scholarly journals Both Determinants of Allosteric and Active Sites Responsible for Catalytic Activity of Delta 12 Fatty Acid Desaturase by Domain Swapping

2019 ◽  
Author(s):  
Haisu Shi ◽  
Jinlong Tian ◽  
Chen Wu ◽  
Mo Li ◽  
Feiyu An ◽  
...  
Keyword(s):  
Amino Acids ◽  
Catalytic Activity ◽  
Fatty Acid ◽  
Amino Acid ◽  
Molecular Mechanism ◽  
Active Sites ◽  
Transmembrane Domain ◽  
Fatty Acid Desaturase ◽  
Domain Swapping ◽  
The Third

AbstractCheese lacks essential fatty acids (EFAs). Delta 12 fatty acid desaturase (FADS12) is a critical enzyme required for EFA biosynthesis in fermentation of the predominant strains of cheese. Previously, we identified the FADS12 gene and characterized its function for the first time in Geotrichum candidum, a dominant strain used to manufacture soft cheese with white rind. In this study, we analyzed the molecular mechanism of FADS12 function by swapping domains from Mortierella alpina and G. candidum that had, respectively, high and low oleic acid conversion rates. The results revealed three regions that are essential to this process, including regions from the end of the second transmembrane domain to the beginning of the third transmembrane domain, from the end of the third transmembrane domain to the beginning of the fourth transmembrane domain, and from the 30-amino acid from the end of the sixth transmembrane domain to the C-terminal end region. Based on our domain swapping analyses, nine pairs of amino acids including H112, S118, H156, Q161, K301, R306, E307, A309 and S323 in MaFADS12 (K123, A129, N167, M172, T302, D307, I308, E310 and D324 in GcFADS12) were identified as having a significantly effect on FADS12 catalytic efficiency, and linoleic acid and its analogues (12,13-cyclopropenoid fatty acid) were found to inhibit the catalytic activity of FADS12 and related recombinant enzymes. Furthermore, the molecular mechanism of FADS12 inhibition was analyzed. The results revealed two allosteric domains, including one domain from the N-terminal region to the beginning of the first transmembrane domain and another from the 31st amino acid from the end of the sixth transmembrane domain to the C terminus. Y4 and F398 amino acid residues from MaFADS12 and eight pairs of amino acids including G56, L60, L344, G10, Q13, S24, K326 and L344 in MaFADS12 (while Y66, F70, F345, F20, Y23, Y34, F327 and F345 in GcFADS12) played a pivotal role in FADS12 inhibition. Finally, we found that both allosteric and active sites were responsible for the catalytic activity of FADS12 at various temperatures, pH, and times. This study offers a solid theoretical basis to develop preconditioning methods to increase the rate at which GcFADS12 converts oleic and linoleic acids to produce higher levels of EFAs in cheese.

scholarly journals Bernard-Soulier Syndrome Caused by a Dinucleotide Deletion and Reading Frameshift in the Region Encoding the Glycoprotein Ibα Transmembrane Domain

Blood ◽  
1997 ◽  
Vol 90 (7) ◽  
pp. 2634-2643 ◽  
Author(s):  
Vahid Afshar-Kharghan ◽  
José A. López
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Transmembrane Domain ◽  
Silent Mutation ◽  
Cell Surface Expression ◽  
Unaffected Individual ◽  
Exon 2 ◽  
The Third ◽  
Bernard Soulier Syndrome

