condensation domain
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2021 ◽  
Author(s):  
Janik Kranz ◽  
Sebastian L. Wenski ◽  
Alexnder A. Dichter ◽  
Helge B. Bode ◽  
Kenan A. J. Bozhueyuek

Many clinically used natural products are produced by non-ribosomal peptide synthetases (NRPSs), which due to their modular nature should be accessible to modification and engineering approaches. While the adenylation domain (A) plays the key role in substrate recognition and activation, the condensation domain (C) which is responsible for substrate linkage and stereochemical filtering recently became the subject of debate - with its attributed role as a "gatekeeper" being called into question. Since we have thoroughly investigated different combinations of C-A didomains in a series of in vitro, in vivo, and in situ experiments suggesting an important role to the C-A interface for the activity and specificity of the downstream A domain and not the C domain as such, we would like to contribute to this discussion. The role of the C-A interface, termed 'extended gatekeeping', due to structural features of the C domains, can also be transferred to other NRPSs by engineering, was finally investigated and characterised in an in silico approach on 30 wild-type and recombinant C-A interfaces. With these data, we not only would like to offer a new perspective on the specificity of C domains, but also to revise our previously established NRPS engineering and construction rules.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Thierry Izoré ◽  
Y. T. Candace Ho ◽  
Joe A. Kaczmarski ◽  
Athina Gavriilidou ◽  
Ka Ho Chow ◽  
...  

AbstractNon-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 that controls access of acceptor substrates to the active site in condensation domains. The structures of this 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.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Lin Zhong ◽  
Xiaotong Diao ◽  
Na Zhang ◽  
Fengwei Li ◽  
Haibo Zhou ◽  
...  

AbstractNonribosomal peptide synthetases containing starter condensation domains direct the biosynthesis of nonribosomal lipopeptides, which generally exhibit wide bioactivities. The acyl chain has strong impacts on bioactivity and toxicity, but the lack of an in-depth understanding of starter condensation domain-mediated lipoinitiation limits the bioengineering of NRPSs to obtain novel derivatives with desired acyl chains. Here, we show that the acyl chains of the lipopeptides rhizomide, holrhizin, and glidobactin were modified by engineering the starter condensation domain, suggesting a workable approach to change the acyl chain. Based on the structure of the mutated starter condensation domain of rhizomide biosynthetic enzyme RzmA in complex with octanoyl-CoA and related point mutation experiments, we identify a set of residues responsible for the selectivity of substrate acyl chains and extend the acyl chains from acetyl to palmitoyl. Furthermore, we illustrate three possible conformational states of starter condensation domains during the reaction cycle of the lipoinitiation process. Our studies provide further insights into the mechanism of lipoinitiation and the engineering of nonribosomal peptide synthetases.


2021 ◽  
Author(s):  
Max Cryle ◽  
Thierry Izore ◽  
Y. T. Ho ◽  
Joe Kaczmarski ◽  
Athina Gavriilidou ◽  
...  

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.


Toxins ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 349
Author(s):  
Mario Alberto Martínez-Núñez ◽  
Zuemy Rodríguez-Escamilla

Prokaryotes represent a source of both biotechnological and pharmaceutical molecules of importance, such as nonribosomal peptides (NRPs). NRPs are secondary metabolites which their synthesis is independent of ribosomes. Traditionally, obtaining NRPs had focused on organisms from terrestrial environments, but in recent years marine and coastal environments have emerged as an important source for the search and obtaining of nonribosomal compounds. In this study, we carried out a metataxonomic analysis of sediment of the coast of Yucatan in order to evaluate the potential of the microbial communities to contain bacteria involved in the synthesis of NRPs in two sites: one contaminated and the other conserved. As well as a metatranscriptomic analysis to discover nonribosomal peptide synthetases (NRPSs) genes. We found that the phyla with the highest representation of NRPs producing organisms were the Proteobacteria and Firmicutes present in the sediments of the conserved site. Similarly, the metatranscriptomic analysis showed that 52% of the sequences identified as catalytic domains of NRPSs were found in the conserved site sample, mostly (82%) belonging to Proteobacteria and Firmicutes; while the representation of Actinobacteria traditionally described as the major producers of secondary metabolites was low. It is important to highlight the prediction of metabolic pathways for siderophores production, as well as the identification of NRPS’s condensation domain in organisms of the Archaea domain. Because this opens the possibility to the search for new nonribosomal structures in these organisms. This is the first mining study using high throughput sequencing technologies conducted in the sediments of the Yucatan coast to search for bacteria producing NRPs, and genes that encode NRPSs enzymes.


