specific isoform (B2t8) reveal both an ancient divergence in metazoan isotypes and structural constraints for beta-tubulin function

1987 ◽  
Vol 7 (6) ◽  
pp. 2231-2242
Author(s):  
J E Rudolph ◽  
M Kimble ◽  
H D Hoyle ◽  
M A Subler ◽  
E C Raff
Keyword(s):  
Amino Acid ◽  
Sequence Divergence ◽  
Amino Acid Sequences ◽  
Single Amino Acid ◽  
Structural Constraints ◽  
Wild Type ◽  
Beta Tubulin ◽  
Developmentally Regulated ◽  
Single Amino Acid Residue ◽  
Beta 2

The genomic DNA sequence and deduced amino acid sequence are presented for three Drosophila melanogaster beta-tubulins: a developmentally regulated isoform beta 3-tubulin, the wild-type testis-specific isoform beta 2-tubulin, and an ethyl methanesulfonate-induced assembly-defective mutation of the testis isoform, B2t8. The testis-specific beta 2-tubulin is highly homologous to the major vertebrate beta-tubulins, but beta 3-tubulin is considerably diverged. Comparison of the amino acid sequences of the two Drosophila isoforms to those of other beta-tubulins indicates that these two proteins are representative of an ancient sequence divergence event which at least preceded the split between lines leading to vertebrates and invertebrates. The intron/exon structures of the genes for beta 2- and beta 3-tubulin are not the same. The structure of the gene for the variant beta 3-tubulin isoform, but not that of the testis-specific beta 2-tubulin gene, is similar to that of vertebrate beta-tubulins. The mutation B2t8 in the gene for the testis-specific beta 2-tubulin defines a single amino acid residue required for normal assembly function of beta-tubulin. The sequence of the B2t8 gene is identical to that of the wild-type gene except for a single nucleotide change resulting in the substitution of lysine for glutamic acid at residue 288. This position falls at the junction between two major structural domains of the beta-tubulin molecule. Although this hinge region is relatively variable in sequence among different beta-tubulins, the residue corresponding to glu 288 of Drosophila beta 2-tubulin is highly conserved as an acidic amino acid not only in all other beta-tubulins but in alpha-tubulins as well.

scholarly journals Site-directed mutation studies of human liver cytochrome P-450 isoenzymes in the CYP2C subfamily

1993 ◽  
Vol 289 (2) ◽  
pp. 533-538 ◽  
Author(s):  
M E Veronese ◽  
C J Doecke ◽  
P I Mackenzie ◽  
M E McManus ◽  
J O Miners ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Human Liver ◽  
Liver Microsomes ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Kinetics Of ◽  
Cytochrome P 450

Evidence from human studies in vivo and in vitro strongly suggests that the methylhydroxylation of tolbutamide and the 4-hydroxylation of phenytoin, the major pathways in the elimination of these two drugs, are catalysed by the same cytochrome P-450 isoenzyme(s). In the present study we used site-directed mutagenesis and cDNA expression in COS cells to characterize in detail the kinetics of tolbutamide and phenytoin hydroxylations by seven CYP2C proteins (2C8, 2C9 and variants, and 2C10) in order to define the effects of small changes in amino acid sequences and the likely proteins responsible in the metabolism of these two drugs in man. Tolbutamide was hydroxylated to varying extents by all expressed cytochrome P-450 isoenzymes, although activity was much lower for the expressed 2C8 protein. While the apparent Km values for the 2C9/10 isoenzymes (71.6-131.7 microM) were comparable with the range of apparent Km values previously observed in human liver microsomes, the apparent Km for 2C8 (650.5 microM) was appreciably higher. The 2C8 enzyme also showed quite different sulphaphenazole inhibition characteristics. The 4-hydroxylation of phenytoin was also more efficiently catalysed by the 2C9/10 enzymes. These enzymes showed similarities in kinetics of phenytoin hydroxylation and sulphaphenazole inhibition compared with human liver phenytoin hydroxylase. Also of interest was the observation that, among the 2C9 variants, small differences in amino acid composition could appreciably affect both tolbutamide and phenytoin hydroxylations. The amino acid substitution Cys-144->Arg increased both the rates of tolbutamide and phenytoin hydroxylations, while the Leu-359->Ile change had a greater effect on phenytoin hydroxylation. We conclude that: (1) although 2C8 and 2C9/10 proteins metabolize tolbutamide. only 2C9/10 proteins play a major role in human liver; (2) 2C9/10 proteins also appear to be chiefly responsible for phenytoin hydroxylation; and (3) subtle differences in the amino acid composition of these 2C9/10 proteins can affect the functional specificities towards both tolbutamide and phenytoin.

scholarly journals Combinations of two amino acids (Ala40 and Phe75 or Ser40 and Tyr75) in the coat protein of apple chlorotic leaf spot virus are crucial for infectivity

2007 ◽  
Vol 88 (9) ◽  
pp. 2611-2618 ◽  
Author(s):  
Hajime Yaegashi ◽  
Masamichi Isogai ◽  
Hiroko Tajima ◽  
Teruo Sano ◽  
Nobuyuki Yoshikawa
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Coat Protein ◽  
Leaf Spot ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Cdna Clones ◽  
Spot Virus ◽  
Chlorotic Leaf

