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  • 1
    Language: English
    In: PLoS ONE, 01 January 2011, Vol.6(11), p.e28170
    Description: The pathway of molybdenum cofactor biosynthesis has been studied in detail by using proteins from Mycobacterium species, which contain several homologs associated with the first steps of Moco biosynthesis. While all Mycobacteria species contain a MoeZR, only some strains have acquired an additional homolog, MoeBR, by horizontal gene transfer. The role of MoeBR and MoeZR was studied in detail for the interaction with the two MoaD-homologs involved in Moco biosynthesis, MoaD1 and MoaD2, in addition to the CysO protein involved in cysteine biosynthesis. We show that both proteins have a role in Moco biosynthesis, while only MoeZR, but not MoeBR, has an additional role in cysteine biosynthesis. MoeZR and MoeBR were able to complement an E. coli moeB mutant strain, but only in conjunction with the Mycobacterial MoaD1 or MoaD2 proteins. Both proteins were able to sulfurate MoaD1 and MoaD2 in vivo, while only MoeZR additionally transferred the sulfur to CysO. Our in vivo studies show that Mycobacteria have acquired several homologs to maintain Moco biosynthesis. MoeZR has a dual role in Moco- and cysteine biosynthesis and is involved in the sulfuration of MoaD and CysO, whereas MoeBR only has a role in Moco biosynthesis, which is not an essential function for Mycobacteria.
    Keywords: Sciences (General)
    E-ISSN: 1932-6203
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  • 2
    Language: English
    In: PLoS ONE, 2011, Vol.6(11), p.e28170
    Description: The pathway of molybdenum cofactor biosynthesis has been studied in detail by using proteins from Mycobacterium species, which contain several homologs associated with the first steps of Moco biosynthesis. While all Mycobacteria species contain a MoeZR, only some strains have acquired an additional homolog, MoeBR, by horizontal gene transfer. The role of MoeBR and MoeZR was studied in detail for the interaction with the two MoaD-homologs involved in Moco biosynthesis, MoaD1 and MoaD2, in addition to the CysO protein involved in cysteine biosynthesis. We show that both proteins have a role in Moco biosynthesis, while only MoeZR, but not MoeBR, has an additional role in cysteine biosynthesis. MoeZR and MoeBR were able to complement an E. coli moeB mutant strain, but only in conjunction with the Mycobacterial MoaD1 or MoaD2 proteins. Both proteins were able to sulfurate MoaD1 and MoaD2 in vivo , while only MoeZR additionally transferred the sulfur to CysO. Our in vivo studies show that Mycobacteria have acquired several homologs to maintain Moco biosynthesis. MoeZR has a dual role in Moco- and cysteine biosynthesis and is involved in the sulfuration of MoaD and CysO, whereas MoeBR only has a role in Moco biosynthesis, which is not an essential function for Mycobacteria .
    Keywords: Research Article ; Biology ; Medicine ; Infectious Diseases ; Biochemistry
    E-ISSN: 1932-6203
    Source: PLoS
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  • 3
    Language: English
    In: Applied Microbiology and Biotechnology, 2011, Vol.92(4), pp.823-833
    Description: Generating sufficient quantities of labeled proteins represents a bottleneck in protein structure determination. A simple protocol for producing heavy isotope as well as selenomethionine (Se-Met)-labeled proteins was developed using T7-based Escherichia coli expression systems. The protocol is applicable for generation of single-, double-, and triple-labeled proteins ( 15 N, 13 C, and 2 H) in shaker flask cultures. Label incorporation into the target protein reached 99% and 97% for 15 N and 13 C, respectively, and 75% of (non-exchangeable) hydrogen for 2 H labeling. The expression yields and final cell densities (OD600 ∼16) were the same as for the production of non-labeled protein. This protocol is also applicable for Se-Met labeling, leading to Se-Met incorporation into the target protein of 70% or 90% using prototrophic or methionine auxotrophic E. coli strains, respectively.
    Keywords: Escherichia coli ; Recombinant protein production ; Stable heavy isotope labeling ; Selenomethionine labeling ; Autoinduction ; Defined medium
    ISSN: 0175-7598
    E-ISSN: 1432-0614
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  • 4
    Language: English
    In: Bioresource Technology, February 2011, Vol.102(3), pp.3316-3321
    Description: enjoys high popularity as an edible mushroom in Asian cuisines. Investigating the secretion of peptidases in nutrient media enriched with gluten, an enzyme was noticed that catalyzed the deamidation of -asparagine and -glutamine. The enzyme was purified to electrophoretic homogeneity by foaming and SEC. PAGE analysis revealed a protein of about 85 kDa with 13 kDa subunits indicating a hexameric protein. Degenerated primers were deduced from peptide fragments and the complete coding sequence of 372 bp was determined. The gene of asparaginase (FvNase) over expressed in achieved an -asparagine-hydrolyzing activity of 16 U/mL in crude extract, which was five times higher than its -glutamine-hydrolyzing ability. The enzyme showed a pH-optimum at pH 7, remarkable tolerance towards elevated temperature and sodium chloride concentration in both the native and recombinant form, and no significant homology to any conserved domains of published asparaginases or glutaminases.
