scholarly journals Identification of Amino Acid Residues Involved in Heme Binding and Hemoprotein Utilization in the Porphyromonas gingivalis Heme Receptor HmuR

2006 ◽  
Vol 74 (2) ◽  
pp. 1222-1232 ◽  
Author(s):  
Xinyan Liu ◽  
Teresa Olczak ◽  
Hwai-Chen Guo ◽  
Dabney W. Dixon ◽  
Caroline Attardo Genco
Keyword(s):  
Amino Acid ◽  
Human Serum ◽  
Porphyromonas Gingivalis ◽  
Three Dimensional ◽  
Point Mutations ◽  
Homology Model ◽  
Site Directed Mutagenesis ◽  
Source Analysis ◽  
Heme Binding ◽  
Amino Acid Residues

ABSTRACT We have previously identified and characterized a heme/hemoglobin receptor, HmuR, in Porphyromonas gingivalis. To analyze the conserved amino acid residues of HmuR that may be involved in hemin/hemoprotein binding and utilization, we constructed a series of P. gingivalis A7436 hmuR mutants with amino acid replacements and characterized the ability of these mutants to utilize hemin and hemoproteins. Site-directed mutagenesis was employed to introduce mutations H95A, H434A, H95A-H434A, YRAP420-423YAAA, and NPDL442-445NAAA into HmuR in both P. gingivalis and Escherichia coli. Point mutations at H95 and H434 and in the NPDL motif of HmuR resulted in decreased binding to hemin, hemoglobin, and human serum albumin-hemin complex. Notably, mutations of these conserved sites and motifs led to reduced growth of P. gingivalis when human serum was used as the heme source. Analysis using a three-dimensional homology model of HmuR indicated that H95, H434, and the NPDL motif are present on apical or extracellular loops of HmuR, while the YRAP motif is present on the barrel wall. Taken together, these results support a role for H95, H434, and the NPDL motif of the P. gingivalis HmuR protein in heme binding and utilization of serum hemoproteins and the HmuR YRAP motif in serum hemoprotein utilization.

scholarly journals Activation of Ha-ras p21 by substitution, deletion, and insertion mutations.

1985 ◽  
Vol 5 (8) ◽  
pp. 1809-1813 ◽  
Author(s):  
R G Chipperfield ◽  
S S Jones ◽  
K M Lo ◽  
R A Weinberg
Keyword(s):  
Amino Acid ◽  
Point Mutations ◽  
Normal Function ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Transforming Activity ◽  
Ras P21 ◽  
Ras Oncogenes ◽  
Insertion Mutations

The transforming activity of naturally arising ras oncogenes results from point mutations that affect residue 12 or 61 of the encoded 21-kilodalton protein (p21). By use of site-directed mutagenesis, we showed that deletions and insertions of amino acid residues in the region of residue 12 are also effective in conferring oncogenic activity on p21. Common to these various alterations is the disruption that they create in this domain of the protein, which we propose results in the inactivation of a normal function of the protein.

scholarly journals Identification and Characterization of Porphyromonas gingivalis Client Proteins That Bind to Streptococcus oralis Glyceraldehyde-3-Phosphate Dehydrogenase

2012 ◽  
Vol 81 (3) ◽  
pp. 753-763 ◽  
Author(s):  
Kazuhiko Maeda ◽  
Hideki Nagata ◽  
Masae Kuboniwa ◽  
Miki Ojima ◽  
Tsukasa Osaki ◽  
...  
Keyword(s):  
Amino Acid ◽  
Porphyromonas Gingivalis ◽  
Interaction Analysis ◽  
Site Directed Mutagenesis ◽  
Receptor Protein ◽  
Oral Streptococci ◽  
Amino Acid Residues ◽  
Content Type ◽  
Streptococcus Oralis ◽  
Client Proteins

