scholarly journals Functional Analysis of the Nicotinate Mononucleotide:5,6-Dimethylbenzimidazole Phosphoribosyltransferase (CobT) Enzyme, Involved in the Late Steps of Coenzyme B12 Biosynthesis in Salmonella enterica

2009 ◽  
Vol 192 (1) ◽  
pp. 145-154 ◽  
Author(s):  
Kathy R. Claas ◽  
J. R. Parrish ◽  
L. A. Maggio-Hall ◽  
J. C. Escalante-Semerena
Keyword(s):  
Salmonella Enterica ◽  
Mutational Analysis ◽  
Residual Activity ◽  
Catalytic Efficiency ◽  
Structural Data ◽  
Site Directed Mutagenesis ◽  
Negative Effects ◽  
Intragenic Complementation

ABSTRACT In Salmonella enterica, the CobT enzyme activates the lower ligand base during the assembly of the nucleotide loop of adenosylcobalamin (AdoCbl) and other cobamides. Previously, mutational analysis identified a class of alleles (class M) that failed to restore AdoCbl biosynthesis during intragenic complementation studies. To learn why class M cobT mutations were deleterious, we determined the nature of three class M cobT alleles and performed in vivo and in vitro functional analyses guided by available structural data on the wild-type CobT (CobTWT) enzyme. We analyzed the effects of the variants CobT(G257D), CobT(G171D), CobT(G320D), and CobT(C160A). The latter was not a class M variant but was of interest because of the potential role of a disulfide bond between residues C160 and C256 in CobT activity. Substitutions G171D, G257D, and G320D had profound negative effects on the catalytic efficiency of the enzyme. The C160A substitution rendered the enzyme fivefold less efficient than CobTWT. The CobT(G320D) protein was unstable, and results of structure-guided site-directed mutagenesis suggest that either variants CobT(G257D) and CobT(G171D) have less affinity for 5,6-dimethylbenzimidazole (DMB) or access of DMB to the active site is restricted in these variant proteins. The reported lack of intragenic complementation among class M cobT alleles is caused in some cases by unstable proteins, and in others it may be caused by the formation of dimers between two mutant CobT proteins with residual activity that is so low that the resulting CobT dimer cannot synthesize sufficient product to keep up with even the lowest demand for AdoCbl.

scholarly journals Mutational Analysis of Ocriplasmin to Reduce Proteolytic and Autolytic Activity in Pichia pastoris

2020 ◽  
Vol 22 (1) ◽  
Author(s):  
Roghayyeh Baghban ◽  
Safar Farajnia ◽  
Younes Ghasemi ◽  
Reyhaneh Hoseinpoor ◽  
Azam Safary ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Proteolytic Activity ◽  
Mutational Analysis ◽  
Expression System ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Vitreomacular Adhesion ◽  
Autolytic Activity

Abstract Background Ocriplasmin (Jetrea) is using for the treatment of symptomatic vitreomacular adhesion. This enzyme undergoes rapid inactivation and limited activity duration as a result of its autolytic nature after injection within the eye. Moreover, the proteolytic activity can cause photoreceptor damage, which may result in visual impairment in more serious cases. Results The present research aimed to reduce the disadvantages of ocriplasmin using site-directed mutagenesis. To reduce the autolytic activity of ocriplasmin in the first variant, lysine 156 changed to glutamic acid and, in the second variant for the proteolytic activity reduction, alanine 59 mutated to threonine. The third variant contained both mutations. Expression of wild type and three mutant variants of ocriplasmin constructs were done in the Pichia pastoris expression system. The mutant variants were analyzed in silico and in vitro and compared to the wild type. The kinetic parameters of ocriplasmin variants showed both variants with K156E substitution were more resistant to autolytic degradation than wild-type. These variants also exhibited reduced Kcat and Vmax values. An increase in their Km values, leading to a decreased catalytic efficiency (the Kcat/Km ratio) of autolytic and mixed variants. Moreover, in the variant with A59T mutation, Kcat and Vmax values have reduced compared to wild type. The mix variants showed the most increase in Km value (almost 2-fold) as well as reduced enzymatic affinity to the substrate. Thus, the results indicated that combined mutations at the ocriplasmin sequence were more effective compared with single mutations. Conclusions The results indicated such variants represent valuable tools for the investigation of therapeutic strategies aiming at the non-surgical resolution of vitreomacular adhesion.

