Biosynthetic Incorporation of Non-native Aryl Acid Building Blocks into Peptide Products Using Engineered Adenylation Domains DOI
Fumihiro Ishikawa,

Maya Nohara,

Akimasa Miyanaga

et al.

ACS Chemical Biology, Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 2, 2024

Nonribosomal peptides (NRPs), one of the most widespread secondary metabolites in nature, with therapeutically significant activities, are biosynthesized by modular nonribosomal peptide synthetases (NRPSs). Aryl acids contribute to structural diversity NRPs as well nonproteinogenic amino and keto acids. We previously confirmed that a single Asn-to-Gly substitution 2,3-dihydroxybenzoic acid-activating adenylation (A) domain EntE involved enterobactin biosynthesis accepts monosubstituted benzoic acid derivatives nitro, cyano, bromo, iodo functionalities at 2 or 3 positions. Here, we showed mutant (N235G) accommodates various disubstituted halogen, methyl, methoxy, cyano positions an alkyne position. Structural analysis nonhydrolyzable aryl-AMP analogues using 3-chloro-2-methylbenzoic 3-prop-2-ynoxybenzoic revealed how bulky clickable recognized enlarging substrate-binding pocket enzyme. When engineered mutants were coupled vibriobactin biosynthetic enzymes, 3-hydroxybenzoic acid-, salicylic 3-bromo-2-fluorobenzoic acid-containing produced early stage intermediates, highlighting potential NRP pathway engineering for constructing diverse aryl metabolites.

Language: Английский

Chemical Logic of Peptide Branching by Iterative Nonlinear Nonribosomal Peptide Synthetases DOI
Jinping Yang, Adam Balutowski, Chinmay Trivedi

et al.

Biochemistry, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 23, 2025

Branch-point syntheses in nonribosomal peptide assembly are rare but useful strategies to generate tripodal peptides with advantageous hexadentate iron-chelating capabilities, as seen siderophores. However, the chemical logic underlying branching by synthetase (NRPS) often remains complex and elusive. Here, we review common for biosynthesis of branched (NRPs) present our biochemical investigation on NRPS-catalyzed fimsbactin A, a mixed-ligand siderophore produced human pathogenic strain Acinetobacter baumannii. We untangled unusual mechanism A through combination bioinformatics, site-directed mutagenesis, vitro reconstitution, molecular modeling, dynamics simulation. Our findings clarify roles NRPS enzymes, uncovering catalytically redundant domains identifying multifunctional nature FbsF cyclization (Cy) domain. demonstrate dynamic interplay between l-serine 2,3-dihydroxybenzoic acid derived dipeptides, partitioning amide ester forms via 1,2-N-to-O-acyl shift orchestrated noncanonical, multichannel Cy The event occurs secondary condensation facilitated this domain two dipeptidyl intermediates, which generates tetrapeptide thioester. Finally, terminal FbsG recruits soluble nucleophile release final product. This study advances understanding intricate biosynthetic pathways employed NRPSs, shedding light mechanisms synthesis peptides.

Language: Английский

Citations

1
The Structural Basis of Substrate Selectivity of the Acinetobactin Biosynthetic Adenylation Domain, BasE DOI Creative Commons
Syed Fardin Ahmed, Andrew M. Gulick

Journal of Biological Chemistry, Journal Year: 2025, Volume and Issue: unknown, P. 108413 - 108413

Published: March 1, 2025

Language: Английский

Citations

1
l-2,3-Diaminopropionate Binding Mode of the SulM Adenylation Domain Limits Engineering Monobactam Analogue Biosynthesis with Larger Substrates DOI Creative Commons
Lukas Kahlert, Ketan D. Patel, Michael S. Lichstrahl

et al.