ABSTRACTCoaggregation ofPorphyromonas gingivalisand oral streptococci is thought to play an important role inP. gingivaliscolonization. Previously, we reported thatP. gingivalismajor fimbriae interacted withStreptococcus oralisglyceraldehyde-3-phosphate dehydrogenase (GAPDH), and that amino acid residues 166 to 183 of GAPDH exhibited strong binding activity towardP. gingivalisfimbriae (H. Nagata, M. Iwasaki, K. Maeda, M. Kuboniwa, E. Hashino, M. Toe, N. Minamino, H. Kuwahara, and S. Shizukuishi, Infect. Immun.77:5130–5138, 2009). The present study aimed to identify and characterizeP. gingivaliscomponents other than fimbriae that interact withS. oralisGAPDH. A pulldown assay was performed to detect potential interactions betweenP. gingivalisclient proteins andS. oralisrecombinant GAPDH with amino acid residues 166 to 183 deleted by site-directed mutagenesis. Seven proteins, namely,tonB-dependent receptor protein (RagA4), arginine-specific proteinase B, 4-hydroxybutyryl-coenzyme A dehydratase (AbfD), lysine-specific proteinase, GAPDH, NAD-dependent glutamate dehydrogenase (GDH), and malate dehydrogenase (MDH), were identified by two-dimensional gel electrophoresis followed by proteomic analysis using tandem mass spectrometry. Interactions between these client proteins andS. oralisGAPDH were analyzed with a biomolecular interaction analysis system.S. oralisGAPDH showed high affinity for five of the seven client proteins (RagA4, AbfD, GAPDH, GDH, and MDH). Interactions betweenP. gingivalisandS. oraliswere measured by a turbidimetric method and fluorescence microscopy. RagA4, AbfD, and GDH enhanced coaggregation, whereas GAPDH and MDH inhibited coaggregation. Furthermore, the expression ofluxSinP. gingivaliswas upregulated by RagA4, AbfD, and GDH but was downregulated by MDH. These results indicate that the fiveP. gingivalisclient proteins function as regulators inP. gingivalisbiofilm formation with oral streptococci.

scholarly journals Effect of Point Mutations on Structural and Allergenic Properties of the Lentil Allergen Len c 3

Membranes ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 939
Author(s):  
Daria N. Melnikova ◽  
Ekaterina I. Finkina ◽  
Ivan V. Bogdanov ◽  
Anastasia A. Ignatova ◽  
Natalia S. Matveevskaya ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Capacity ◽  
Point Mutations ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Lipid Transfer ◽  
Ige Binding ◽  
Allergenic Potential ◽  
Binding Epitope ◽  
Clinically Significant

Plant lipid transfer proteins (LTPs) are known to be clinically significant allergens capable of binding various lipid ligands. Recent data showed that lipid ligands affected the allergenic properties of plant LTPs. In this work, we checked the assumption that specific amino acid residues in the Len c 3 structure can play a key role both in the interaction with lipid ligands and IgE-binding capacity of the allergen. The recombinant analogues of Len c 3 with the single or double substitutions of Thr41, Arg45 and/or Tyr80 were obtained by site-directed mutagenesis. All these amino acid residues are located near the “bottom” entrance to the hydrophobic cavity of Len c 3 and are likely included in the IgE-binding epitope of the allergen. Using a bioinformatic approach, circular dichroism and fluorescence spectroscopies, ELISA, and experiments mimicking the allergen Len c 3 gastroduodenal digestion we showed that the substitution of all the three amino acid residues significantly affected structural organization of this region and led both to a change of the ligand-binding capacity and the allergenic potential of Len c 3.

scholarly journals A l-Lysine Transporter of High Stereoselectivity of the Amino Acid-Polyamine-Organocation (APC) Superfamily

2013 ◽  
Vol 289 (3) ◽  
pp. 1377-1387 ◽  
Author(s):  
Jagdeep Kaur ◽  
Elena Olkhova ◽  
Viveka Nand Malviya ◽  
Ernst Grell ◽  
Hartmut Michel
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ligand Binding ◽  
Isothermal Titration Calorimetry ◽  
Functional Characterization ◽  
Homology Model ◽  
Docking Studies ◽  
Site Directed Mutagenesis ◽  
Titration Calorimetry ◽  
Amino Acid Residues

Membrane proteins of the amino acid-polyamine-organocation (APC) superfamily transport amino acids and amines across membranes and play an important role in the regulation of cellular processes. We report the heterologous production of the LysP-related transporter STM2200 from Salmonella typhimurium in Escherichia coli, its purification, and functional characterization. STM2200 is assumed to be a proton-dependent APC transporter of l-lysine. The functional interaction between basic amino acids and STM2200 was investigated by thermoanalytical methods, i.e. differential scanning and isothermal titration calorimetry. Binding of l-lysine to STM2200 in its solubilized monomer form is entropy-driven. It is characterized by a dissociation constant of 40 μm at pH 5.9 and is highly selective; no evidence was found for the binding of l-arginine, l-ornithine, l-2,4-diaminobutyric acid, and l-alanine. d-Lysine is bound 45 times more weakly than its l-chiral form. We thus postulate that STM2200 functions as a specific transport protein. Based on the crystal structure of ApcT (Shaffer, P. L., Goehring, A., Shankaranarayanan, A., and Gouaux, E. (2009) Science 325, 1010–1014), a proton-dependent amino acid transporter of the APC superfamily, a homology model of STM2200 was created. Docking studies allowed identification of possible ligand binding sites. The resulting predictions indicated that Glu-222 and Arg-395 of STM2200 are markedly involved in ligand binding, whereas Lys-163 is suggested to be of structural and functional relevance. Selected variants of STM2200 where these three amino acid residues were substituted using single site-directed mutagenesis showed no evidence for l-lysine binding by isothermal titration calorimetry, which confirmed the predictions. Molecular aspects of the observed ligand specificity are discussed.

