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 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 Regulation of enzyme turnover during tissue differentiation. Interactions of insulin, prolactin and cortisol in controlling the turnover of fatty acid synthetase in rabbit mammary gland in organ culture

1976 ◽  
Vol 154 (2) ◽  
pp. 359-370 ◽  
Author(s):  
Brian K. Speake ◽  
Raymond Dils ◽  
R. John Mayer
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Fatty Acid ◽  
Amino Acid ◽  
Culture Medium ◽  
Fatty Acid Synthetase ◽  
Amino Acid Pool ◽  
Acid Pool ◽  
Intracellular Amino Acid ◽  
Enzyme Degradation

1. Explants of mammary gland from mid-pregnant rabbits were cultured in Medium 199 containing combinations of insulin, prolactin and cortisol. With hormone combinations which included prolactin, a sustained increase in the apparent rate of synthesis and in the amount of fatty acid synthetase was measurable immunologically. Maximum increase was produced with insulin, prolactin and cortisol present together. 2. With prolactin present alone, synthetase activity in the explants decreased to undetectable values after 1 day in culture, whereas the incorporation of l-[U-14C]leucine into immunodetectable material increased. Prolactin may therefore direct the synthesis of immunologically cross-reactive precursors of fatty acid synthetase which are enzymically inactive. 3. Culture with dibutyryl cyclic AMP plus theophylline in the presence of insulin, prolactin and cortisol delayed the increase in the rate of synthesis and accumulation of the synthetase. These compounds may also prevent the apparent decrease in the rate of degradation of the synthetase which occurs on day 2 of culture. 4. A large decrease in the apparent rate of degradation of the synthetase on day 2 of culture occurs during culture with hormone combinations which include prolactin. The protein obtained by centrifugation of explant homogenates for 6min at 14000gav. is degraded continuously throughout the culture period. 5. This decrease in the apparent rate of degradation of the synthetase was measured by radio-immunological precipitation. It is probably part of a regulated programme of enzyme degradation and not a reflexion of the reutilization of radioactive amino acids for the following reasons. (a) The calculated increase in the amount of the synthetase in explants on day 2 of culture with insulin, prolactin and cortisol was approximately equal to the measured increase of the enzyme complex which accumulates in the explants. This suggests little or no enzyme degradation has occurred. (b) Explants were cultured for 24h with insulin, prolactin and cortisol. They were then incubated with l-[U-14C]leucine, washed and incubated again for up to 4½h. l-[U-14C]Leucine rapidly equilibrated with the intracellular amino acid pool. Within 10min of incubation after washing explants to remove endogenous l-[U-14C]leucine the previously linear incorporation of l-[U-14C]-leucine into total explant protein ceased. This suggests that protein is synthesized from an amino acid pool which rapidly equilibrates with amino acids in the culture medium. (c) Explants were cultured for 24h as described in (b) but after washing they were cultured with insulin, prolactin and cortisol for 24h. Approx. 90% of the radioactivity lost from the ‘free’ intracellular amino acid pool and from amino acids derived from the degradation of explant protein in this period was detected in the culture medium. This suggests that the ‘free’ intracellular amino acids and amino acids derived from protein degradation can equilibrate with amino acids in the medium. A residual ‘free’ radioactive amino acid pool was present in the tissue. (d) Casein represents approx. 20% of the protein synthesized after 1 day in culture with insulin, prolactin and cortisol. Histological evidence suggests that on day 2 of culture, casein is unlikely to be degraded in the tissue. No increase in the radioactivity incorporated into casein can be measured in the 23h after incubation of explants with l-[U-14C]leucine as described in (b). This suggests that the incorporation of radioactivity into proteins during culture after incubation with l-[U-14C]leucine is minimal. (e) Inhibition of protein synthesis in explants by cycloheximide after incubation with l-[U-14C]leucine does not reveal a latent continuous degradation of fatty acid synthetase on day 2 of culture which might have been masked by the high rates of protein synthesis and therefore the accumulation of the enzyme. 6. The conclusion is discussed that there is a real decrease (or even cessation) in the rate of degradation of fatty acid synthetase during the period when the enzyme accumulates in explants cultured with hormone combinations which contain prolactin.

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.

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.

Mouse protamine 2 is synthesized as a precursor whereas mouse protamine 1 is not

1987 ◽  
Vol 7 (6) ◽  
pp. 2173-2179
Author(s):  
P C Yelick ◽  
R Balhorn ◽  
P A Johnson ◽  
M Corzett ◽  
J A Mazrimas ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Nucleotide Sequence ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Nucleotide Sequence Analysis ◽  
Cdna Clones ◽  
Protamine 1 ◽  
Protamine 2

