2019
DOI: 10.1021/acschembio.9b00532
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Directed Evolution Reveals the Functional Sequence Space of an Adenylation Domain Specificity Code

Abstract: Nonribosomal peptides are important natural products biosynthesized by nonribosomal peptide synthetases (NRPSs). Adenylation (A) domains of NRPSs are highly specific for the substrate they recognize. This recognition is determined by 10 residues in the substrate-binding pocket, termed the specificity code. This finding led to the proposal that nonribosomal peptides could be altered by specificity code swapping. Unfortunately, this approach has proven, with few exceptions, to be unproductive; changing the speci… Show more

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Cited by 23 publications
(23 citation statements)
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“…substrates [37]. Together, these studies show that although it remains to be seen whether the redesign rules inferred from separate NRPS systems can be applied more generally [38], novel high-throughput screens in conjunction with screening of a larger sequence space, might result in more substantial changes in peptide structure and improved variants [39].…”
Section: A Domain Re-engineeringmentioning
confidence: 88%
“…substrates [37]. Together, these studies show that although it remains to be seen whether the redesign rules inferred from separate NRPS systems can be applied more generally [38], novel high-throughput screens in conjunction with screening of a larger sequence space, might result in more substantial changes in peptide structure and improved variants [39].…”
Section: A Domain Re-engineeringmentioning
confidence: 88%
“…OA-specific AraC variants were predicted using the Iterative Protein Redesign and Optimization suite of programs -IPRO (Pantazes et al 2015); refer to Methods section). IPRO has been previously used to successfully tune substrate and cofactor specificities of several enzymes including bacterial thioestereases (HernĂĄndez Lozada et al 2018, Grisewood et al 2017 and non-ribosomal peptide synthases (Throckmorton et al 2019). The top five designs (OA/TAL interaction energy ratios) as identified by IPRO, named AraC-OA1 through AraC-OA5, are listed in Table S3.…”
Section: Design Of a Sensor With Enhanced Specificity Toward Orsellinmentioning
confidence: 99%
“…A salient feature of many nonribosomal adenylation domains is a high degree of substrate promiscuity which can serve as a springboard for natural enzyme evolution 22 and engineering in the laboratory. [23][24][25][26][27][28] Given the availability of vast numbers of structurally related amino acid substrates, adenylation enzymes are highly suitable for studying enzyme promiscuity. 29 However, specicity proles of adenylating enzymes must be measured one substrate at a time 24,27,30 which poorly reects the situation in a cell where numerous amino acids and carboxylic acids compete for the active site.…”
mentioning
confidence: 99%