scholarly journals Mutational Analysis of Ocriplasmin to Reduce Proteolytic and Autolytic Activity in Pichia Pastoris

2020 ◽  
Author(s):  
Roghayyeh Baghban ◽  
Safar Farajnia ◽  
Younes Ghasemi ◽  
Reyhaneh Hoseinpoor ◽  
Azam Safary ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Mutational Analysis ◽  
Expression System ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Vitreomacular Adhesion ◽  
Km Value ◽  
Autolytic Activity

Abstract Background: Ocriplasmin (Jetrea) is using for the treatment of symptomatic vitreomacular adhesion. This enzyme undergoes rapid inactivation and limited activities duration as a result of its autolytic and proteolytic nature after injection within the eye. Moreover, the proteolytic activities can cause photoreceptor damage, which may result in visual impairment in the more serious cases.Results: The present research aimed to reduce the disadvantages of ocriplasmin using site-directed mutagenesis. To reduce the autolytic activity of ocriplasmin in the first variant, lysine 156 changed to glutamic acid and in the second variant for the proteolytic activity reduction, alanine 59 mutated to threonine. The third variant contained both the mutations. Expression of wild type and three mutant variants of ocriplasmin constructs were done in Pichia pastoris expression system. The mutant variants analyzed in silico and in vitro and compared to the wild type. The kinetic parameters of ocriplasmin variants showed both variants with K156E substitution were more resistant to autolytic degradation than wild-type. These variants also exhibited reduced Kcat and Vmax values. An increase in their Km values, leading to a decreased catalytic efficiency (the Kcat/Km ratio) of autolytic and mix variants. Moreover, in variant with A59T mutation, Kcat and Vmax values have reduced compared to wild type. The mix variants showed the most increase in Km value (almost 2-fold) as well as reduced enzymatic affinity to the substrate. Thus, the results indicated combine mutations at ocriplasmin sequence were more effective compared with single mutations. Conclusions: The results indicated such variants represent valuable tools for the investigation of therapeutic strategies aiming at non-surgical resolution of vitreomacular adhesion.

scholarly journals Rapid Oligonucleotide-Based Recombineering of the Chromosome of Salmonella enterica

2009 ◽  
Vol 75 (6) ◽  
pp. 1575-1580 ◽  
Author(s):  
Roman G. Gerlach ◽  
Daniela Jäckel ◽  
Stefanie U. Hölzer ◽  
Michael Hensel
Keyword(s):  
Salmonella Enterica ◽  
Mutational Analysis ◽  
Bacterial Species ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Synthetic Dna ◽  
Chromosomal Genes ◽  
Bacterial Chromosomes ◽  
Chromosomal Loci

ABSTRACT Recombinant engineering using Red recombinase-based approaches offers efficient and rapid approaches to deletion and modification of genes. Here we describe a novel application of Red recombinant engineering that enables direct manipulation of chromosomal loci by electroporation with short synthetic DNA molecules. We demonstrate the use of this approach for the generation of scarless in-frame deletions in chromosomal genes of Salmonella enterica. Furthermore, we describe rapid site-directed mutagenesis within bacterial chromosomes without any requirement for cloning and mutating genes in vitro or for reintroducing mutant alleles into the chromosome. This approach can be expected to facilitate mutational analysis in S. enterica and in other bacterial species able to support Red-mediated recombination.

scholarly journals Identification of the In Vivo Casein Kinase II Phosphorylation Site within the Homeodomain of the Cardiac Tisue-Specifying Homeobox Gene Product Csx/Nkx2.5

1999 ◽  
Vol 19 (1) ◽  
pp. 526-536 ◽  
Author(s):  
Hideko Kasahara ◽  
Seigo Izumo
Keyword(s):  
Mutational Analysis ◽  
Intracellular Localization ◽  
Phosphorylation Site ◽  
Homeobox Gene ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Site Directed Mutagenesis ◽  
Phosphopeptide Mapping

