scholarly journals A Glutamate Mutase Is Involved in the Biosynthesis of the Lipopeptide Antibiotic Friulimicin in Actinoplanes friuliensis

2003 ◽  
Vol 47 (2) ◽  
pp. 447-457 ◽  
Author(s):  
E. Heinzelmann ◽  
S. Berger ◽  
O. Puk ◽  
B. Reichenstein ◽  
W. Wohlleben ◽  
...  
Keyword(s):  
Amino Acids ◽  
Genetic Manipulation ◽  
Active Form ◽  
Ring Structure ◽  
Vector System ◽  
Acyl Residue ◽  
Biosynthetic Genes ◽  
Glutamate Mutase ◽  
Glutamate Fermentation ◽  
Lipopeptide Antibiotic

ABSTRACT Actinoplanes friuliensis produces the lipopeptide antibiotic friulimicin. This antibiotic is active against gram-positive bacteria such as multiresistant Enterococcus and Staphylococcus strains. It consists of 10 amino acids that form a ring structure and 1 exocyclic amino acid to which an acyl residue is attached. By a reverse genetic approach, biosynthetic genes were identified that are required for the nonribosomal synthesis of the antibiotic. In close proximity two genes (glmA and glmB) were found which are involved in the production of methylaspartate, one of the amino acids of the peptide core. Methylaspartate is synthesized by a glutamate mutase mechanism, which was up to now only described for glutamate fermentation in Clostridium sp. or members of the family Enterobacteriaceae. The active enzyme consists of two subunits, and the corresponding genes overlap each other. To demonstrate enzyme activity in a heterologous host, it was necessary to genetically fuse glmA and glmB. The resulting gene was overexpressed in Streptomyces lividans, and the fusion protein was purified in an active form. For gene disruption mutagenesis, a host-vector system was established which enables genetic manipulation of Actinoplanes spp. for the first time. Thus, targeted inactivation of biosynthetic genes was possible, and their involvement in friulimicin biosynthesis was demonstrated.

Investigation of Optimum Conformations and Structure Analysis of RL and LR Nests using Ramachandran Plot

2015 ◽  
pp. 209-224
Author(s):  
Sumukh Deshpande ◽  
Saikat Kumar Basu ◽  
Pooja Purohit
Keyword(s):  
Amino Acids ◽  
Ring Structure ◽  
Ramachandran Plot ◽  
Binding Motifs ◽  
Automated Algorithm ◽  
Automation And Control ◽  
The Common ◽  
And Control ◽  
Common Anion ◽  
Unique Application

We have surveyed polypeptides with the optimal conformations of nests which are the common anion-binding motifs comprising 8% of the amino acids which are characterized by a structural depression or a hole. Using automated bioinformatics algorithm, novel ring structure of the nest has been found. Using automated algorithm, models of polypeptides were made in-silico (computationally) and oxygen atoms are inserted along the extension of the NH groups. These sophisticated algorithms allow insertion of atoms along the NH group at the correct distance which causes extension of the group thus forming hydrogen bond. Optimal conformations of these structures are found from these customized models. This study chapter provides a demonstration of an important discovery of optimum conformations of RL and LR nests by the use of sophisticated bioinformatics automation pipeline and a unique application of automation and control in bioinformatics.

An In-Frame Deletion of Six Amino Acids and a Point Mutation in the Disintegrin Domain of ADAMTS-13 Associates with a Case of Congenital Thrombotic Thromocytopenic Purpura.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 854-854
Author(s):  
Zhenyin Tao ◽  
Leticia Nolasco ◽  
Bernardo Aubrey ◽  
Lawrence Rice ◽  
Joel F. Moake ◽  
...  
Keyword(s):  
Amino Acids ◽  
Point Mutation ◽  
Active Form ◽  
White Blood Cells ◽  
In Vitro Mutagenesis ◽  
Severe Thrombocytopenia ◽  
Direct Dna Sequencing ◽  
Disintegrin Domain ◽  
The Impact