scholarly journals Characterization of the Critical Amino Acids of anAspergillus parasiticus Cytochrome P-450 Monooxygenase Encoded by ordA That Is Involved in the Biosynthesis of Aflatoxins B1, G1, B2, and G2

1998 ◽  
Vol 64 (12) ◽  
pp. 4834-4841 ◽  
Author(s):  
Jiujiang Yu ◽  
Perng-Kuang Chang ◽  
Kenneth C. Ehrlich ◽  
Jeffrey W. Cary ◽  
Beverly Montalbano ◽  
...  
Keyword(s):  
Amino Acid ◽  
Point Mutations ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Oxidoreductase Activity ◽  
Monooxygenase Activity ◽  
Pathway Gene ◽  
Chromosomal Walking ◽  
Aflatoxin B ◽  
Cytochrome P 450

The conversion of O-methylsterigmatocystin (OMST) and dihydro-O-methylsterigmatocystin to aflatoxins B1, G1, B2, and G2requires a cytochrome P-450 type of oxidoreductase activity.ordA, a gene adjacent to the omtA gene, was identified in the aflatoxin-biosynthetic pathway gene cluster by chromosomal walking in Aspergillus parasiticus. The ordA gene was a homolog of the Aspergillus flavus ord1 gene, which is involved in the conversion of OMST to aflatoxin B1. Complementation of A. parasiticus SRRC 2043, an OMST-accumulating strain, with theordA gene restored the ability to produce aflatoxins B1, G1, B2, and G2. TheordA gene placed under the control of the GAL1promoter converted exogenously supplied OMST to aflatoxin B1 in Saccharomyces cerevisiae. In contrast, the ordA gene homolog in A. parasiticus SRRC 2043, ordA1, was not able to carry out the same conversion in the yeast system. Sequence analysis revealed that theordA1 gene had three point mutations which resulted in three amino acid changes (His-400→Leu-400, Ala-143→Ser-143, and Ile-528→Tyr-528). Site-directed mutagenesis studies showed that the change of His-400 to Leu-400 resulted in a loss of the monooxygenase activity and that Ala-143 played a significant role in the catalytic conversion. In contrast, Ile-528 was not associated with the enzymatic activity. The involvement of the ordA gene in the synthesis of aflatoxins G1, and G2 inA. parasiticus suggests that enzymes required for the formation of aflatoxins G1 and G2 are not present in A. flavus. The results showed that in addition to the conserved heme-binding and redox reaction domains encoded by ordA, other seemingly domain-unrelated amino acid residues are critical for cytochrome P-450 catalytic activity. TheordA gene has been assigned to a new cytochrome P-450 gene family named CYP64 by The Cytochrome P450 Nomenclature Committee.

scholarly journals The C-Terminal Region of Bacteroides fragilis Toxin Is Essential to Its Biological Activity

2006 ◽  
Vol 74 (10) ◽  
pp. 5595-5601 ◽  
Author(s):  
Cynthia L. Sears ◽  
Simy L. Buckwold ◽  
Jai W. Shin ◽  
Augusto A. Franco
Keyword(s):  
Biological Activity ◽  
Amino Acid ◽  
Point Mutations ◽  
Bacteroides Fragilis ◽  
Terminal Region ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
C Terminus

ABSTRACT To evaluate the role of the C-terminal region in Bacteroides fragilis toxin (BFT) activity, processing, and secretion, sequential C-terminal truncation and point mutations were created by site-directed mutagenesis. Determination of BFT activity on HT29/C1 cells, cleavage of E-cadherin, and the capacity to induce interleukin-8 secretion by wild-type BFT and C-terminal deletion mutants showed that deletion of only 2 amino acid residues at the C terminus significantly reduced BFT biological activity and deletion of eight or more amino acid residues obliterated BFT biologic activity. Western blot and reverse transcription-PCR analyses indicated that BFT mutants lacking seven or fewer amino acid residues in the C-terminal region are processed and expressed similar to wild-type BFT. However, BFT mutants lacking eight or more amino acids at the C terminus are expressed similar to wild-type BFT but are unstable. We concluded that the C terminus of BFT is not tolerant of modest amino acid deletions, suggesting that it is biologically important for BFT activity.