ABSTRACT Csx/Nkx2.5, a member of the homeodomain-containing transcription factors, serves critical developmental functions in heart formation in vertebrates and nonvertebrates. In this study the putative nuclear localization signal (NLS) of Csx/Nkx2.5 was identified by site-directed mutagenesis to the amino terminus of the homeodomain, which is conserved in almost all homeodomain proteins. When the putative NLS of Csx/Nkx2.5 was mutated a significant amount of the cytoplasmically localized Csx/Nkx2.5 was unphosphorylated, in contrast to the nuclearly localized Csx/Nkx2.5, which is serine- and threonine-phosphorylated, suggesting that Csx/Nkx2.5 phosphorylation is regulated, at least in part, by intracellular localization. Tryptic phosphopeptide mapping indicated that Csx/Nkx2.5 has at least five phosphorylation sites. Using in-gel kinase assays, we detected a Csx/Nkx2.5 kinase whose molecular mass is approximately 40 kDa in both cytoplasmic and nuclear extracts. Mutational analysis and in vitro kinase assays suggested that this 40-kDa Csx/Nkx2.5 kinase is a catalytic subunit of casein kinase II (CKII) that phosphorylates the serine residue between the first and second helix of the homeodomain. This CKII site is phosphorylated in vivo. CKII-dependent phosphorylation of the homeodomain increased Csx/Nkx2.5 DNA binding. Serine-to-alanine mutation at the CKII phosphorylation site reduced transcriptional activity when the carboxyl-terminal repressor domain was deleted. Although the precise biological function of Csx/Nkx2.5 phosphorylation by CKII remains to be determined, it may play an important role, as this CKII phosphorylation site within the homeodomain is fully conserved in all known members of the NK2 family of the homeobox genes.

scholarly journals Mutational Analysis of the MutH Protein fromEscherichia coli

2000 ◽  
Vol 276 (15) ◽  
pp. 12113-12119 ◽  
Author(s):  
Tamalette Loh ◽  
Kenan C. Murphy ◽  
Martin G. Marinus
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Mutational Analysis ◽  
Site Directed Mutagenesis ◽  
Mutant Proteins ◽  
Flexible Loop ◽  
Loop Regions ◽  
Binding Residues

Site-directed mutagenesis was performed on several areas of MutH based on the similarity of MutH andPvuII structural models. The aims were to identify DNA-binding residues; to determine whether MutH has the same mechanism for DNA binding and catalysis asPvuII; and to localize the residues responsible for MutH stimulation by MutL. No DNA-binding residues were identified in the two flexible loop regions of MutH, although similar loops inPvuII are involved in DNA binding. Two histidines in MutH are in a similar position as two histidines (His-84 and His-85) inPvuII that signal for DNA binding and catalysis. These MutH histidines (His-112 and His-115) were changed to alanines, but the mutant proteins had wild-type activity bothin vivoandin vitro. The results indicate that the MutH signal for DNA binding and catalysis remains unknown. Instead, a lysine residue (Lys-48) was found in the first flexible loop that functions in catalysis together with the three presumed catalytic amino acids (Asp-70, Glu-77, and Lys-79). Two deletion mutations (MutHΔ224 and MutHΔ214) in the C-terminal end of the protein, localized the MutL stimulation region to five amino acids (Ala-220, Leu-221, Leu-222, Ala-223, and Arg-224).

scholarly journals Mutational Analysis of the Residue at Position 48 in the Salmonella enterica Serovar Typhimurium PhoQ Sensor Kinase

2003 ◽  
Vol 185 (6) ◽  
pp. 1935-1941 ◽  
Author(s):  
Sarah Sanowar ◽  
Alexandre Martel ◽  
Hervé Le Moual
Keyword(s):  
Amino Acid ◽  
Phosphatase Activity ◽  
Salmonella Enterica ◽  
Mutational Analysis ◽  
Regulatory System ◽  
Single Amino Acid ◽  
Serovar Typhimurium ◽  
Constitutively Active