Abstract Thrombotic thrombocytopenic purpura (TTP) is characterized by severe thrombocytopenia, hemolytic anemia, and diffuse and non-focal neurological findings. Microthrombi found in these patients are predominantly composed of platelets and von Willebrand factor (VWF). Recent studies suggest that the systemic thrombosis in TTP is mostly due to the congenital or acquired deficiency of the VWF-cleaving metalloprotease ADAMTS-13, which cleaves the ultra-large and hyperreactive VWF to smaller and less active form found in plasma. Most congenital cases of TTP have so far been identified in children. Here, we report a 60-year-old Caucasian man with a history of chronic relapsing TTP over thirty years, requiring plasma transfusion every 24 days in recent years. Repeat assays showed no ADAMTS13 activity in patient’s plasma under both static and flow conditions without inhibitors being detected. We therefore examined the possible genetic defects in the ADAMTS-13 gene of this patient. Genomic DNA was extracted from patient’s white blood cells and exons of ADAMTS-13 gene were amplified by polymerase chain reaction. The amplified DNA fragments were then screened for mutations by direct DNA sequencing. We identified a deletion of 18 base pairs from G1095 to G1112 (GTGCTCCAAGGGTCGCTG) in the exon10 of ADAMTS-13 gene, resulting in a deletion of six amino acids (C366 to C371) in the disintegrin domain of the metalloprotease. A point mutation (W365C) occurred immediately before the deletion due to a nucleotide realignment. The patient is heterozygote for the deletion. This is the first report of a deletion mutant (without frame shift and truncations) in the disintegrin region that has previously been demonstrated as critical for the ADAMTS-13 function by in vitro mutagenesis, epitope mapping of autoantibody to the metalloprotease in patients with adult acquired TTP, and identification of natural occurring mutations in patients with congenital TTP. Our ongoing studies are to determine the impact of these two mutations on the synthesis, release, and cleavage of ADAMTS-13.

CD38 Expression Marks CLL Cells with Invasive Properties

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2422-2422
Author(s):  
Tiziana Vaisitti ◽  
Cinzia Bologna ◽  
Sara Serra ◽  
Giovanni D'Arena ◽  
Davide Rossi ◽  
...  
Keyword(s):  
Prognostic Marker ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Genetic Manipulation ◽  
Active Form ◽  
Gelatin Zymography ◽  
Integrin Subunit ◽  
Proliferating Cells ◽  
Neoplastic Cells

Abstract Abstract 2422 CLL is characterized by a dynamic balance between proliferating cells in the lymphoid organs and circulating cells resisting programmed cell death. Regulating this equilibrium entails complex interactions between tumor and host, modulated by a set of surface molecules expressed by the CLL cell according to environmental conditions. CD38 is a negative prognostic marker, involved in the homing process. We previously reported that CD38+ cells are significantly more sensitive to CXCL12, a critical chemokine for the recirculation of neoplastic cells, both in vitro and in vivo. Activation of CD38 by means of agonistic mAbs promotes chemotaxis, while block of the molecule impairs it. CD38 is also co-expressed with CD49d, the alpha4 integrin subunit and a further independent negative prognostic marker for CLL. The two molecules appear to be intertwined in a dynamic loop which involves CD31 (the CD38 ligand predominantly expressed by endothelial cells) and VCAM-1 (the CD49d ligand). Attention has now been focused on MMP-9, the main matrix metalloproteinase expressed by CLL cells, due to the relevance of extra-vasation in the homing process. The final aim is to clarify whether CD38 may represent a key player, taking part to all the main steps of CLL cells recirculation. Results obtained analyzing a large cohort of CLL patients indicate that i) CD38+ cases are characterized by a higher expression and activity of MMP-9, as measured by gelatin zymography. Moreover, ii) the analysis of CLL patients with a bimodal expression of CD38 indicate that the CD38+ fraction of the clone is the one expressing higher levels of MMP-9 compared to the negative one. A formal proof of the connection between CD38 and this gelatinase has been obtained using a lentiviral technique that allows genetic manipulation of CLL cells. iii) De novo expression of CD38 is followed by secretion of high amounts of the active form of MMP-9, suggesting that de novo CD38+ cells digest extracellular matrix more readily. Furthermore, iv) the engagement of CD38 by means of an agonistic antibody is followed by an increased MMP-9 activation, while blocking anti-CD38 mAbs are highly effective in the modulation of MMP-9 secretion by CLL cells. Finally, v) CD38 appears to co-localize with MMP-9 in the same membrane areas, as inferred by confocal microscopy analysis. Considered together, these information pinpoint CD38 as a connecting element between chemokines, adhesion molecules and matrix metalloproteinases. The finding of a physical proximity of all these molecules suggests that they form a large supramolecular complex, with the characteristics of the invadosome, a podosome of neoplastic cells that controls diffusion and metastasis. If confirmed, these results would link the ability to migrate and invade tissues with an inferior clinical outcome also in leukemia and not only in solid tumors. Disclosures: No relevant conflicts of interest to declare.

