Recent advances in the structural analysis of adenylation domains in natural product biosynthesis

Miyanaga, Akimasa; Kudo, Fumitaka; Eguchi, Tadashi

doi:10.1016/j.cbpa.2022.102212

Cited by 18 publications

(14 citation statements)

References 47 publications

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“…Mutations were targeted to an A‐domain of the NRPS. A‐domains act as “gatekeepers” and primarily dictate the sequence of peptide products [38–42] . Two positions in the binding pocket of the GrsB1 A‐domain (Figure 3A) were simultaneously mutated using degenerate NNK codons (Table S1).…”

Section: Resultsmentioning

confidence: 99%

“…A-domains act as "gatekeepers" and primarily dictate the sequence of peptide products. [38][39][40][41][42] Two positions in the binding pocket of the GrsB1 A-domain (Figure 3A) were simultaneously mutated using degenerate NNK codons (Table S1). We positions I729 and A731 (Table S2) because these are located in direct vicinity of the substrate and thus part of the "specificity code" [39,40] (Figure 3B, Table S3) but not fully conserved.…”

Section: Screening a Library Of Megasynthetase Mutantsmentioning

confidence: 99%

See 1 more Smart Citation

Analysing Megasynthetase Mutants at High Throughput Using Droplet Microfluidics**

Pourmasoumi,

Hengoju,

Beck

et al. 2023

ChemBioChem

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Nonribosomal peptide synthetases (NRPSs) are giant enzymatic assembly lines that deliver many pharmaceutically valuable natural products, including antibiotics. As the search for new antibiotics motivates attempts to redesign nonribosomal metabolic pathways, more robust and rapid sorting and screening platforms are needed. Here, we establish a microfluidic platform that reliably detects production of the model nonribosomal peptide gramicidin S. The detection is based on calcein‐filled sensor liposomes yielding increased fluorescence upon permeabilization. From a library of NRPS mutants, the sorting platform enriches the gramicidin S producer 14.5‐fold, decreases internal stop codons 250‐fold, and generates enrichment factors correlating with enzyme activity. Screening for NRPS activity with a reliable non‐binary sensor will enable more sophisticated structure‐activity studies and new engineering applications in the future.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Screening a Library Of Megasynthetase Mutantsmentioning

confidence: 99%

Analysing Megasynthetase Mutants at High Throughput Using Droplet Microfluidics**

Pourmasoumi,

Hengoju,

Beck

et al. 2023

ChemBioChem

View full text Add to dashboard Cite

show abstract

“…This structure (PDB 6M01) showed a similar overall conformation to those seen previously and highlighted the largely conserved nature of the interactions of the adenylation and carrier domains. 127 Finally, several carboxylic acid reductase (CAR) enzymes have been used for structural characterization of adenylation-PCP complexes. 91 In one example, a CAR (PDB 5MSS) retained similar interactions as other didomains, but did not require the common adenosine vinylsulfonamide inhibitor to trap the complex into a thiolation state.…”

Section: Bmdbcmentioning

confidence: 99%

Structural advances toward understanding the catalytic activity and conformational dynamics of modular nonribosomal peptide synthetases

et al. 2023

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show abstract

“…A-domains act as "gatekeepers" and primarily dictate the sequence of peptide products. [33][34][35][36][37] Two positions in the binding pocket of the GrsB1 A-domain (Figure 3A) were simultaneously mutated using degenerate NNK codons (Table S1). We chose positions I729 and A731 (Table S2) because these are located in direct vicinity of the substrate and thus part of the "specificity code" [34,35] (Figure 3B, Table S3) but not too strictly conserved.…”

Section: Sorting For Gramicidin S Productionmentioning

confidence: 99%

Screening megasynthetase mutants at high throughput using droplet microfluidics

Pourmasoumi¹,

Hengoju

Beck

et al. 2023

Preprint

View full text Add to dashboard Cite

Nonribosomal peptide synthetases (NRPSs) are giant enzymatic assembly lines that deliver many pharmaceutically valuable natural products, including antibiotics. As the search for new antibiotics motivates attempts to redesign nonribosomal metabolic pathways, more robust and rapid sorting and screening platforms are needed. Here, we establish a microfluidic platform that reliably detects production of the model nonribosomal peptide gramicidin S. The detection is based on calcein-filled sensor liposomes yielding increased fluorescence upon permeabilization. From a library of NRPS mutants, the sorting platform enriches the gramicidin S producer 14.5-fold, decreases the number of stop codons 250-fold, and generates enrichment factors correlating with enzyme activity. Screening for NRPS activity with a reliable non-binary sensor will enable more sophisticated structure-activity studies and new engineering applications in the future.

show abstract

Recent advances in the structural analysis of adenylation domains in natural product biosynthesis

Cited by 18 publications

References 47 publications

Analysing Megasynthetase Mutants at High Throughput Using Droplet Microfluidics**

Analysing Megasynthetase Mutants at High Throughput Using Droplet Microfluidics**

Structural advances toward understanding the catalytic activity and conformational dynamics of modular nonribosomal peptide synthetases

Screening megasynthetase mutants at high throughput using droplet microfluidics

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