ABSTRACT The PhoP/PhoQ two-component regulatory system of Salmonella enterica serovar Typhimurium plays an essential role in controlling virulence by mediating the adaptation to Mg2+ depletion. The pho-24 allele of phoQ harbors a single amino acid substitution (T48I) in the periplasmic domain of the PhoQ histidine kinase sensor. This mutation has been shown to increase net phosphorylation of the PhoP response regulator. We analyzed the effect on signaling by PhoP/PhoQ of various amino acid substitutions at this position (PhoQ-T48X [X = A, S, V, I, or L]). Mutations T48V, T48I, and T48L were found to affect signaling by PhoP/PhoQ both in vivo and in vitro. Mutations PhoQ-T48V and PhoQ-T48I increased both the expression of the mgtA::lacZ transcriptional fusion and the net phosphorylation of PhoP, conferring to cells a PhoP constitutively active phenotype. In contrast, mutation PhoQ-T48L barely responded to changes in the concentration of external Mg2+, in vivo and in vitro, conferring to cells a PhoP constitutively inactive phenotype. By analyzing in vitro the individual catalytic activities of the PhoQ-T48X sensors, we found that the PhoP constitutively active phenotype observed for the PhoQ-T48V and PhoQ-T48I proteins is solely due to decreased phosphatase activity. In contrast, the PhoP constitutively inactive phenotype observed for the PhoQ-T48L mutant resulted from both decreased autokinase activity and increased phosphatase activity. Our data are consistent with a model in which the residue at position 48 of PhoQ contributes to a conformational switch between kinase- and phosphatase-dominant states.

scholarly journals A Phosphohexomutase from the Archaeon Sulfolobus solfataricus Is Covalently Modified by Phosphorylation on Serine

2005 ◽  
Vol 187 (12) ◽  
pp. 4270-4275 ◽  
Author(s):  
W. Keith Ray ◽  
Sabrina M. Keith ◽  
Andrea M. DeSantis ◽  
Jeremy P. Hunt ◽  
Timothy J. Larson ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Sulfolobus Solfataricus ◽  
Catalytic Efficiency ◽  
Protein Product ◽  
Open Reading Frame ◽  
Site Directed Mutagenesis ◽  
Phosphoryl Group ◽  
Reading Frame

ABSTRACT A phosphoserine-containing peptide was identified from tryptic digests from Sulfolobus solfataricus P1 by liquid chromatography-tandem mass spectrometry. Its amino acid sequence closely matched that bracketing Ser-309 in the predicted protein product of open reading frame sso0207, a putative phosphohexomutase, in the genome of S. solfataricus P2. Open reading frame sso0207 was cloned, and its protein product expressed in Escherichia coli. The recombinant protein proved capable of interconverting mannose 1-phosphate and mannose 6-phosphate, as well as glucose 1-phosphate and glucose 6-phosphate, in vitro. It displayed no catalytic activity toward glucosamine 6-phosphate or N-acetylglucosamine 6-phosphate. Models constructed using the X-ray crystal structure of a homologous phosphohexomutase from Pseudomonas aeruginosa predicted that Ser-309 of the archaeal protein lies within the substrate binding site. The presence of a phosphoryl group at this location would be expected to electrostatically interfere with the binding of negatively charged phosphohexose substrates, thus attenuating the catalytic efficiency of the enzyme. Using site-directed mutagenesis, Ser-309 was substituted by aspartic acid to mimic the presence of a phosphoryl group. The V max of the mutationally altered protein was only 4% that of the unmodified form. Substitution of Ser-309 with larger, but uncharged, amino acids, including threonine, also decreased catalytic efficiency, but to a lesser extent—three- to fivefold. We therefore predict that phosphorylation of the enzyme in vivo serves to regulate its catalytic activity.

scholarly journals Mutational analysis of ocriplasmin to reduce proteolytic and autolytic activity in Pichia pastoris

2020 ◽  
Author(s):  
Roghayyeh Baghban ◽  
safar farajnia ◽  
Younes Ghasemi ◽  
Reyhaneh Hoseinpoor ◽  
Azam Safary ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Proteolytic Activity ◽  
Mutational Analysis ◽  
Expression System ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Vitreomacular Adhesion ◽  
Autolytic Activity

