scholarly journals Mutagenesis of NosM Leader Peptide Reveals Important Elements in Nosiheptide Biosynthesis

2016 ◽  
Vol 83 (4) ◽  
Author(s):  
Liang Jin ◽  
Xuri Wu ◽  
Yanjiu Xue ◽  
Yue Jin ◽  
Shuzhen Wang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gene Cluster ◽  
Genetic Manipulation ◽  
Fold Increase ◽  
Biosynthetic Gene Cluster ◽  
Leader Peptide ◽  
Amino Acid Residues ◽  
Precursor Peptide

ABSTRACT Nosiheptide, a typical member of the ribosomally synthesized and posttranslationally modified peptides (RiPPs), exhibits potent activity against multidrug-resistant Gram-positive bacterial pathogens. The precursor peptide of nosiheptide (NosM) is comprised of a leader peptide with 37 amino acids and a core peptide containing 13 amino acids. To pinpoint elements in the leader peptide that are essential for nosiheptide biosynthesis, a collection of mutants with unique sequence features, including N- and C-terminal motifs, peptide length, and specific sites in the leader peptide, was generated by mutagenesis in vivo. The effects of various mutants on nosiheptide biosynthesis were evaluated. In addition to the necessity of a conserved motif LEIS box, native length and the N-terminal 12 amino acid residues were indispensable, and single-site substitutions of these 12 amino acid residues resulted in changes ranging from a greater-than-5-fold decrease to a 2-fold increase of nosiheptide production, depending on the sites and substituted residues. Moreover, although the C-terminal motif is not conservative, significant effects of this portion on nosiheptide production were also evident. Taken together, the present results further highlight the importance of the leader peptide in nosiheptide biosynthesis, and provide new insights into the diversity and specificity of leader peptides in the biosynthesis of various RiPPs. IMPORTANCE As a representative thiopeptide, nosiheptide exhibits excellent antibacterial activity. Although the biosynthetic gene cluster and several modification steps have been revealed, the presence and roles of the leader peptide within the precursor peptide of the nosiheptide gene cluster remain elusive. Thus, identification of specific elements in the leader peptide can significantly facilitate the genetic manipulation of the gene cluster for increasing nosiheptide production or generating diverse analogues. Given the complexity of the biosynthetic process, the instability of the leader peptide, and the unavailability of intermediates, cocrystallization of intermediates, leader peptide, and modification enzymes is currently not feasible. Therefore, a mutagenesis approach was used to construct a series of leader peptide mutants to uncover a number of crucial and characteristic elements affecting nosiheptide biosynthesis, which moves a considerable distance toward a thorough understanding of the biosynthetic machinery for thiopeptides.

scholarly journals The enduracidin biosynthetic gene cluster from Streptomyces fungicidicus

Microbiology ◽  
2006 ◽  
Vol 152 (10) ◽  
pp. 2969-2983 ◽  
Author(s):  
Xihou Yin ◽  
T. Mark Zabriskie
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gene Cluster ◽  
Genetic Locus ◽  
Biosynthetic Gene Cluster ◽  
Antibiotic Biosynthesis ◽  
Peptide Antibiotic ◽  
Amino Acid Residues ◽  
Biosynthetic Gene ◽  
Condensation Domain

The biosynthetic gene cluster for the 17 aa peptide antibiotic enduracidin has been cloned and sequenced from Streptomyces fungicidicus ATCC 21013. The 84 kb gene cluster contains 25 ORFs and is located within a 116 kb genetic locus that was fully sequenced. Targeted disruption of non-ribosomal peptide synthetase (NRPS) genes in the cluster abolished enduracidin production and confirmed function. The cluster includes four genes, endA-D, encoding two-, seven-, eight- and one-module NRPSs, respectively, and includes unique modules for the incorporation of citrulline and enduracididine. The NRPS organization generally follows the collinearity principle, and starts with a condensation domain (C domain) similar to those found in other lipopeptide systems for the coupling of an acyl group to the starting amino acid. The sixth module of EndB, corresponding to Thr8, is missing an adenylation domain (A domain) and this module is presumed to be loaded in trans by the single module protein EndD. The most striking feature of the NRPS organization is the lack of epimerization domains (E domains) in light of the fact that the product has seven d-amino acid residues. Sequence analysis reveals that C domains following modules corresponding to d-amino acids belong to a unique subset of C domains able to catalyse both epimerization and condensation reactions. Other genes directing lipid modification and activation, and formation of the non-proteinogenic amino acids 4-hydroxyphenylglycine and enduracididine are readily identified, as are genes possibly involved in regulation of antibiotic biosynthesis and export. These findings provide the basis to further genetically manipulate and improve lipodepsipeptide antibiotics via combinatorial and chemical methods.