We investigated the molecular genetic and biosynthetic basis of Bernard-Soulier syndrome in a severely affected white woman. Flow cytometric analysis showed a severe deficiency of glycoprotein (GP) Ib, GP IX, and GP V on the surface of her platelets. Similarly, GP Ibα was undetectable by immunoblot analysis of platelet lysates. Surprisingly, a large quantity of a 70-kD protein (which probably represents a GP Ibα degradation product) was found in the patient's plasma in much greater quantities than in the plasma of an unaffected individual. To analyze the molecular lesion responsible for the disorder, we amplified and sequenced gene segments corresponding to the entire coding regions of the GP Ibα, GP Ibβ, and GP IX genes. The patient was homozygous for a specific GP Ibα allele that contained two tandem VNTR repeats in the region encoding the macroglycopeptide (C variant) and three differences from the published GP Ibα gene sequence. Two mutations were unlikely to be involved in the disorder: the substitution of a single base (T → C) in the second nucleotide of exon 2, which is in the 5′ untranslated region of the GP Ibα transcript, and a silent mutation in the third base of the codon for Arg342 (A → G) that does not change the amino acid sequence. The third mutation was a deletion of the last two bases of the codon for Tyr492 (TAT). This mutation causes a frameshift that alters the GP Ibα amino acid sequence, beginning within its transmembrane region. The mutant polypeptide contains 81 novel amino acids and is 38 amino acids shorter than its wild-type counterpart. The new sequence changes the hydrophobic nature of the transmembrane domain and greatly decreases the net positive charge of what had been the cytoplasmic domain. The deletion mutation was introduced into the GP Ibα cDNA, alone and in combination with the 5′ mutation, and expressed in Chinese hamster ovary (CHO) cells. The deletion alone severely reduced GP Ibα expression on the cell surface. Expression was not decreased further by addition of the 5′ mutation, confirming that the deletion was the cause of the Bernard-Soulier phenotype. Stable cell lines expressing the mutant polypeptide secreted large amounts of the polypeptide into the medium, suggesting that the mutant anchors poorly in the plasma membrane. Nevertheless, a fraction of the mutant was able to associate with GP Ibβ, as demonstrated by their coimmunoprecipitation with a GP Ibβ antibody.

scholarly journals Bernard-Soulier Syndrome Caused by a Dinucleotide Deletion and Reading Frameshift in the Region Encoding the Glycoprotein Ibα Transmembrane Domain

Blood ◽  
1997 ◽  
Vol 90 (7) ◽  
pp. 2634-2643 ◽  
Author(s):  
Vahid Afshar-Kharghan ◽  
José A. López
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Transmembrane Domain ◽  
Silent Mutation ◽  
Cell Surface Expression ◽  
Unaffected Individual ◽  
Exon 2 ◽  
The Third ◽  
Bernard Soulier Syndrome

Abstract We investigated the molecular genetic and biosynthetic basis of Bernard-Soulier syndrome in a severely affected white woman. Flow cytometric analysis showed a severe deficiency of glycoprotein (GP) Ib, GP IX, and GP V on the surface of her platelets. Similarly, GP Ibα was undetectable by immunoblot analysis of platelet lysates. Surprisingly, a large quantity of a 70-kD protein (which probably represents a GP Ibα degradation product) was found in the patient's plasma in much greater quantities than in the plasma of an unaffected individual. To analyze the molecular lesion responsible for the disorder, we amplified and sequenced gene segments corresponding to the entire coding regions of the GP Ibα, GP Ibβ, and GP IX genes. The patient was homozygous for a specific GP Ibα allele that contained two tandem VNTR repeats in the region encoding the macroglycopeptide (C variant) and three differences from the published GP Ibα gene sequence. Two mutations were unlikely to be involved in the disorder: the substitution of a single base (T → C) in the second nucleotide of exon 2, which is in the 5′ untranslated region of the GP Ibα transcript, and a silent mutation in the third base of the codon for Arg342 (A → G) that does not change the amino acid sequence. The third mutation was a deletion of the last two bases of the codon for Tyr492 (TAT). This mutation causes a frameshift that alters the GP Ibα amino acid sequence, beginning within its transmembrane region. The mutant polypeptide contains 81 novel amino acids and is 38 amino acids shorter than its wild-type counterpart. The new sequence changes the hydrophobic nature of the transmembrane domain and greatly decreases the net positive charge of what had been the cytoplasmic domain. The deletion mutation was introduced into the GP Ibα cDNA, alone and in combination with the 5′ mutation, and expressed in Chinese hamster ovary (CHO) cells. The deletion alone severely reduced GP Ibα expression on the cell surface. Expression was not decreased further by addition of the 5′ mutation, confirming that the deletion was the cause of the Bernard-Soulier phenotype. Stable cell lines expressing the mutant polypeptide secreted large amounts of the polypeptide into the medium, suggesting that the mutant anchors poorly in the plasma membrane. Nevertheless, a fraction of the mutant was able to associate with GP Ibβ, as demonstrated by their coimmunoprecipitation with a GP Ibβ antibody.