Abstract Background: Ocriplasmin (Jetrea) is using for the treatment of symptomatic vitreomacular adhesion. This enzyme undergoes rapid inactivation and limited activity duration as a result of its autolytic nature after injection within the eye. Moreover, the proteolytic activity can cause photoreceptor damage, which may result in visual impairment in more serious cases.Results: The present research aimed to reduce the disadvantages of ocriplasmin using site-directed mutagenesis. To reduce the autolytic activity of ocriplasmin in the first variant, lysine 156 changed to glutamic acid and, in the second variant for the proteolytic activity reduction, alanine 59 mutated to threonine. The third variant contained both mutations. Expression of wild type and three mutant variants of ocriplasmin constructs were done in the Pichia pastoris expression system. The mutant variants were analyzed in silico and in vitro and compared to the wild type. The kinetic parameters of ocriplasmin variants showed both variants with K156E substitution were more resistant to autolytic degradation than wild-type. These variants also exhibited reduced Kcat and Vmax values. An increase in their Km values, leading to a decreased catalytic efficiency (the Kcat/Km ratio) of autolytic and mixed variants. Moreover, in the variant with A59T mutation, Kcat and Vmax values have reduced compared to wild type. The mix variants showed the most increase in Km value (almost 2-fold) as well as reduced enzymatic affinity to the substrate. Thus, the results indicated that combined mutations at the ocriplasmin sequence were more effective compared with single mutations. Conclusions: The results indicated such variants represent valuable tools for the investigation of therapeutic strategies aiming at the non-surgical resolution of vitreomacular adhesion.

Endogenous hydrogen sulfide sulfhydrates IKKβ at cysteine 179 to control pulmonary artery endothelial cell inflammation

2019 ◽  
Vol 133 (20) ◽  
pp. 2045-2059 ◽  
Author(s):  
Da Zhang ◽  
Xiuli Wang ◽  
Siyao Chen ◽  
Selena Chen ◽  
Wen Yu ◽  
...  
Keyword(s):  
Hydrogen Sulfide ◽  
Endothelial Cell ◽  
Pulmonary Artery ◽  
Cell Model ◽  
Site Directed Mutagenesis ◽  
Critical Event ◽  
Hypertensive Rats ◽  
Pulmonary Artery Endothelial Cell

Abstract Background: Pulmonary artery endothelial cell (PAEC) inflammation is a critical event in the development of pulmonary arterial hypertension (PAH). However, the pathogenesis of PAEC inflammation remains unclear. Methods: Purified recombinant human inhibitor of κB kinase subunit β (IKKβ) protein, human PAECs and monocrotaline-induced pulmonary hypertensive rats were employed in the study. Site-directed mutagenesis, gene knockdown or overexpression were conducted to manipulate the expression or activity of a target protein. Results: We showed that hydrogen sulfide (H2S) inhibited IKKβ activation in the cell model of human PAEC inflammation induced by monocrotaline pyrrole-stimulation or knockdown of cystathionine γ-lyase (CSE), an H2S generating enzyme. Mechanistically, H2S was proved to inhibit IKKβ activity directly via sulfhydrating IKKβ at cysteinyl residue 179 (C179) in purified recombinant IKKβ protein in vitro, whereas thiol reductant dithiothreitol (DTT) reversed H2S-induced IKKβ inactivation. Furthermore, to demonstrate the significance of IKKβ sulfhydration by H2S in the development of PAEC inflammation, we mutated C179 to serine (C179S) in IKKβ. In purified IKKβ protein, C179S mutation of IKKβ abolished H2S-induced IKKβ sulfhydration and the subsequent IKKβ inactivation. In human PAECs, C179S mutation of IKKβ blocked H2S-inhibited IKKβ activation and PAEC inflammatory response. In pulmonary hypertensive rats, C179S mutation of IKKβ abolished the inhibitory effect of H2S on IKKβ activation and pulmonary vascular inflammation and remodeling. Conclusion: Collectively, our in vivo and in vitro findings demonstrated, for the first time, that endogenous H2S directly inactivated IKKβ via sulfhydrating IKKβ at Cys179 to inhibit nuclear factor-κB (NF-κB) pathway activation and thereby control PAEC inflammation in PAH.

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