JACS Au, Journal Year: 2025, Volume and Issue: 5(4), P. 1992 - 2003

Published: April 16, 2025

The simple but essential azetidinone core of the β-lactam antibiotics is uniquely N-sulfonated in monobactam subfamily. This feature confers both target binding specificity to inactivate bacterial cell wall biosynthesis (antibiosis) and structural differentiation elude destruction by metallo-β-lactamases (MBLs). recent FDA approval Emblaveo treat serious infections combines an established synthetic aztreonam avibactam, which additionally blocks serine β-lactamases, create a broadly effective antibacterial therapeutic. Here we report experiments capture native biosynthetic steps natural product sulfazecin with aim accessing new monobactams reprogramming its machinery. In biosynthesis, ring formed nonribosomal peptide synthetase SulM that incorporates l-2,3-diaminopropionate (Dap), then trans efficiently cyclized fully elaborated unusual thioesterase (TE) domain. We describe improved synthesis (2S,3R)-vinylDap support rational structure-based engineering obtain corresponding (4R)-vinyl sulfazecin. While these were initially based on AlphaFold model adenylation domain Dap (SulM A3), further high-resolution X-ray crystal structures l-Dap substrate accurate analogue activated (3R)-methyl-Dap adenylate bound. ligand-bound rationalize inability SulA3 incorporate larger substrates. Comparisons other diamino acid-activating domains identify alternate modes may be more suitable for production analogues. impact relation recently structurally characterized SulTE are discussed.

Language: Английский

Citations

0
Fimsbactin Siderophores From a South African Marine Sponge Symbiont, Marinomonas sp. PE14‐40 DOI Creative Commons

Nompumelelo Philile Praiseworth Ikegwuoha,

Thea Hanekom,

Elzaan Booysen

et al.

Microbial Biotechnology, Journal Year: 2025, Volume and Issue: 18(5)

Published: May 1, 2025

ABSTRACT Low iron levels in marine habitats necessitate the production of structurally diverse siderophores by many bacterial species for acquisition. Siderophores exhibit bioactivities ranging from chelation reduction hemochromatosis sufferers to antimicrobial activity either their own right or when coupled known antibiotics targeted delivery molecular imaging. Thus, environments are a sought‐after resource novel that could have pharmaceutical industrial application. The fimsbactins A‐F ( 1–6 ) mixed catechol‐hydroxamate only been reported be produced Acinetobacter with fimsbactin biosynthetic gene clusters (BGCs) widespread among within this genus. Here, we identified putative BGC an uncharacterized isolate, Marinomonas sp. PE14‐40. Not was synteny not conserved comparing pathway PE14‐40 sp., but five core genes found canonical located elsewhere on genome and do form part cluster PE14‐40, four these, fbsBCDL, colocalized. Through ESI‐MS/MS analysis extracts analogues 1 6 were identified, as well two new analogues, 7 8 , containing previously unreported L‐lysine‐derived hydroxamate moiety, N ‐acetyl‐ ‐hydroxycadaverine. Feeding experiments using stable isotope‐label L‐lysine provided further evidence ‐hydroxycadaverine moiety . study demonstrates functional conservation seemingly disparate pathways enzyme promiscuity's role producing compounds.

Language: Английский

Citations

0
Biosynthetic Incorporation of Non-native Aryl Acid Building Blocks into Peptide Products Using Engineered Adenylation Domains DOI
Fumihiro Ishikawa,

Maya Nohara,

Akimasa Miyanaga

et al.

ACS Chemical Biology, Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 2, 2024

Nonribosomal peptides (NRPs), one of the most widespread secondary metabolites in nature, with therapeutically significant activities, are biosynthesized by modular nonribosomal peptide synthetases (NRPSs). Aryl acids contribute to structural diversity NRPs as well nonproteinogenic amino and keto acids. We previously confirmed that a single Asn-to-Gly substitution 2,3-dihydroxybenzoic acid-activating adenylation (A) domain EntE involved enterobactin biosynthesis accepts monosubstituted benzoic acid derivatives nitro, cyano, bromo, iodo functionalities at 2 or 3 positions. Here, we showed mutant (N235G) accommodates various disubstituted halogen, methyl, methoxy, cyano positions an alkyne position. Structural analysis nonhydrolyzable aryl-AMP analogues using 3-chloro-2-methylbenzoic 3-prop-2-ynoxybenzoic revealed how bulky clickable recognized enlarging substrate-binding pocket enzyme. When engineered mutants were coupled vibriobactin biosynthetic enzymes, 3-hydroxybenzoic acid-, salicylic 3-bromo-2-fluorobenzoic acid-containing produced early stage intermediates, highlighting potential NRP pathway engineering for constructing diverse aryl metabolites.

Language: Английский

Citations

2