Amino acid sequences of apple chlorotic leaf spot virus (ACLSV) coat protein (CP) were compared between 12 isolates from apple, plum and cherry, and 109 cDNA clones that were amplified directly from infected apple tissues. Phylogenetic analysis based on the amino acid sequences of CP showed that the isolates and cDNA clones were separated into two major clusters in which the combinations of the five amino acids at positions 40, 59, 75, 130 and 184 (Ala40-Val59-Phe75-Ser130-Met184 or Ser40-Leu59-Tyr75-Thr130-Leu184) were highly conserved within each cluster. Site-directed mutagenesis using an infectious cDNA clone of ACLSV indicated that the combinations of two amino acids (Ala40 and Phe75 or Ser40 and Tyr75) are necessary for infectivity to Chenopodium quinoa plants by mechanical inoculation. Moreover, an agroinoculation assay indicated that the substitution of a single amino acid (Ala40 to Ser40 or Phe75 to Tyr75) resulted in extreme reduction in the accumulation of viral genomic RNA, double-stranded RNAs and viral proteins (movement protein and CP) in infiltrated tissues, suggesting that the combinations of the two amino acids at positions 40 and 75 are important for effective replication in host plant cells.

scholarly journals Combination of Amino Acid Substitutions Leading to CTX-M-15-Mediated Resistance to the Ceftazidime-Avibactam Combination

2018 ◽  
Vol 62 (9) ◽  
Author(s):  
Fabrice Compain ◽  
Delphine Dorchène ◽  
Michel Arthur
Keyword(s):  
Amino Acid ◽  
Rare Variants ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Content Type ◽  
Single Amino Acid Polymorphisms ◽  
Emergence Of Resistance ◽  
Encoding Genes

ABSTRACTSingle amino acid substitutions in the Ω loop of KPC β-lactamases are known to lead to resistance to the ceftazidime-avibactam combination. Here, we investigate this mechanism of resistance in CTX-M enzymes, which are the most widely spread extended-spectrum β-lactamases worldwide. Nine single amino acid polymorphisms were identified in the Ω loop of the 172 CTX-M sequences present in the Lahey database of β-lactamases. The corresponding modifications were introduced in CTX-M-15 by site-directed mutagenesis. None of the nine substitutions was associated with ceftazidime-avibactam resistance inEscherichia coliTOP10. However, two substitutions led to 4-fold (P167S) and 16-fold (L169Q) increases in the MIC of ceftazidime. We determined whether these substitutions favor thein vitroselection of mutants resistant to ceftazidime-avibactam. The selection provided mutants for the L169Q substitution but not for the P167S substitution or for the parental enzyme CTX-M-15. Resistance to the drug combination (MIC of ceftazidime, 16 μg/ml in the presence of 4 μg/ml of avibactam) resulted from the acquisition of the S130G substitution by CTX-M-15 L169Q. Purified CTX-M-15 with the two substitutions, L169Q and S130G, was only partially inhibited by avibactam at concentrations as high as 50,000 μM but retained ceftazidime hydrolysis activity with partially compensatory decreases inkcatandKm. These results indicate that emergence of resistance to the ceftazidime-avibactam combination requires more than one mutation in most CTX-M-encoding genes. Acquisition of resistance could be restricted to rare variants harboring predisposing polymorphisms such as Q at position 169 detected in a single naturally occurring CTX-M enzyme (CTX-M-93).

Triclabendazole-resistant Fasciola hepatica: β-tubulin and response to in vitro treatment with triclabendazole

Parasitology ◽  
2002 ◽  
Vol 124 (3) ◽  
pp. 325-338 ◽  
Author(s):  
M. W. ROBINSON ◽  
A. TRUDGETT ◽  
E. M. HOEY ◽  
I. FAIRWEATHER
Keyword(s):  
Amino Acid ◽  
Fasciola Hepatica ◽  
Amino Acid Sequences ◽  
Single Amino Acid ◽  
Parasitic Nematodes ◽  
Transmission Electron ◽  
Primary Amino ◽  
Triclabendazole Resistance ◽  
Β Tubulin

Resistance in Fasciola hepatica to triclabendazole (‘Fasinex’) has emerged in several countries. Benzimidazole resistance in parasitic nematodes has been linked to a single amino acid substitution (phenylalanine to tyrosine) at position 200 on the β-tubulin molecule. Sequencing of β-tubulin cDNAs from triclabendazole-susceptible and triclabendazole-resistant flukes revealed no amino acid differences between their respective primary amino acid sequences. In order to investigate the mechanism of triclabendazole resistance, triclabendazole-susceptible and triclabendazole-resistant flukes were incubated in vitro with triclabendazole sulphoxide (50 μg/ml). Scanning and transmission electron microscopy revealed extensive damage to the tegument of triclabendazole-susceptible F. hepatica, whereas triclabendazole-resistant flukes showed only localized and relatively minor disruption of the tegument covering the spines. Immunocytochemical studies, using an anti-tubulin antibody, showed that tubulin organization was disrupted in the tegument of triclabendazole-susceptible flukes. No such disruption was evident in triclabendazole-resistant F. hepatica. The significance of these findings is discussed with regard to the mechanism of triclabendazole resistance in F. hepatica.

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