Dataset Information

Understanding condensation domain selectivity in non-ribosomal peptide biosynthesis: structural characterization of the acceptor bound state

ABSTRACT: Non-ribosomal peptide synthetases are important enzymes for the assembly of complex peptide natural products. Within these multi-modular assembly lines, condensation domains perform the central function of chain assembly, typically by forming a peptide bond between two peptidyl carrier protein (PCP)-bound substrates. In this work, we report the first structural snapshots of a condensation domain in complex with an aminoacyl-PCP acceptor substrate. These structures allow the identification of a mechanism that controls access of acceptor substrates to the active site in condensation domains. The structures of this previously uncharacterized complex also allow us to demonstrate that condensation domain active sites do not contain a distinct pocket to select the side chain of the acceptor substrate during peptide assembly but that residues within the active site motif can instead serve to tune the selectivity of these central biosynthetic domains.

INSTRUMENT(S): micrOTOF II

ORGANISM(S): Escherichia Coli

SUBMITTER: david Steer

LAB HEAD: Max Cryle

PROVIDER: PXD024004 | Pride | 2021-05-28

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	1_cal_dil_pepMSMS_1000_8ul_1-D_4_01_19585.d.zip	Other
	2_cal_dil_pepMSMS_1000_8ul_1-D_5_01_19586.d.zip	Other
	3_cal_dil_pepMSMS_1000_8ul_1-D_6_01_19587.d.zip	Other
	DMOH1_MSMS_4ul_1-A_7_01_19925.d.zip	Other
	DMOH2_MSMS_4ul_1-A_8_01_19926.d.zip	Other

Items per page:

1 - 5 of 14

Publications

Structures of a non-ribosomal peptide synthetase condensation domain suggest the basis of substrate selectivity.

Izoré Thierry T Candace Ho Y T YT Kaczmarski Joe A JA Gavriilidou Athina A Chow Ka Ho KH Steer David L DL Goode Robert J A RJA Schittenhelm Ralf B RB Tailhades Julien J Tosin Manuela M Challis Gregory L GL Krenske Elizabeth H EH Ziemert Nadine N Jackson Colin J CJ Cryle Max J MJ

Nature communications 20210504 1

Non-ribosomal peptide synthetases are important enzymes for the assembly of complex peptide natural products. Within these multi-modular assembly lines, condensation domains perform the central function of chain assembly, typically by forming a peptide bond between two peptidyl carrier protein (PCP)-bound substrates. In this work, we report structural snapshots of a condensation domain in complex with an aminoacyl-PCP acceptor substrate. These structures allow the identification of a mechanism t ...[more]

PMID: 33947858

Similar Datasets

Project description:MUC5B is the predominant glycoprotein in saliva and is instrumental in the establishment and maintenance of multi-species eubiotic biofilms in the oral cavity. Investigations of the aciduric Lactobacillaceae family, and its role in biofilms emphasizes the diversity across different genera of the proteolytic systems involved in the nutritional utilization of mucins. We have characterized a cysteine protease from Limosilactobacillus fermentum, MdpL (Mucin degrading protease from Limosilactobacillus) with a high protein backbone similarity with commensals that exploit mucins for attachment and nutrition. MdpL was shown to be associated with the bacterial cell surface, in close proximity to MUC5B, which was sequentially degraded into low molecular weight fragments. Mapping the substrate preference revealed multiple hydrolytic sites of proteins with a high O-glycan occurrence, although hydrolysis was not dependent on the presence of O-glycans. However, since proteolysis of immunoglobulins was absent, and general protease activity was low, a preference for glycoproteins similar to MUC5B in terms of glycosylation and structure is suggested. MdpL preferentially hydrolyzed C-terminally located hydrophobic residues in peptides larger than 20 amino acids, which hinted at a limited sequence preference. To secure proper enzyme folding and optimal conditions for activity, L. fermentum incorporates a complex system that establishes a reducing environment. The importance of overall reducing conditions was confirmed by the activity boosting effect of the added reducing agents L-cysteine and DTT. High activity was retained in low to neutral pH 5.5-7.0, but the enzyme was completely inhibited in the presence of Zn2+. Here we have characterized a highly conserved mucin degrading protease from L. fermentum. MdpL, that together with the recently discovered O-glycanase and O-glycoprotease enzyme groups, increases our understanding of mucin degradation and complex biofilm dynamics.

Project description:Perturbations in the expression profiles of microRNAs (miRNAs) have been reported for a variety of different cancers. Basal cell carcinoma (BCC) of the skin has not been systematically evaluated regarding differentially expressed miRNAs. Patients with BCC (n=7) were included in the study. Punch biopsies were harvested from the center of the tumors (lesional, n=7) and from sites of adjacent non-lesional skin (intraindividual control, n=7). Microarray miRNA expression profiles were detected based on an Agilent platform and miRBase 16. For validation purposes the expression of a subset of dysregulated miRNAs were measured by means of TaqMan real-time reverse transcription polymerase chain reaction (RT-PCR). Seven patients with basal cell carcinoma (BCC) were enrolled in the present study. While BCCs were excised with cold steel under local anaesthesia, a 4-mm punch biopsy was taken from the center of the tumor and from adjacent non-lesional epithelial skin (control), immediately stored in RNAlater (Qiagen, HIlden, Germany) and stored at -80 M-BM-0C. miRNAs were isolated with miRNeasy Mini Kit (Quiagen, Hilden, Germany) according to the manufacturerM-bM-^@M-^Ys protocol. RNA concentration, purity and RNA integrity number (RIN) were determined with NanoDrop ND-1000 spectral photometer (Peqlab, Erlangen, Germany), Agilent 2100 Bioanalyzer and the RNA 6000 NanoLabCHip Kits (both Agilent Technologies, Santa Clara, USA). total RNA samples were spiked with the MicroRNA Spike-In Kit (Agilent Technologies, Santa Clara, USA) for assessment of labeling and hybridization efficiencies. After the spiked total RNA was treated with alkaline calf intestine phosphatase (CIP), a labeling reaction was started with 100 ng total-RNA per sample. T4 RNA ligase, incorporating Cyanine 3-Cytidine biphosphate (miRNA Complete Labeling and Hyb Kit, Agilent Technologies, Santa Clara, USA) was used to label the dephosphorylated RNA. Cyanine-3-labeled miRNA samples were subsequently prepared for One-Color based hybridization (Complete miRNA Labeling and Hyb Kit, Agilent Technologies, Santa Clara, USA). Hybridization of the labeled miRNA samples with Human miRNA Microarrays Release 16.0 (Agilent Technologies, Santa Clara, USA) was done at 55M-BM-0C for 20 hrs. After washing microarray slides with increasing stringency (Gene Expression Wash Buffers, Agilent Technologies, Santa Clara) they were dried with acetonitrile (Sigma-Aldich, St.Louis, USA). Agilent DNA Microarray Scanner (Agilent Technologies, Santa Clara, USA) was used detect fluorescent signal intensities. They were detected with the Scan Control A.8.4.1 Software (Agilent Technologies, Santa Clara, USA) and extracted from images by using the Feature Extraction 10.7.3.1 Software (Agilent Technologies, Santa Clara, USA). All the steps described were carried out according to the manufacturerM-BM-4s instructions.