scholarly journals A cDNA clone for human glucosamine-6-sulphatase reveals differences between arylsulphatases and non-arylsulphatases

1992 ◽  
Vol 288 (2) ◽  
pp. 539-544 ◽  
Author(s):  
D A Robertson ◽  
C Freeman ◽  
C P Morris ◽  
J J Hopwood
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Sequence Similarity ◽  
Genetic Disorder ◽  
Heparan Sulphate ◽  
Leader Peptide ◽  
Cdna Clones ◽  
Amino Acid Residues ◽  
Lysosomal Storage ◽  
Glycosylation Sites

Glucosamine-6-sulphatase is an exo-hydrolase required for the lysosomal degradation of heparan sulphate and keratan sulphate. Deficiency of glucosamine-6-sulphatase activity leads to the lysosomal storage of the glycosaminoglycan, heparan sulphate and the monosaccharide sulphate N-acetylglucosamine 6-sulphate and the autosomal recessive genetic disorder mucopolysaccharidosis type IIID. Glucosamine-6-sulphatase can be classified as a non-arylsulphatase since, relative to arylsulphatase B, it shows negligible activity toward 4-methylumbelliferyl sulphate. We have isolated human cDNA clones and derived amino acid sequence coding for the entire glucosamine-6-sulphatase protein. The predicted sequence has 552 amino acids with a leader peptide of 36 amino acids and contains 13 potential N-glycosylation sites, of which it is likely that 10 are used. Glucosamine-6-sulphatase shows strong sequence similarity to other sulphatases such as the family of arylsulphatases, although the degree of similarity is not as high as that between members of the arylsulphatase family. This pattern of inter- and intra-family similarity delineates regions and amino acid residues that may be critical for sulphatase function and substrate specificity.

scholarly journals Biosynthesis of cittilins, unusual ribosomally synthesized and post-translationally modified peptides from Myxococcus xanthus

2020 ◽  
Author(s):  
Joachim J. Hug ◽  
Jan Dastbaz ◽  
Sebastian Adam ◽  
Ole Revermann ◽  
Jesko Koehnke ◽  
...  
Keyword(s):  
Amino Acid ◽  
Gene Cluster ◽  
Biochemical Characterization ◽  
Biosynthetic Gene Cluster ◽  
Cytochrome P450 Enzyme ◽  
Biosynthetic Gene ◽  
Aryl Ether ◽  
Precursor Peptide ◽  
Carbon Storage Regulator

AbstractCittilins are secondary metabolites from myxobacteria comprised of three L-tyrosines and one L-isoleucine forming a bicyclic tetrapeptide scaffold with biaryl and aryl-oxygen-aryl ether bonds. Here we reveal that cittilins belong to the ribosomally synthesized and post-translationally modified peptide (RiPP) family of natural products, for which only the crocagins have been reported from myxobacteria. A 27 amino acid precursor peptide harbors a C-terminal four amino acid core peptide, which is enzymatically modified and finally exported to yield cittilins. The small biosynthetic gene cluster responsible for cittilin biosynthesis also encodes a cytochrome P450 enzyme and a methyltransferase, whereas a gene encoding a prolyl endopeptidase for the cleavage of the precursor peptide is located outside of the cittilin biosynthetic gene cluster. We confirm the roles of the biosynthetic genes responsible for the formation of cittilins using targeted gene inactivation and heterologous expression in Streptomyces. We also report first steps towards the biochemical characterization of the proposed biosynthetic pathway in vitro. An investigation of the cellular uptake properties of cittilin A connected it to a potential biological function as an inhibitor of the prokaryotic carbon storage regulator A (CsrA).Abstract Figure

scholarly journals Peb1p (Pas7p) is an intraperoxisomal receptor for the NH2-terminal, type 2, peroxisomal targeting sequence of thiolase: Peb1p itself is targeted to peroxisomes by an NH2-terminal peptide.

