scholarly journals In vivo and in vitro reconstitution of unique key steps in cystobactamid antibiotic biosynthesis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sebastian Groß ◽  
Bastien Schnell ◽  
Patrick A. Haack ◽  
David Auerbach ◽  
Rolf Müller
Keyword(s):  
Biosynthetic Gene Cluster ◽  
Antibiotic Biosynthesis ◽  
Gene Deletions ◽  
Aminobenzoic Acids ◽  
In Vitro Reconstitution ◽  
Peptide Synthetase ◽  
Nrps Module ◽  
Key Steps

AbstractCystobactamids are myxobacteria-derived topoisomerase inhibitors with potent anti-Gram-negative activity. They are formed by a non-ribosomal peptide synthetase (NRPS) and consist of tailored para-aminobenzoic acids, connected by a unique α-methoxy-l-isoasparagine or a β-methoxy-l-asparagine linker moiety. We describe the heterologous expression of the cystobactamid biosynthetic gene cluster (BGC) in Myxococcus xanthus. Targeted gene deletions produce several unnatural cystobactamids. Using in vitro experiments, we reconstitute the key biosynthetic steps of linker formation and shuttling via CysB to the NRPS. The biosynthetic logic involves a previously uncharacterized bifunctional domain found in the stand-alone NRPS module CysH, albicidin biosynthesis and numerous BGCs of unknown natural products. This domain performs either an aminomutase (AM) or an amide dehydratase (DH) type of reaction, depending on the activity of CysJ which hydroxylates CysH-bound l-asparagine. Furthermore, CysQ O-methylates hydroxyl-l-(iso)asparagine only in the presence of the AMDH domain. Taken together, these findings provide direct evidence for unique steps in cystobactamid biosynthesis.

scholarly journals Author Correction: In vivo and in vitro reconstitution of unique key steps in cystobactamid antibiotic biosynthesis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sebastian Groß ◽  
Bastien Schnell ◽  
Patrick A. Haack ◽  
David Auerbach ◽  
Rolf Müller
Keyword(s):  
Antibiotic Biosynthesis ◽  
In Vitro Reconstitution ◽  
Key Steps

scholarly journals Pentaminomycins C–E: Cyclic Pentapeptides as Autophagy Inducers from a Mealworm Beetle Gut Bacterium

2020 ◽  
Vol 8 (9) ◽  
pp. 1390 ◽  
Author(s):  
Sunghoon Hwang ◽  
Ly Thi Huong Luu Le ◽  
Shin-Il Jo ◽  
Jongheon Shin ◽  
Min Jae Lee ◽  
...  
Keyword(s):  
Amino Acid ◽  
2D Nmr ◽  
Biosynthetic Gene Cluster ◽  
Amino Acid Residues ◽  
Structural Variations ◽  
Nonribosomal Peptide ◽  
Peptide Synthetase ◽  
Nrps Module ◽  
Mealworm Beetle

Pentaminomycins C–E (1–3) were isolated from the culture of the Streptomyces sp. GG23 strain from the guts of the mealworm beetle, Tenebrio molitor. The structures of the pentaminomycins were determined to be cyclic pentapeptides containing a modified amino acid, N5-hydroxyarginine, based on 1D and 2D NMR and mass spectroscopic analyses. The absolute configurations of the amino acid residues were assigned using Marfey’s method and bioinformatics analysis of their nonribosomal peptide biosynthetic gene cluster (BGC). Detailed analysis of the BGC enabled us to propose that the structural variations in 1–3 originate from the low specificity of the adenylation domain in the nonribosomal peptide synthetase (NRPS) module 1, and indicate that macrocyclization can be catalyzed noncanonically by penicillin binding protein (PBP)-type TE. Furthermore, pentaminomycins C and D (1 and 2) showed significant autophagy-inducing activities and were cytoprotective against oxidative stress in vitro.

scholarly journals Biosynthesis of isonitrile lipopeptides by conserved nonribosomal peptide synthetase gene clusters in Actinobacteria