scholarly journals The phtE Locus in the Phaseolotoxin Gene Cluster Has ORFs with Homologies to Genes Encoding Amino Acid Transferases, the AraC Family of Transcriptional Factors, and Fatty Acid Desaturases

1997 ◽  
Vol 10 (8) ◽  
pp. 947-960 ◽  
Author(s):  
Yuan Xin Zhang ◽  
Suresh S. Patil
Keyword(s):  
Amino Acids ◽  
Fatty Acid ◽  
Amino Acid ◽  
Binding Site ◽  
Pseudomonas Syringae ◽  
Pyridoxal Phosphate ◽  
Fatty Acid Desaturase ◽  
Transcriptional Factors ◽  
Copper Binding ◽  
Arac Family

A cluster of genes involved in the production of phaseolotoxin, a phytotoxin produced by Pseudomonas syringae pv. phaseolicola, contains eight (phtA through phtH) complementation groups (Y. X. Zhang, K. B. Rowley, and S. S. Patil, J. Bacteriol., 175:6451–6458, 1993). In this study, sequencing of the region encompassing the phtE locus revealed six putative open reading frames (ORFs), each preceded by a putative ribosomal binding site, and all oriented in the same direction. Reverse transcription-polymerase chain reaction suggested that the phtE locus is transcribed as one large (6.4 kb) transcript, indicating that the ORFs constitute an operon. Primer extension analysis showed that the transcript begins at a T, located 31 bp upstream of the ATG codon of ORF1. Comparison of the sequences of the putative ORFs with the sequences of known genes revealed that ORF3, encoding a protein containing 395 amino acids, has 55% similarity to the acetylornithine aminotransferase gene from Escherichia coli, and the ornithine aminotransferase genes from other organisms. A lysine residue that is a binding site for pyridoxal phosphate and an arginine residue that is a binding site for the α-carboxylate group of the substrate are conserved in ORF3. These data suggest that ORF3 encodes a protein involved in the biosynthesis of ornithine, a constituent of phaseolotoxin. ORF5, encoding a peptide of 378 amino acid residues, possesses a helix-turn-helix motif at the C-terminal end that is characteristic of the AraC family of transcriptional factors, and there is a possible leucine zipper at the N-terminal end of this peptide. ORF6, encoding a protein of 327 amino acids, has about 40% similarity with the fatty acid desaturase gene, desA, of Synechocystis Pcc6803 and considerable similarity with fatty acid desaturase genes from other organisms. ORF6 and desA show very similar hydropathy profiles and both contain a copper binding signature. Computer searches did not discover significant homologies in the data base for the other ORFs, but hydropathy analysis showed that all of them contain one to several hydrophobic domains, suggesting that the gene products of these ORFs may be membrane associated.

Isolation and sequencing of a genomic DNA clone containing the 3′ terminus of the 6-methylsalicylic acid polyketide synthetase gene of Penicillium urticae

1991 ◽  
Vol 37 (1) ◽  
pp. 86-95 ◽  
Author(s):  
Ing-Kae Wang ◽  
C. Reeves ◽  
G. M. Gaucher
Keyword(s):  
Amino Acids ◽  
Fatty Acid ◽  
Amino Acid ◽  
Genomic Dna ◽  
Active Sites ◽  
Acyl Carrier Protein ◽  
Fatty Acid Synthetase ◽  
Protein Domain ◽  
Nucleotide Sequence Analysis ◽  
Functional Domains