1996 ◽  
Vol 132 (3) ◽  
pp. 325-334 ◽  
Author(s):  
J W Zhang ◽  
P B Lazarow
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Fusion Protein ◽  
Equilibrium Density ◽  
Amino Acid Residues ◽  
Loss Of Function ◽  
Peroxisomal Targeting ◽  
Digitonin Permeabilization

Peb1 is a peroxisome biogenesis mutant isolated in Saccharomyces cerevisiae that is selectively defective in the import of thiolase into peroxisomes but has a normal ability to package catalase, luciferase and acyl-CoA oxidase (Zhang, J. W., C. Luckey, and P. B. Lazarow. 1993. Mol. Biol. Cell. 4:1351-1359). Thiolase differs from these other peroxisomal proteins in that it is targeted by an NH2-terminal, 16-amino acid peroxisomal targeting sequence type 2 (PTS 2). This phenotype suggests that the PEB1 protein might function as a receptor for the PTS2. The PEB1 gene has been cloned by functional complementation. It encodes a 42,320-D, hydrophilic protein with no predicted transmembrane segment. It contains six WD repeats that comprise the entire protein except for the first 55 amino acids. Peb1p was tagged with hemagglutinin epitopes and determined to be exclusively within peroxisomes by digitonin permeabilization, immunofluorescence, protease protection and immuno-electron microscopy (Zhang, J. W., and P. B. Lazarow. 1995. J. Cell Biol. 129:65-80). Peb1p is identical to Pas7p (Marzioch, M., R. Erdmann, M. Veenhuis, and W.-H. Kunau. 1994. EMBO J. 13: 4908-4917). We have now tested whether Peb1p interacts with the PTS2 of thiolase. With the two-hybrid assay, we observed a strong interaction between Peb1p and thiolase that was abolished by deleting the first 16 amino acids of thiolase. An oligopeptide consisting of the first 16 amino acids of thiolase was sufficient for the affinity binding of Peb1p. Binding was reduced by the replacement of leucine with arginine at residue five, a change that is known to reduce thiolase targeting in vivo. Finally, a thiolase-Peb1p complex was isolated by immunoprecipitation. To investigate the topogenesis of Peb1p, its first 56-amino acid residues were fused in front of truncated thiolase lacking the NH2-terminal 16-amino acid PTS2. The fusion protein was expressed in a thiolase knockout strain. Equilibrium density centrifugation and immunofluorescence indicated that the fusion protein was located in peroxisomes. Deletion of residues 6-55 from native Peb1p resulted in a cytosolic location and the loss of function. Thus the NH2-terminal 56-amino acid residues of Peb1p are necessary and sufficient for peroxisomal targeting. Peb1p is found in peroxisomes whether thiolase is expressed or not. These results suggest that Peb1p (Pas7p) is an intraperoxisomal receptor for the type 2 peroxisomal targeting signal.

The impact of specific oxidized amino acids on protein turnover in J774 cells

2008 ◽  
Vol 410 (1) ◽  
pp. 131-140 ◽  
Author(s):  
Rachael A. Dunlop ◽  
Roger T. Dean ◽  
Kenneth J. Rodgers
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Oxidative Modification ◽  
Amino Acid Residues ◽  
Protein Deposition ◽  
In Situ Modification ◽  
Age Related ◽  
The Impact

Oxidized protein deposition and accumulation have been implicated in the aetiology of a wide variety of age-related pathologies. Protein oxidation in vivo commonly results in the in situ modification of amino acid side chains, generating new oxidized amino acid residues in proteins. We have demonstrated previously that certain oxidized amino acids can be (mis)incorporated into cell proteins in vitro via protein synthesis. In the present study, we show that incorporation of o- and m-tyrosine resulted in increased protein catabolism, whereas dopa incorporation generated proteins that were inefficiently degraded by cells. Incorporation of higher levels of L-dopa into proteins resulted in an increase in the activity of lysosomal cathepsins, increased autofluorescence and the generation of high-molecular-mass SDS-stable complexes, indicative of protein aggregation. These effects were due to proteins containing incorporated L-dopa, since they were not seen with the stereoisomer D-dopa, which enters the cell and generates the same reactive species as L-dopa, but cannot be incorporated into proteins. The present study highlights how the nature of the oxidative modification to the protein can determine the efficiency of its removal from the cell by proteolysis. Protection against the generation of dopa and other species that promote resistance to proteolysis might prove to be critical in preventing toxicity from oxidative stress in pathologies associated with protein deposition, such as atherosclerosis, Alzheimer's disease and Parkinson's disease.