2017 ◽  
Vol 114 (27) ◽  
pp. 7025-7030 ◽  
Author(s):  
Nicholas C. Harris ◽  
Michio Sato ◽  
Nicolaus A. Herman ◽  
Frederick Twigg ◽  
Wenlong Cai ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Acyl Chain ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Nonribosomal Peptide Synthetase ◽  
Biosynthetic Gene ◽  
Nonribosomal Peptide ◽  
Peptide Synthetase

A putative lipopeptide biosynthetic gene cluster is conserved in many species of Actinobacteria, including Mycobacterium tuberculosis and M. marinum, but the specific function of the encoding proteins has been elusive. Using both in vivo heterologous reconstitution and in vitro biochemical analyses, we have revealed that the five encoding biosynthetic enzymes are capable of synthesizing a family of isonitrile lipopeptides (INLPs) through a thio-template mechanism. The biosynthesis features the generation of isonitrile from a single precursor Gly promoted by a thioesterase and a nonheme iron(II)-dependent oxidase homolog and the acylation of both amino groups of Lys by the same isonitrile acyl chain facilitated by a single condensation domain of a nonribosomal peptide synthetase. In addition, the deletion of INLP biosynthetic genes in M. marinum has decreased the intracellular metal concentration, suggesting the role of this biosynthetic gene cluster in metal transport.

Identification of the immunophilins capable of mediating inhibition of signal transduction by cyclosporin A and FK506: roles of calcineurin binding and cellular location

1993 ◽  
Vol 13 (8) ◽  
pp. 4760-4769
Author(s):  
R J Bram ◽  
D T Hung ◽  
P K Martin ◽  
S L Schreiber ◽  
G R Crabtree
Keyword(s):  
Signal Transduction ◽  
T Cell ◽  
Cyclosporin A ◽  
Cell Function ◽  
Inhibitory Effects ◽  
Gene Deletions ◽  
Cyclophilin B ◽  
Intracellular Vesicles

The immunosuppressants cyclosporin A (CsA) and FK506 appear to block T-cell function by inhibiting the calcium-regulated phosphatase calcineurin. While multiple distinct intracellular receptors for these drugs (cyclophilins and FKBPs, collectively immunophilins) have been characterized, the functionally active ones have not been discerned. We found that overexpression of cyclophilin A or B or FKBP12 increased T-cell sensitivity to CsA or FK506, respectively, demonstrating that they are able to mediate the inhibitory effects of their respective immunosuppressants in vivo. In contrast, cyclophilin C, FKBP13, and FKBP25 had no effect. Direct comparison of the Ki of each drug-immunophilin complex for calcineurin in vitro revealed that although calcineurin binding was clearly necessary, it was not sufficient to explain the in vivo activity of the immunophilin. Subcellular localization was shown also to play a role, since gene deletions of cyclophilins B and C which changed their intracellular locations altered their activities significantly. Cyclophilin B has been shown previously to be located within calcium-containing intracellular vesicles; its ability to mediate CsA inhibition implies that certain components of the signal transduction machinery are also spatially restricted within the cell.

scholarly journals Intermediate step of cohesin’s ATPase cycle allows cohesin to entrap DNA

2018 ◽  
Vol 115 (39) ◽  
pp. 9732-9737 ◽  
Author(s):  
Gamze Ö. Çamdere ◽  
Kristian K. Carlborg ◽  
Douglas Koshland
Keyword(s):  
Atp Hydrolysis ◽  
Intermediate Step ◽  
Mutant Form ◽  
In Vitro Reconstitution ◽  
Chromatid Cohesion ◽  
The Family ◽  
Structural Maintenance Of Chromosomes ◽  
Dna Substrate