Activation of Ha-ras p21 by substitution, deletion, and insertion mutations

1985 ◽  
Vol 5 (8) ◽  
pp. 1809-1813
Author(s):  
R G Chipperfield ◽  
S S Jones ◽  
K M Lo ◽  
R A Weinberg
Keyword(s):  
Amino Acid ◽  
Point Mutations ◽  
Normal Function ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Transforming Activity ◽  
Ras P21 ◽  
Ras Oncogenes ◽  
Insertion Mutations

The transforming activity of naturally arising ras oncogenes results from point mutations that affect residue 12 or 61 of the encoded 21-kilodalton protein (p21). By use of site-directed mutagenesis, we showed that deletions and insertions of amino acid residues in the region of residue 12 are also effective in conferring oncogenic activity on p21. Common to these various alterations is the disruption that they create in this domain of the protein, which we propose results in the inactivation of a normal function of the protein.

scholarly journals In Vivo and In Vitro Studies of Bacillus subtilis Ferrochelatase Mutants Suggest Substrate Channeling in the Heme Biosynthesis Pathway

2002 ◽  
Vol 184 (14) ◽  
pp. 4018-4024 ◽  
Author(s):  
Ulf Olsson ◽  
Annika Billberg ◽  
Sara Sjövall ◽  
Salam Al-Karadaghi ◽  
Mats Hansson
Keyword(s):  
Bacillus Subtilis ◽  
Amino Acid ◽  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Three Dimensional Structure ◽  
Activity Measurements

ABSTRACT Ferrochelatase (EC 4.99.1.1) catalyzes the last reaction in the heme biosynthetic pathway. The enzyme was studied in the bacterium Bacillus subtilis, for which the ferrochelatase three-dimensional structure is known. Two conserved amino acid residues, S54 and Q63, were changed to alanine by site-directed mutagenesis in order to detect any function they might have. The effects of these changes were studied in vivo and in vitro. S54 and Q63 are both located at helix α3. The functional group of S54 points out from the enzyme, while Q63 is located in the interior of the structure. None of these residues interact with any other amino acid residues in the ferrochelatase and their function is not understood from the three-dimensional structure. The exchange S54A, but not Q63A, reduced the growth rate of B. subtilis and resulted in the accumulation of coproporphyrin III in the growth medium. This was in contrast to the in vitro activity measurements with the purified enzymes. The ferrochelatase with the exchange S54A was as active as wild-type ferrochelatase, whereas the exchange Q63A caused a 16-fold reduction in V max. The function of Q63 remains unclear, but it is suggested that S54 is involved in substrate reception or delivery of the enzymatic product.

scholarly journals Mutation of Key Residues in β-Glycosidase LXYL-P1-2 for Improved Activity

Catalysts ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 1042
Author(s):  
Jing-Jing Chen ◽  
Xiao Liang ◽  
Tian-Jiao Chen ◽  
Jin-Ling Yang ◽  
Ping Zhu
Keyword(s):  
Amino Acid ◽  
Lentinula Edodes ◽  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Recent Success ◽  
Key Residues ◽  
Positive Effect ◽  
Candidate Strain

The β-glycosidase LXYL-P1-2 identified from Lentinula edodes can be used to hydrolyze 7-β-xylosyl-10-deacetyltaxol (XDT) into 10-deacetyltaxol (DT) for the semi-synthesis of Taxol. Recent success in obtaining the high-resolution X-ray crystal of LXYL-P1-2 and resolving its three-dimensional structure has enabled us to perform molecular docking of LXYL-P1-2 with substrate XDT and investigate the roles of the three noncatalytic amino acid residues located around the active cavity in LXYL-P1-2. Site-directed mutagenesis results demonstrated that Tyr268 and Ser466 were essential for maintaining the β-glycosidase activity, and the L220G mutation exhibited a positive effect on increasing activity by enlarging the channel that facilitates the entrance of the substrate XDT into the active cavity. Moreover, introducing L220G mutation into the other LXYL-P1-2 mutant further increased the enzyme activity, and the β-d-xylosidase activity of the mutant EP2-L220G was nearly two times higher than that of LXYL-P1-2. Thus, the recombinant yeast GS115-EP2-L220G can be used for efficiently biocatalyzing XDT to DT for the semi-synthesis of Taxol. Our study provides not only the prospective candidate strain for industrial production, but also a theoretical basis for exploring the key amino acid residues in LXYL-P1-2.

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