Molecular approaches to the manipulation of carbon allocation in plants

1993 ◽  
Vol 71 (6) ◽  
pp. 765-778 ◽  
Author(s):  
S. D. Blakeley ◽  
D. T. Dennis
Keyword(s):  
Amino Acids ◽  
Fatty Acids ◽  
Metabolic Pathways ◽  
Carbon Allocation ◽  
Genetic Manipulation ◽  
Biosynthetic Pathways ◽  
Molecular Approaches ◽  
Carbon Substrates ◽  
Storage Organs ◽  
Storage Products

In plants, sucrose is the end product of photosynthesis and is converted to a wide variety of storage compounds in tissues such as seeds and tubers. The allocation of carbon from sucrose to the various metabolic pathways leading to these products will determine the quantity of each synthesized in the respective storage organs. If the level of the enzymes involved in the allocation of carbon could be changed by genetic manipulation, it is probable that the relative yields of the various storage products can also be altered. The initial breakdown of sucrose occurs in the cytosol of the cell. Many biosynthetic pathways, however, including those involved in the synthesis of storage products such as fatty acids, starch, and amino acids, occur in the plastid. The distribution of carbon substrates for these processes will be determined, to a large extent, by the flux of carbon through the glycolytic pathways found in both the cytosolic and plastid compartments. This article will discuss the importance and consequences of compartmentation, review the extent of our understanding of glycolysis and other enzymes and pathways regulating carbon allocation, and will speculate on the potential for the genetic manipulation of these pathways. Key words: genetic manipulation, carbon allocation, metabolism, glycolysis.

scholarly journals Systematic Analysis of a Conserved Region of the Aminoglycoside 6′-N-Acetyltransferase Type Ib

2001 ◽  
Vol 45 (12) ◽  
pp. 3287-3292 ◽  
Author(s):  
Ali Shmara ◽  
Natalia Weinsetel ◽  
Ken J. Dery ◽  
Ramona Chavideh ◽  
Marcelo E. Tolmasky
Keyword(s):  
Amino Acids ◽  
Active Form ◽  
Major Change ◽  
Cysteine Residue ◽  
Alanine Scanning Mutagenesis ◽  
Systematic Analysis ◽  
C Terminus ◽  
Acetyl Group ◽  
Semisynthetic Derivative ◽  
High Level

ABSTRACT Alanine-scanning mutagenesis was applied to the aminoglycoside 6′-N-acetyltransferase type Ib conserved motif B, and the effects of the substitutions were analyzed by measuring the MICs of kanamycin (KAN) and its semisynthetic derivative, amikacin (AMK). Several substitutions resulted in no major change in MICs. E167A and F171A resulted in derivatives that lost the ability to confer resistance to KAN and AMK. P155A, P157A, N159A, L160A, I163A, K168A, and G170A conferred intermediate levels of resistance. Y166A resulted in an enzyme derivative with a modified specificity; it conferred a high level of resistance to KAN but lost the ability to confer resistance to AMK. Although not as pronounced, the resistance profiles conferred by substitutions N159A and G170A were related to that conferred by Y166A. These phenotypes, taken together with previous results indicating that mutant F171L could not catalyze acetylation of AMK when the assays were carried out at 42°C (D. Panaite and M. Tolmasky, Plasmid 39:123–133, 1998), suggest that some motif B amino acids play a direct or indirect role in acceptor substrate specificity. MICs of AMK and KAN for cells harboring the substitution C165A were high, suggesting that the active form of the enzyme may not be a dimer formed through a disulfide bond. Furthermore, this result indicated that the acetylation reaction occurs through a direct mechanism rather than a ping-pong mechanism that includes a transient transfer of the acetyl group to a cysteine residue. Deletion of fragments at the C terminus demonstrated that up to 10 amino acids could be deleted without a loss of activity.

scholarly journals Inactivation of rat adipocyte pyruvate dehydrogenase by palmitate. Protection against this effect by insulin in the presence of glucose

1979 ◽  
Vol 184 (1) ◽  
pp. 59-62 ◽  
Author(s):  
S R Sooranna ◽  
E D Saggerson
Keyword(s):  
Amino Acids ◽  
Fatty Acids ◽  
Dehydrogenase Activity ◽  
Pyruvate Dehydrogenase ◽  
Protective Action ◽  
Active Form ◽  
Rat Plasma ◽  
Epididymal Fat ◽  
Fat Pads