2019 ◽  
Vol 116 (40) ◽  
pp. 19805-19814 ◽  
Author(s):  
Zachary L. Reitz ◽  
Clifford D. Hardy ◽  
Jaewon Suk ◽  
Jean Bouvet ◽  
Alison Butler

Genome mining of biosynthetic pathways streamlines discovery of secondary metabolites but can leave ambiguities in the predicted structures, which must be rectified experimentally. Through coupling the reactivity predicted by biosynthetic gene clusters with verified structures, the origin of the β-hydroxyaspartic acid diastereomers in siderophores is reported herein. Two functional subtypes of nonheme Fe(II)/α-ketoglutarate–dependent aspartyl β-hydroxylases are identified in siderophore biosynthetic gene clusters, which differ in genomic organization—existing either as fused domains (IβHAsp) at the carboxyl terminus of a nonribosomal peptide synthetase (NRPS) or as stand-alone enzymes (TβHAsp)—and each directs opposite stereoselectivity of Asp β-hydroxylation. The predictive power of this subtype delineation is confirmed by the stereochemical characterization of β-OHAsp residues in pyoverdine GB-1, delftibactin, histicorrugatin, and cupriachelin. The l-threo (2S, 3S) β-OHAsp residues of alterobactin arise from hydroxylation by the β-hydroxylase domain integrated into NRPS AltH, while l-erythro (2S, 3R) β-OHAsp in delftibactin arises from the stand-alone β-hydroxylase DelD. Cupriachelin contains both l-threo and l-erythro β-OHAsp, consistent with the presence of both types of β-hydroxylases in the biosynthetic gene cluster. A third subtype of nonheme Fe(II)/α-ketoglutarate–dependent enzymes (IβHHis) hydroxylates histidyl residues with l-threo stereospecificity. A previously undescribed, noncanonical member of the NRPS condensation domain superfamily is identified, named the interface domain, which is proposed to position the β-hydroxylase and the NRPS-bound amino acid prior to hydroxylation. Through mapping characterized β-OHAsp diastereomers to the phylogenetic tree of siderophore β-hydroxylases, methods to predict β-OHAsp stereochemistry in silico are realized.


2019 ◽  
Vol 116 (8) ◽  
pp. 2913-2918 ◽  
Author(s):  
Thierry Izoré ◽  
Julien Tailhades ◽  
Mathias Henning Hansen ◽  
Joe A. Kaczmarski ◽  
Colin J. Jackson ◽  
...  

The protein Ebony from Drosophila melanogaster plays a central role in the regulation of histamine and dopamine in various tissues through condensation of these amines with β-alanine. Ebony is a rare example of a nonribosomal peptide synthetase (NRPS) from a higher eukaryote and contains a C-terminal sequence that does not correspond to any previously characterized NRPS domain. We have structurally characterized this C-terminal domain and have discovered that it adopts the aryl-alkylamine-N-acetyl transferase (AANAT) fold, which is unprecedented in NRPS biology. Through analysis of ligand-bound structures, activity assays, and binding measurements, we have determined how this atypical condensation domain is able to provide selectivity for both the carrier protein-bound amino acid and the amine substrates, a situation that remains unclear for standard condensation domains identified to date from NRPS assembly lines. These results demonstrate that the C terminus of Ebony encodes a eukaryotic example of an alternative type of NRPS condensation domain; they also illustrate how the catalytic components of such assembly lines are significantly more diverse than a minimal set of conserved functional domains.


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