A 7.7-kilobase (kb) Penicillium urticae genomic DNA fragment containing the 3′ terminus of the 6-methylsalicylic acid polyketide synthetase gene was cloned using a 41-mer mixed oligodeoxynucleotide probe which was based on a cyanogen bromide cleavage peptide of 35 amino acids obtained from pure synthetase. Nucleotide sequence analysis of a 2.2-kb region of the cloned fragment revealed a large open reading frame of 1866 bases which was devoid of introns and which corresponded to amino acids of the carboxyl terminus of the enzyme. This was followed by a putative transcription termination–polyadenylation signal. A putative acyl carrier protein domain at the 3′ terminus was preceded by a β-ketoreductase domain. These functionalities were identified by amino acid sequences known to be characteristic of the active sites of fatty acid synthetase functional domains. Their relative positions contrast with those in yeast and P. urticae fatty acid synthetase genes where the two functional domains are located at the 5′ terminus and in reverse order. Furthermore, amino acid sequence identities indicated a striking homology with vertebrate rather than either yeast or P. urticae fatty acid synthetases. Key words: multifunctional enzyme, mycotoxin, Pencillium, polyketide synthetase, secondary metabolism.

scholarly journals Characteristics of Amino Acid, Fatty Acid and Mineral of Sea Hare

2016 ◽  
Vol 19 (2) ◽  
pp. 168 ◽  
Author(s):  
Benny Manulang ◽  
Sri Purwaningsih ◽  
Azrifitria Azrifitria
Keyword(s):  
Amino Acids ◽  
Fatty Acids ◽  
Fatty Acid ◽  
Amino Acid ◽  
Saturated Fatty Acids ◽  
Monounsaturated Fatty Acids ◽  
Chemical Characteristic ◽  
Essential Amino Acids ◽  
Limited Information ◽  
Sea Hare

Dolabella auricularia are found in the waters of Indo - Pacific and has active compound in health, which until now is still limited information about nutritional content from sea hare. The aim of this research were to determine morphometric and chemical characteristic D. auricularia which includes the proximate, amino acids, fatty acids and minerals. The composition of fatty acid were measured by gas chromatography (GC), amino acids were measured by high performanced liquid chromatography (HPLC), and mineral was measured by atomic absorption spectrophotometer (AAS). The sea hare contained 9 essential amino acids and 6 non essential amino acids. The highest essential amino acid was arginine (1.61%) while the highest non essential amino acids was glycine (3.02%). Sea hare contained 26 fatty acids such as saturated fatty acids 5.33%, monounsaturated fatty acids 2.11% and polyunsaturated fatty acids 4.10%. The high mineral was calcium 68100 mg/kg.

scholarly journals Multiple Transmembrane Amino Acid Requirements Suggest a Highly Specific Interaction between the Bovine Papillomavirus E5 Oncoprotein and the Platelet-Derived Growth Factor Beta Receptor

2002 ◽  
Vol 76 (16) ◽  
pp. 7976-7986 ◽  
Author(s):  
Valerie M. Nappi ◽  
Lisa M. Petti
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Growth Factor ◽  
Transmembrane Domain ◽  
Specific Interaction ◽  
Platelet Derived Growth Factor ◽  
Helical Conformation ◽  
Bovine Papillomavirus ◽  
E5 Protein ◽  
Amino Acid Requirements

ABSTRACT The bovine papillomavirus E5 protein activates the cellular platelet-derived growth factor β receptor (PDGFβR) tyrosine kinase in a ligand-independent manner. Evidence suggests that the small transmembrane E5 protein homodimerizes and physically interacts with the transmembrane domain of the PDGFβR, thereby inducing constitutive dimerization and activation of this receptor. Amino acids in the receptor previously found to be required for the PDGFβR-E5 interaction are a transmembrane Thr513 and a juxtamembrane Lys499. Here, we sought to determine if these are the only two receptor amino acids required for an interaction with the E5 protein. Substitution of large portions of the PDGFβR transmembrane domain indicated that additional amino acids in both the amino and carboxyl halves of the receptor transmembrane domain are required for a productive interaction with the E5 protein. Indeed, individual amino acid substitutions in the receptor transmembrane domain identified roles for the extracellular proximal transmembrane residues in the interaction. These data suggest that multiple amino acids within the transmembrane domain of the PDGFβR are required for a stable interaction with the E5 protein. These may be involved in direct protein-protein contacts or may support the proper transmembrane alpha-helical conformation for optimal positioning of the primary amino acid requirements.