    Keywords: Asparaginase ; Basidiomycete ; Flammulina Velutipes ; Heterologous Expression ; Escherichia Coli ; Agriculture ; Engineering ; Chemistry
    ISSN: 0960-8524
    E-ISSN: 1873-2976
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  • 5
    Language: English
    In: Journal of Bacteriology, March, 2012, Vol.194(5-6), p.1401(16)
    Description: The specific aminoacylation of the phospholipid phosphatidylglycerol (PG) with alanine or with lysine catalyzed by aminoacyl-phosphatidylglycerol synthases (aaPGS) was shown to render various organisms less susceptible to antibacterial agents. This study makes use of Pseudomonas aeruginosa chimeric mutant strains producing lysyl-phosphatidylglycerol (L-PG) instead of the naturally occurring alanyl-phosphatidylglycerol (A-PG) to study the resulting impact on bacterial resistance. Consequences of such artificial phospholipid composition were studied in the presence of an overall of seven antimicrobials ([beta]-1actams, a lipopeptide antibiotic, cationic antimicrobial peptides [CAMPs]) to quantitatively assess the effect of A-PG substitution (with L-PG, L-PG and A-PG, increased A-PG levels). For the employed Gram-negative P. aeruginosa model system, an exclusive charge repulsion mechanism does not explain the attenuated antimicrobial susceptibility due to PG modification. Additionally, the specificity of nine orthologous aaPGS enzymes was experimentally determined. The newly characterized protein sequences allowed for the establishment of a significant group of A-PG synthase sequences which were bioinformatically compared to the related group of L-PG synthesizing enzymes. The analysis revealed a diverse origin for the evolution of A-PG and L-PG synthases, as the specificity of an individual enzyme is not reflected in terms of a characteristic sequence motif. This finding is relevant for future development of potential aaPGS inhibitors. doi: 10.1128/JB.06576-11
    ISSN: 0021-9193
    Source: Cengage Learning, Inc.
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  • 6
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 09 January 2018, Vol.115(2), pp.E273-E282
    Description: AAA+ disaggregases solubilize aggregated proteins and confer heat tolerance to cells. Their disaggregation activities crucially depend on partner proteins, which target the AAA+ disaggregases to protein aggregates while concurrently stimulating their ATPase activities. Here, we report on two potent ClpG disaggregase homologs acquired through horizontal gene transfer by the species and subsequently abundant clone C. ClpG exhibits high, stand-alone disaggregation potential without involving any partner cooperation. Specific molecular features, including high basal ATPase activity, a unique aggregate binding domain, and almost exclusive expression in stationary phase distinguish ClpG from other AAA+ disaggregases. Consequently, ClpG largely contributes to heat tolerance of primarily in stationary phase and boosts heat resistance 100-fold when expressed in This qualifies ClpG as a potential persistence and virulence factor in .
    Keywords: AAA+ Protein ; Hsp100 ; Heat Tolerance ; Mobile Genetic Element ; Protein Disaggregation ; Adaptation, Physiological ; Hot Temperature ; Bacterial Proteins -- Metabolism ; Pseudomonas Aeruginosa -- Enzymology
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 7
    Language: English
    In: Journal of agricultural and food chemistry, 28 September 2011, Vol.59(18), pp.10311-6
    Description: The first enzyme of the basidiomycete Piptoporus soloniensis, a peptidase (PsoP1), was characterized after isolation from submerged cultures, purification by fractional precipitation, and preparative native-polyarylamide gel electrophoresis (PAGE). The native molecular mass of PsoP1 was 38 kDa with an isoelectric point of 3.9. Similar to chymosin from milk calves, PsoP1 showed a maximum milk-clotting activity (MCA) at 35-40 °C and was most stable at pH 6 and below 40 °C. The complete inhibition by pepstatin A identified this enzyme as an aspartic peptidase. Electrospray ionization-tandem MS showed an amino acid partial sequence that was more homologous to mammalian milk clotting peptidases than to the chymosin substitute from a fungal species, such as the Zygomycete Mucor miehei. According to sodium dodecyl sulfate-PAGE patterns, the peptidase cleaved κ-casein in a way similar to chymosin and hydrolyzed β-casein slowly, as it would be expected from an efficient chymosin substitute.