The nuclei of mouse spermatozoa contain two protamine variants, mouse protamine 1 (mP1) and mouse protamine 2 (mP2). The amino acid sequence predicted from mP1 cDNAs demonstrates that mP1 is a 50-amino-acid protein with strong homology to other mammalian P1 protamines. Nucleotide sequence analysis of independently isolated, overlapping cDNA clones indicated that mP2 is initially synthesized as a precursor protein which is subsequently processed into the spermatozoan form of mP2. The existence of the mP2 precursor was confirmed by amino acid composition and sequence analysis of the largest of a set of four basic proteins isolated from late-step spermatids whose synthesis is coincident with that of mP1. The sequence of the first 10 amino acids of this protein, mP2 precursor 1, exactly matches that predicted from the nucleotide sequence of cDNA and genomic mP2 clones. The amino acid composition of isolated mP2 precursor 1 very closely matches that predicted from the mP2 cDNA nucleotide sequence. Sequence analysis of the amino terminus of isolated mature mP2 identified the final processing point within the mP2 precursor. These studies demonstrated that mP2 is synthesized as a precursor containing 106 amino acids which is processed into the mature, 63-amino-acid form found in spermatozoa.

scholarly journals Cloning and characterization of platelet factor 4 cDNA derived from a human erythroleukemic cell line

Blood ◽  
1987 ◽  
Vol 69 (1) ◽  
pp. 219-223 ◽  
Author(s):  
M Poncz ◽  
S Surrey ◽  
P LaRocco ◽  
MJ Weiss ◽  
EF Rappaport ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cell Line ◽  
Genomic Dna ◽  
Cdna Probe ◽  
Leader Sequence ◽  
Platelet Factor 4 ◽  
Coding Region ◽  
Platelet Factor ◽  
Amino Acid Homology

Abstract We report the isolation of a platelet factor 4 (PF4) cDNA clone from a lambda gt11 expression cDNA library which was derived from a human erythroleukemic (HEL) cell line. The sequence of the DNA insert includes the 3′-untranslated region, the entire amino acid coding region for the mature PF4 protein, and a 5′ region containing coding information for an additional 18 amino acids. In addition, supplemental genomic DNA sequencing shows that the full-length leader sequence is 30 amino acids long plus an initial methionine and codes for a hydrophobic signal-like sequence which is probably involved in transmembrane transport. A single species mRNA of approximately 800 nucleotides was detected on blots of HEL cell poly(A) + RNA using a labeled PF4 cDNA probe. The human PF4 leader sequence shares some DNA, but no amino acid, homology with the 15 amino acids at the N-terminus of mature bovine PF4, suggesting rapid divergence in this region of PF4 between these two species. Sequence comparison of the coding regions of mature PF4 and gamma IP-10, a protein induced in a variety of cells following treatment with gamma-interferon, shows a corrected divergence of 76%. The divergence of a common ancestor protein into PF4 and gamma IP-10 may have accompanied the development of sophisticated immune and coagulation systems in vertebrates. The availability of cDNA and genomic DNA information for these genes in other species will be useful in studying the evolution of the coagulation and immune systems.

scholarly journals Targeted Metabolomic Analysis of a Mucopolysaccharidosis IIIB Mouse Model Reveals an Imbalance of Branched-Chain Amino Acid and Fatty Acid Metabolism

2020 ◽  
Vol 21 (12) ◽  
pp. 4211 ◽  
Author(s):  
Valeria De Pasquale ◽  
Marianna Caterino ◽  
Michele Costanzo ◽  
Roberta Fedele ◽  
Margherita Ruoppolo ◽  
...  
Keyword(s):  
Amino Acids ◽  
Fatty Acid ◽  
Amino Acid ◽  
Mouse Model ◽  
Molecular Mechanisms ◽  
Clinical Manifestations ◽  
Neurological Impairment ◽  
Genetic Mutation ◽  
Inherited Disorders ◽  
Branched Chain

Mucopolysaccharidoses (MPSs) are inherited disorders of the glycosaminoglycan (GAG) metabolism. The defective digestion of GAGs within the intralysosomal compartment of affected patients leads to a broad spectrum of clinical manifestations ranging from cardiovascular disease to neurological impairment. The molecular mechanisms underlying the progression of the disease downstream of the genetic mutation of genes encoding for lysosomal enzymes still remain unclear. Here, we applied a targeted metabolomic approach to a mouse model of PS IIIB, using a platform dedicated to the diagnosis of inherited metabolic disorders, in order to identify amino acid and fatty acid metabolic pathway alterations or the manifestations of other metabolic phenotypes. Our analysis highlighted an increase in the levels of branched-chain amino acids (BCAAs: Val, Ile, and Leu), aromatic amino acids (Tyr and Phe), free carnitine, and acylcarnitines in the liver and heart tissues of MPS IIIB mice as compared to the wild type (WT). Moreover, Ala, Met, Glu, Gly, Arg, Orn, and Cit amino acids were also found upregulated in the liver of MPS IIIB mice. These findings show a specific impairment of the BCAA and fatty acid catabolism in the heart of MPS IIIB mice. In the liver of affected mice, the glucose-alanine cycle and urea cycle resulted in being altered alongside a deregulation of the BCAA metabolism. Thus, our data demonstrate that an accumulation of BCAAs occurs secondary to lysosomal GAG storage, in both the liver and the heart of MPS IIIB mice. Since BCAAs regulate the biogenesis of lysosomes and autophagy mechanisms through mTOR signaling, impacting on lipid metabolism, this condition might contribute to the progression of the MPS IIIB disease.

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