Daptomycin biosynthesis in Streptomyces roseosporus: cloning and analysis of the gene cluster and revision of peptide stereochemistry

Microbiology ◽  
2005 ◽  
Vol 151 (5) ◽  
pp. 1507-1523 ◽  
Author(s):  
Vivian Miao ◽  
Marie-Françoise Coëffet-LeGal ◽  
Paul Brian ◽  
Renee Brost ◽  
Julia Penn ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gene Cluster ◽  
Binding Pocket ◽  
Biosynthetic Gene Cluster ◽  
Streptomyces Roseosporus ◽  
Cyclic Lipopeptide ◽  
Peptide Synthetase ◽  
Amino Acid Binding ◽  
Condensation Domain

Daptomycin is a 13 amino acid, cyclic lipopeptide produced by a non-ribosomal peptide synthetase (NRPS) mechanism in Streptomyces roseosporus. A 128 kb region of S. roseosporus DNA was cloned and verified by heterologous expression in Streptomyces lividans to contain the daptomycin biosynthetic gene cluster (dpt). The cloned region was completely sequenced and three genes (dptA, dptBC, dptD) encoding the three subunits of an NRPS were identified. The catalytic domains in the subunits, predicted to couple five, six or two amino acids, respectively, included a novel activation domain and amino-acid-binding pocket for incorporating the unusual amino acid l-kynurenine (Kyn), three types of condensation domains and an extra epimerase domain (E-domain) in the second module. Novel genes (dptE, dptF) whose products likely work in conjunction with a unique condensation domain to acylate the first amino acid, as well as other genes (dptI, dptJ) probably involved in supply of the non-proteinogenic amino acids l-3-methylglutamic acid and Kyn, were located next to the NRPS genes. The unexpected E-domain suggested that daptomycin would have d-Asn, rather than l-Asn, as originally assigned, and this was confirmed by comparing stereospecific synthetic peptides and the natural product both chemically and microbiologically.

scholarly journals Streptomyces lividans Blasticidin S Deaminase and Its Application in Engineering a Blasticidin S-Producing Strain for Ease of Genetic Manipulation

2013 ◽  
Vol 79 (7) ◽  
pp. 2349-2357 ◽  
Author(s):  
Li Li ◽  
Jun Wu ◽  
Zixin Deng ◽  
T. Mark Zabriskie ◽  
Xinyi He
Keyword(s):  
Gene Cluster ◽  
Genetic Manipulation ◽  
Biosynthetic Gene Cluster ◽  
Streptomyces Lividans ◽  
Dna Uptake ◽  
Biosynthetic Gene ◽  
Content Type ◽  
Blasticidin S

ABSTRACTBlasticidin S is a peptidyl nucleoside antibiotic produced byStreptomyces griseochromogenesthat exhibits strong fungicidal activity. To circumvent an effective DNA uptake barrier system in the native producer and investigate its biosynthesisin vivo, the blasticidin S biosynthetic gene cluster (bls) was engrafted to the chromosome ofStreptomyces lividans. However, the resulting mutant, LL2, produced the inactive deaminohydroxyblasticidin S instead of blasticidin S. Subsequently, a blasticidin S deaminase (SLBSD, forS. lividansblasticidin S deaminase) was identified inS. lividansand shown to govern thisin vivoconversion. Purified SLBSD was found to be capable of transforming blasticidin S to deaminohydroxyblasticidin Sin vitro. It also catalyzed deamination of the cytosine moiety of cytosylglucuronic acid, an intermediate in blasticidin S biosynthesis. Disruption of the SLBSD gene inS. lividansLL2 led to successful production of active blasticidin S in the resultant mutant,S. lividansWJ2. To demonstrate the easy manipulation of the blasticidin S biosynthetic gene cluster,blsE,blsF, andblsL, encoding a predicted radicalS-adenosylmethionine (SAM) protein, an unknown protein, and a guanidino methyltransferase, were individually inactivated to access their role in blasticidin S biosynthesis.