Cohesin is a four-subunit ATPase in the family of structural maintenance of chromosomes (SMC). Cohesin promotes sister chromatid cohesion, chromosome condensation, DNA repair, and transcription regulation. Cohesin performs these functions as a DNA tether and potentially a DNA-based motor. At least one of its DNA binding activities involves entrapment of DNA within a lumen formed by its subunits. This activity can be reconstituted in vitro by incubating cohesin with DNA, ATP, and cohesin loader. Previously we showed that a mutant form of cohesin (DE-cohesin) possesses the ability to bind and tether DNA in vivo. Using in vitro reconstitution assays, we show that DE-cohesin can form stable complexes with DNA without ATP hydrolysis. We show that wild-type cohesin with ADP aluminum fluoride (cohesinADP/AlFx) can also form stable cohesin–DNA complexes. These results suggest that an intermediate nucleotide state of cohesin, likely cohesinADP-Pi, is capable of initially dissociating one interface between cohesin subunits to allow DNA entry into a cohesin lumen and subsequently interacting with the bound DNA to stabilize DNA entrapment. We also show that cohesinADP/AlFx binding to DNA is enhanced by cohesin loader, suggesting a function for loader other than stimulating the ATPase. Finally, we show that loader remains stably bound to cohesinADP/AlFx after DNA entrapment, potentially revealing a function for loader in tethering the second DNA substrate. These results provide important clues on how SMC complexes like cohesin can function as both DNA tethers and motors.

scholarly journals Regulation of peripheral lipogenesis by glucagon. Inability of the hormone to inhibit lipogenesis in rat mammary acini in vitro in the presence or absence of agents which alter its effects on adipocytes

1984 ◽  
Vol 217 (3) ◽  
pp. 743-749 ◽  
Author(s):  
N A Robson ◽  
R A Clegg ◽  
V A Zammit
Keyword(s):  
Concentration Ratio ◽  
Response Curve ◽  
Potent Inhibitor ◽  
Specific Binding ◽  
Insulin Dose ◽  
Mammary Glands ◽  
Key Steps ◽  
Glucagon Concentration

The rate of lipogenesis in acini isolated from mammary glands of mid-lactating rats was studied by measuring the rate of incorporation of 3H from 3H2O into total lipid and fatty acids, with glucose as substrate. Glucagon did not affect the rate of lipogenesis in acini. Glucagon did not antagonize the maximal stimulatory effect of insulin, nor did it alter the insulin dose-response curve. Theophylline, at concentrations up to 20 mM, was a potent inhibitor of lipogenesis in acini. Glucagon did not augment the degree of inhibition of lipogenesis induced by 5 mM-theophylline. The results suggest that mammary-gland acini do not respond to glucagon in vitro under conditions in which the hormone induces inhibition of lipogenesis (the present paper) and of individual key steps in the lipogenic pathway in adipocytes [Zammit & Corstorphine (1982) Biochem. J. 208, 783-788; Green (1983) Biochem. J. 212, 189-195]. In agreement with these observations, we could detect only a minimal degree of specific binding of 125I-labelled glucagon to acini which bound insulin normally. This difference in responsiveness of mammary and adipose cell preparations in vitro to glucagon suggests that the two tissues may be differentially responsive to changes in the circulating insulin/glucagon concentration ratio in vivo. The significance of these findings for the regulation of substrate utilization for lipogenesis in the two tissues during lactation is discussed.

scholarly journals Crystallizing the 6S and 8S spliceosomal assembly intermediates: a complex project

2015 ◽  
Vol 71 (10) ◽  
pp. 2040-2053 ◽  
Author(s):  
Jann-Patrick Pelz ◽  
Hermann Schindelin ◽  
Katharina van Pee ◽  
Jochen Kuper ◽  
Caroline Kisker ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Protein Complexes ◽  
Sm Proteins ◽  
Entropy Reduction ◽  
Multimeric Protein ◽  
Complex Project ◽  
Key Steps ◽  
Small Nuclear Ribonucleoproteins

The small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/6 and U5 are major constituents of the pre-mRNA processing spliceosome. They contain a common RNP core that is formed by the ordered binding of Sm proteins onto the single-stranded Sm site of the snRNA. Although spontaneousin vitro, assembly of the Sm core requires assistance from the PRMT5 and SMN complexesin vivo. To gain insight into the key steps of the assembly process, the crystal structures of two assembly intermediates of U snRNPs termed the 6S and 8S complexes have recently been reported. These multimeric protein complexes could only be crystallized after the application of various rescue strategies. The developed strategy leading to the crystallization and solution of the 8S crystal structure was subsequently used to guide a combination of rational crystal-contact optimization with surface-entropy reduction of crystals of the related 6S complex. Conversely, the resulting high-resolution 6S crystal structure was used during the restrained refinement of the 8S crystal structure.

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