    Keywords: Aspartic Acid Proteases -- Isolation & Purification ; Basidiomycota -- Enzymology ; Milk -- Metabolism
    ISSN: 00218561
    E-ISSN: 1520-5118
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  • 8
    Language: English
    In: Molecular microbiology, 2010, Vol.77(3), pp.771-786
    Description: The transcriptional regulator CsgD of Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major regulator of biofilm formation required for the expression of csgBA, which encodes curli fimbriae, and adrA, coding for a diguanylate cyclase. CsgD is a response regulator with an N-terminal receiver domain with a conserved aspartate (D59) as a putative target site for phosphorylation and a C-terminal LuxR-like helix-turn-helix DNA binding motif, but the mechanisms of target gene activation remained unclear. To study the DNA-binding properties of CsgD we used electrophoretic mobility shift assays and DNase I footprint analysis to show that unphosphorylated CsgD-His₆ binds specifically to the csgBA and adrA promoter regions. In vitro transcription analysis revealed that CsgD-His₆ is crucial for the expression of csgBA and adrA. CsgD-His₆ is phosphorylated by acetyl phosphate in vitro, which decreases its DNA-binding properties. The functional impact of D59 in vivo was demonstrated as S. Typhimurium strains expressing modified CsgD protein (D59E and D59N) were dramatically reduced in biofilm formation due to decreased protein stability and DNA-binding properties in the case of D59E. In summary, our findings suggest that the response regulator CsgD functions in its unphosphorylated form under the conditions of biofilm formation investigated in this study. ; Includes references ; p. 771-786.
    Keywords: Salmonella -- Analysis ; Phosphates -- Analysis ; Aspartate -- Analysis ; Nucleases -- Analysis;
    ISSN: 0950-382X
    E-ISSN: 13652958
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  • 9
    Language: English
    In: PLoS ONE, 2012, Vol.7(1), p.e29672
    Description: Natural killer (NK) cells contribute to the defense against infected and transformed cells through the engagement of multiple germline-encoded activation receptors. Stimulation of the Fc receptor CD16 alone is sufficient for NK cell activation, whereas other receptors, such as 2B4 (CD244) and DNAM-1 (CD226), act synergistically. After receptor engagement, protein kinases play a major role in signaling networks controlling NK cell effector functions. However, it has not been characterized systematically which of all kinases encoded by the human genome (kinome) are involved in NK cell activation. ; A kinase-selective phosphoproteome approach enabled the determination of 188 kinases expressed in human NK cells. Crosslinking of CD16 as well as 2B4 and DNAM-1 revealed a total of 313 distinct kinase phosphorylation sites on 109 different kinases. Phosphorylation sites on 21 kinases were similarly regulated after engagement of either CD16 or co-engagement of 2B4 and DNAM-1. Among those, increased phosphorylation of FYN, KCC2G (CAMK2), FES, and AAK1, as well as the reduced phosphorylation of MARK2, were reproducibly observed both after engagement of CD16 and co-engagement of 2B4 and DNAM-1. Notably, only one phosphorylation on PAK4 was differentally regulated. ; The present study has identified a significant portion of the NK cell kinome and defined novel phosphorylation sites in primary lymphocytes. Regulated phosphorylations observed in the early phase of NK cell activation imply these kinases are involved in NK cell signaling. Taken together, this study suggests a largely shared signaling pathway downstream of distinct activation receptors and constitutes a valuable resource for further elucidating the regulation of NK cell effector responses.
    Keywords: Research Article ; Biology ; Immunology ; Biochemistry
    E-ISSN: 1932-6203
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  • 10
    Language: English
    In: Journal of Bacteriology, 2012, Vol. 194(6), p.1401
    Description: The specific aminoacylation of the phospholipid phosphatidylglycerol (PG) with alanine or with lysine catalyzed by aminoacyl-phosphatidylglycerol synthases (aaPGS) was shown to render various organisms less susceptible to antibacterial agents. This study makes use of Pseudomonas aeruginosa chimeric mutant strains producing lysyl-phosphatidylglycerol (L-PG) instead of the naturally occurring alanyl-phosphatidylglycerol (A-PG) to study the resulting impact on bacterial resistance. Consequences of such artificial phospholipid composition were studied in the presence of an overall of seven antimicrobials (β-lactams, a lipopeptide antibiotic, cationic antimicrobial peptides [CAMPs]) to quantitatively assess the effect of A-PG substitution (with L-PG, L-PG and A-PG, increased A-PG levels). For the employed Gram-negative P. aeruginosa model system, an exclusive charge repulsion mechanism does not explain the attenuated antimicrobial susceptibility due to PG modification. Additionally, the specificity of nine orthologous aaPGS enzymes was experimentally determined. The newly characterized protein sequences allowed for the establishment of a significant group of A-PG synthase sequences which were bioinformatically compared to the related group of L-PG synthesizing enzymes. The analysis revealed a diverse origin for the evolution of A-PG and L-PG synthases, as the specificity of an individual enzyme is not reflected in terms of a characteristic sequence motif. This finding is relevant for future development of potential aaPGS inhibitors. ; p. 1401-1416.
    Keywords: Antibiotics ; Alanine ; Amino Acid Sequences ; Lysine ; Antimicrobial Cationic Peptides ; Phenotype ; Enzymes ; Chimerism ; Evolution ; Mutants ; Phospholipids ; Beta-Lactams ; Aminoacylation ; Pseudomonas Aeruginosa ; Lipopeptides ; Bacteriology;
    ISSN: 1098-5530
    ISSN: 10985530
    ISSN: 00219193
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