scholarly journals The expansive library of jadomycins

2018 ◽  
Vol 96 (6) ◽  
pp. 495-501 ◽  
Author(s):  
Jeanna M. MacLeod ◽  
Stephanie M. Forget ◽  
David L. Jakeman
Keyword(s):  
Amino Acids ◽  
Natural Products ◽  
Amino Acid ◽  
Heterologous Expression ◽  
Gene Cluster ◽  
Gene Deletion ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Synthetic Derivatives ◽  
Subsequent Identification

The jadomycin family of natural products was discovered from Streptomyces venezuelae ISP5230 in the 1990s. Subsequent identification of the biosynthetic gene cluster along with synthetic efforts established that incorporation of an amino acid into the polyaromatic angucycline core occurs non-enzymatically. Over two decades, the precursor-directed biosynthetic potential of the jadomycins has been heavily exploited, generating a library exceeding 70 compounds. This review compiles the jadomycins that have been isolated and characterized to date; these include jadomycins incorporating proteinogenic and non-proteinogenic amino acids, semi-synthetic derivatives, biosynthetic shunt products, compounds isolated in structural gene deletion studies, and deoxysugar sugar variant jadomycins produced by deletion or heterologous expression of sugar biosynthetic genes.

Oxytocin analogues with non-coded amino acid residues in position 8: [8-Neopentylglycine]oxytocin and [8-cycloleucine]oxytocin

1987 ◽  
Vol 52 (9) ◽  
pp. 2317-2325 ◽  
Author(s):  
Jan Hlaváček ◽  
Jan Pospíšek ◽  
Jiřina Slaninová ◽  
Walter Y. Chan ◽  
Victor J. Hruby
Keyword(s):  
Amino Acid ◽  
Solid Phase Synthesis ◽  
Solid Phase ◽  
The Other ◽  
Amino Acid Residues ◽  
Phase Synthesis ◽  
Other Hand ◽  
Fragment Condensation

[8-Neopentylglycine]oxytocin (II) and [8-cycloleucine]oxytocin (III) were prepared by a combination of solid-phase synthesis and fragment condensation. Both analogues exhibited decreased uterotonic potency in vitro, each being about 15-30% that of oxytocin. Analogue II also displayed similarly decreased uterotonic potency in vivo and galactogogic potency. On the other hand, analogue III exhibited almost the same potency as oxytocin in the uterotonic assay in vivo and in the galactogogic assay.

Synthesis of Fragments of the Vasoactive Intestinal Peptide (VIP) and Analysis of Their Residual Biological Activities

1995 ◽  
Vol 60 (7) ◽  
pp. 1229-1235 ◽  
Author(s):  
Ivana Zoulíková ◽  
Ivan Svoboda ◽  
Jiří Velek ◽  
Václav Kašička ◽  
Jiřina Slaninová ◽  
...  
Keyword(s):  
Amino Acid ◽  
Biological Activities ◽  
Residual Activity ◽  
Detailed Examination ◽  
Amino Acid Residues ◽  
Linear Peptide ◽  
Zone Electrophoresis ◽  
Capillary Zone

The vasoactive intestinal (poly)peptide (VIP) is a linear peptide containing 28 amino acid residues, whose primary structure indicates a low metabolic stability. The following VIP fragments, as potential metabolites, and their analogues were prepared by synthesis on a solid: [His(Dnp)1]VIP(1-10), VIP(11-14), [D-Arg12]VIP(11-14), [Lys(Pac)15,21,Arg20]VIP(15-22), and VIP(23-28). After purification, the peptides were characterized by amino acid analysis, mass spectrometry, RP HPLC, and capillary zone electrophoresis. In some tests, detailed examination of the biological activity of the substances in vivo and in vitro gave evidence of a low, residual activity of some fragments, viz. a depressoric activity in vivo for [His(Dnp)1]VIP(1-10) and a stimulating activity for the release of α-amylase in vitro and in vivo for [Lys(Pac)15,21,Arg20]VIP(15-22) and VIP(23-28).

Sign in / Sign up

Export Citation Format

Share Document