Composition of amino acids and fatty acids on Luwak coffee processing

Food Research ◽  
2021 ◽  
Vol 5 (3) ◽  
pp. 60-64
Author(s):  
Fitri ◽  
A. Laga ◽  
Z. Dwyana ◽  
A.B. Tawali
Keyword(s):  
Amino Acids ◽  
Fatty Acids ◽  
Fatty Acid ◽  
Amino Acid ◽  
Acid Content ◽  
Green Bean ◽  
Total Amino Acid ◽  
Coffee Bean ◽  
Roasted Coffee ◽  
Coffee Beans

The processing carried out on coffee beans such as fermentation and roasting can affect the contents of amino and fatty acids of coffee beans. This study aimed to determine the amount of amino acid and fatty acid content in Luwak coffee bean during processing. The amino acids of coffee were analyzed using HPLC, while fatty acids of coffee were analyzed using GC-MS. The results showed a change in total amino acid content in raw coffee bean (3.04%), green bean coffee (6.93%), and roasted coffee (6.83%). The total fatty acid of raw coffee bean (1199.86 mg/100 g), green bean coffee (3147.56 mg/100 g), and roasted coffee (6282.4 mg/100 g) also experienced significant changes

Metabolic analysis of infants with bronchopulmonary dysplasia under early nutrition therapy: An observational cohort study

2021 ◽  
pp. 153537022110605
Author(s):  
Li Wang ◽  
Wen Hua Zhong ◽  
Dan Yang Liu ◽  
Hai Qing Shen ◽  
Zhen Juan He
Keyword(s):  
Amino Acids ◽  
Fatty Acid ◽  
Amino Acid ◽  
Bronchopulmonary Dysplasia ◽  
Transitional Period ◽  
Preterm Neonates ◽  
Acid Metabolite ◽  
Early Nutrition ◽  
Fatty Acid Metabolite ◽  
Observational Cohort

To assess the amino acid and fatty acid metabolite patterns between infants with and without bronchopulmonary dysplasia in different nutritional stages after birth and identify metabolic indicators of bronchopulmonary dysplasia. This was an observational cohort of preterm infants born at a gestational age ≤32 + 6 weeks and with a body weight ≤2000 g. Amino acid and carnitine profiles were measured in dried blood spots (DBSs) during the early nutrition transitional phase using tandem mass spectrometry. Bronchopulmonary dysplasia was defined as oxygen dependence at 36 weeks of postmenstrual age or 28 days after birth. Metabolomic analysis was employed to define metabolites with significant differences, map significant metabolites into pathways, and identify metabolic indicators of bronchopulmonary dysplasia. We evaluated 45 neonates with and 40 without bronchopulmonary dysplasia. Four amino acids and three carnitines showed differences between the groups. Three carnitines (C0, C2, and C6:1) were high in the bronchopulmonary dysplasia group mostly; conversely, all four amino acids (threonine, arginine, methionine, and glutamine (Gln)) were low in the bronchopulmonary dysplasia group. Pathway analysis of these metabolites revealed two pathways with significant changes (p < 0.05). ROC analysis showed Gln/C6:1 at total parenteral nutrition phase had both 80% sensitivity and specificity for predicting the development of bronchopulmonary dysplasia, with an area under the curve of 0.81 (95% confidence interval 0.71–0.89). Amino acid and fatty acid metabolite profiles changed in infants with bronchopulmonary dysplasia after birth during the nutrition transitional period, suggesting that metabolic dysregulation may participate in the development of bronchopulmonary dysplasia. Our findings demonstrate that metabolic indicators are promising for forecasting the occurrence of bronchopulmonary dysplasia among preterm neonates.

Sign in / Sign up

Export Citation Format

Share Document