1. Adipocytes from rat epididymal fat-pads were incubated for 30 min with 5 mM-glucose and concentrations of lactate, pyruvate and amino acids typical of those found in rat plasma. 2. PDHa (active form of pyruvate dehydrogenase) activity was significantly increased after incubation of the cells with insulin (200 micro-i.u./ml), and decreased by incubation with palmitate (0.5–2 mM). 3. In the presence of insulin, palmitate did not decrease PDHa activity. 4. Dichloroacetate (1 mM) increased PDHa activity in the absence of palmitate to the same extent as did insulin. In the presence of dichloroacetate but the absence of insulin, palmitate decreased PDHa activity. In the presence of dichloroacetate and insulin, palmitate again did not decrease PDHa activity. 5. It is concluded that, in the presence of glucose, insulin has a strong protective action against inactivation of adipocyte PDHa by fatty acids.

scholarly journals The cmaR gene of Corynebacterium ammoniagenes performs a novel regulatory role in the metabolism of sulfur-containing amino acids

Microbiology ◽  
2009 ◽  
Vol 155 (6) ◽  
pp. 1878-1889 ◽  
Author(s):  
Seok-Myung Lee ◽  
Byung-Joon Hwang ◽  
Younhee Kim ◽  
Heung-Shick Lee
Keyword(s):  
Amino Acids ◽  
Target Genes ◽  
Sulfur Metabolism ◽  
Regulatory Gene ◽  
Positive Control ◽  
Negative Control ◽  
Biosynthetic Genes ◽  
Corynebacterium Ammoniagenes ◽  
Essential Function

A novel regulatory gene, which performs an essential function in sulfur metabolism, has been identified in Corynebacterium ammoniagenes and was designated cmaR (cysteine and methionine regulator in C. ammoniagenes). The cmaR-disrupted strain (ΔcmaR) lost the ability to grow on minimal medium, and was identified as a methionine and cysteine double auxotroph. The mutant strain proved unable to convert cysteine to methionine (and vice versa), and lost the ability to assimilate and reduce sulfate to sulfide. In the ΔcmaR strain, the mRNAs of the methionine biosynthetic genes metYX, metB and metFE were significantly reduced, and the activities of the methionine biosynthetic enzymes cystathionine γ-synthase, O-acetylhomoserine sulfhydrylase, and cystathionine β-lyase were relatively low, thereby suggesting that the cmaR gene exerts a positive regulatory effect on methionine biosynthetic genes. In addition, with the exception of cysK, reduced transcription levels of the sulfur-assimilatory genes cysIXYZ and cysHDN were noted in the cmaR-disrupted strain, which suggests that sulfur assimilation is also under the positive control of the cmaR gene. Furthermore, the expression of the cmaR gene itself was strongly induced via the addition of cysteine or methionine alone, but not the introduction of both amino acids together to the growth medium. In addition, the expression of the cmaR gene was enhanced in an mcbR-disrupted strain, which suggests that cmaR is under the negative control of McbR, which has been identified as a global regulator of sulfur metabolism. DNA binding of the purified CmaR protein to the promoter region of its target genes could be demonstrated in vitro. No metabolite effector was required for the protein to bind DNA. These results demonstrated that the cmaR gene of C. ammoniagenes plays a role similar to but distinct from that of the functional homologue cysR of Corynebacterium glutamicum.

scholarly journals The Uniqueness of Tryptophan in Biology: Properties, Metabolism, Interactions and Localization in Proteins

2020 ◽  
Vol 21 (22) ◽  
pp. 8776 ◽  
Author(s):  
Sailen Barik
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Structural Features ◽  
Ring Structure ◽  
Side Chain ◽  
Aromatic Side Chain ◽  
Single Ring ◽  
Energy Consuming ◽  
Animal Diet ◽  
Abundant Amino Acid

Tryptophan (Trp) holds a unique place in biology for a multitude of reasons. It is the largest of all twenty amino acids in the translational toolbox. Its side chain is indole, which is aromatic with a binuclear ring structure, whereas those of Phe, Tyr, and His are single-ring aromatics. In part due to these elaborate structural features, the biosynthetic pathway of Trp is the most complex and the most energy-consuming among all amino acids. Essential in the animal diet, Trp is also the least abundant amino acid in the cell, and one of the rarest in the proteome. In most eukaryotes, Trp is the only amino acid besides Met, which is coded for by a single codon, namely UGG. Due to the large and hydrophobic π-electron surface area, its aromatic side chain interacts with multiple other side chains in the protein, befitting its strategic locations in the protein structure. Finally, several Trp derivatives, namely tryptophylquinone, oxitriptan, serotonin, melatonin, and tryptophol, have specialized functions. Overall, Trp is a scarce and precious amino acid in the cell, such that nature uses it parsimoniously, for multiple but selective functions. Here, the various aspects of the uniqueness of Trp are presented in molecular terms.

