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 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 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 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 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.

Chitosanase from Streptomyces sp. strain N174: a comparative review of its structure and function

1997 ◽  
Vol 75 (6) ◽  
pp. 687-696 ◽  
Author(s):  
Tamo Fukamizo ◽  
Ryszard Brzezinski
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Substrate Binding ◽  
Structure And Function ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Streptomyces Sp ◽  
Binding Cleft ◽  
And Function

Novel information on the structure and function of chitosanase, which hydrolyzes the beta -1,4-glycosidic linkage of chitosan, has accumulated in recent years. The cloning of the chitosanase gene from Streptomyces sp. strain N174 and the establishment of an efficient expression system using Streptomyces lividans TK24 have contributed to these advances. Amino acid sequence comparisons of the chitosanases that have been sequenced to date revealed a significant homology in the N-terminal module. From energy minimization based on the X-ray crystal structure of Streptomyces sp. strain N174 chitosanase, the substrate binding cleft of this enzyme was estimated to be composed of six monosaccharide binding subsites. The hydrolytic reaction takes place at the center of the binding cleft with an inverting mechanism. Site-directed mutagenesis of the carboxylic amino acid residues that are conserved revealed that Glu-22 and Asp-40 are the catalytic residues. The tryptophan residues in the chitosanase do not participate directly in the substrate binding but stabilize the protein structure by interacting with hydrophobic and carboxylic side chains of the other amino acid residues. Structural and functional similarities were found between chitosanase, barley chitinase, bacteriophage T4 lysozyme, and goose egg white lysozyme, even though these proteins share no sequence similarities. This information can be helpful for the design of new chitinolytic enzymes that can be applied to carbohydrate engineering, biological control of phytopathogens, and other fields including chitinous polysaccharide degradation. Key words: chitosanase, amino acid sequence, overexpression system, reaction mechanism, site-directed mutagenesis.

scholarly journals Identification and characterization of amphibian SLC26A5 using RNA-Seq

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Zhongying Wang ◽  
Qixuan Wang ◽  
Hao Wu ◽  
Zhiwu Huang
Keyword(s):  
Amino Acid ◽  
Inner Ear ◽  
Hair Cells ◽  
Rana Catesbeiana ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Rna Seq ◽  
American Bullfrog ◽  
Frog Species

Abstract Background Prestin (SLC26A5) is responsible for acute sensitivity and frequency selectivity in the vertebrate auditory system. Limited knowledge of prestin is from experiments using site-directed mutagenesis or domain-swapping techniques after the amino acid residues were identified by comparing the sequence of prestin to those of its paralogs and orthologs. Frog prestin is the only representative in amphibian lineage and the studies of it were quite rare with only one species identified. Results Here we report a new coding sequence of SLC26A5 for a frog species, Rana catesbeiana (the American bullfrog). In our study, the SLC26A5 gene of Rana has been mapped, sequenced and cloned successively using RNA-Seq. We measured the nonlinear capacitance (NLC) of prestin both in the hair cells of Rana’s inner ear and HEK293T cells transfected with this new coding gene. HEK293T cells expressing Rana prestin showed electrophysiological features similar to that of hair cells from its inner ear. Comparative studies of zebrafish, chick, Rana and an ancient frog species showed that chick and zebrafish prestin lacked NLC. Ancient frog’s prestin was functionally different from Rana. Conclusions We mapped and sequenced the SLC26A5 of the Rana catesbeiana from its inner ear cDNA using RNA-Seq. The Rana SLC26A5 cDNA was 2292 bp long, encoding a polypeptide of 763 amino acid residues, with 40% identity to mammals. This new coding gene could encode a functionally active protein conferring NLC to both frog HCs and the mammalian cell line. While comparing to its orthologs, the amphibian prestin has been evolutionarily changing its function and becomes more advanced than avian and teleost prestin.

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