scholarly journals Proteinase-Polymerase Precursor as the Active Form of Feline Calicivirus RNA-Dependent RNA Polymerase

2001 ◽  
Vol 75 (3) ◽  
pp. 1211-1219 ◽  
Author(s):  
Lai Wei ◽  
Jason S. Huhn ◽  
Aaron Mory ◽  
Harsh B. Pathak ◽  
Stanislav V. Sosnovtsev ◽  
...  
Keyword(s):  
Amino Acids ◽  
Rna Polymerase ◽  
Active Form ◽  
Polymerase Activity ◽  
Feline Calicivirus ◽  
Amino Terminus ◽  
Rna Dependent Rna Polymerase ◽  
Reading Frame ◽  
Catalytically Active ◽  
Catalytic Cysteine

ABSTRACT The objective of this study was to identify the active form of the feline calicivirus (FCV) RNA-dependent RNA polymerase (RdRP). Multiple active forms of the FCV RdRP were identified. The most active enzyme was the full-length proteinase-polymerase (Pro-Pol) precursor protein, corresponding to amino acids 1072 to 1763 of the FCV polyprotein encoded by open reading frame 1 of the genome. Deletion of 163 amino acids from the amino terminus of Pro-Pol (the Val-1235 amino terminus) caused a threefold reduction in polymerase activity. Deletion of an additional one (the Thr-1236 amino terminus) or two (the Ala-1237 amino terminus) amino acids produced derivatives that were 7- and 175-fold, respectively, less active than Pro-Pol. FCV proteinase-dependent processing of Pro-Pol in the interdomain region preceding Val-1235 was not observed in the presence of a catalytically active proteinase; however, processing within the polymerase domain was observed. Inactivation of proteinase activity by changing the catalytic cysteine-1193 to glycine permitted the production and purification of intact Pro-Pol. Biochemical analysis of Pro-Pol showed that this enzyme has properties expected of a replicative polymerase, suggesting that Pro-Pol is an active form of the FCV RdRP.

scholarly journals A novel serine protease predominately expressed in macrophages

2003 ◽  
Vol 374 (1) ◽  
pp. 97-107 ◽  
Author(s):  
Cailin CHEN ◽  
Andrew L. DARROW ◽  
Jian-shen QI ◽  
Michael R. D'ANDREA ◽  
Patricia ANDRADE-GORDON
Keyword(s):  
Amino Acids ◽  
In Situ Hybridization ◽  
Serine Protease ◽  
Mrna Level ◽  
Active Form ◽  
Northern Blotting ◽  
Specific Marker ◽  
U937 Cells ◽  
Arginine Residues

We have identified a novel serine protease designated EOS by sequence identity searches. The deduced protein contains 284 amino gcids with an active form containing 248 amino acids starting from an Ile-Val-Gly-Gly motif. The active form comprises a catalytic triad of conserved amino acids: His77, Asp126 and Ser231. It shares 44% identity with β-tryptase and belongs to the S1 trypsin-like serine-protease family. Interestingly, this gene also maps to human chromosome 16p13.3. The purified protease showed amidolytic activity, cleaving its substrates before arginine residues. Tissue distribution by immunohistochemistry analysis demonstrated that EOS is highly expressed in spleen and moderately expressed in intestine, colon, lung and brain. We confirmed this expression pattern at the mRNA level by performing in situ hybridization. The results from both immunohistochemistry and in situ hybridization indicate that EOS is associated with macrophages. We corroborated this observation by double immunofluorescence using the anti-EOS antibody and an anti-CD68 antibody, a macrophage specific marker. Furthermore, we have detected a dramatic increase in immune staining of EOS in cultured U937 cells treated with PMA, which represent activated macrophages. This up-regulation is also reflected by elevated EOS mRNA in the PMA-treated U937 cells detected by Northern blotting. Since macrophages have important roles in various pathological conditions, such as wound healing, atherosclerosis and numerous inflammatory diseases, the localization of this novel serine protease to active macrophages may help to further the elucidation of the roles of this gene product in modulating these disorders.

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