Structural, biochemical and bioinformatic analyses of nonribosomal peptide synthetase adenylation domains

Heard, Stephanie C.; Winter, Jaclyn M.

doi:10.1039/d3np00064h

Cited by 4 publications

(1 citation statement)

References 196 publications

(295 reference statements)

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“…Adenylation enzymes are responsible for the selective incorporation of aminoacyl building blocks in natural product biosynthesis. [1,2] The adenylation enzyme activates a specific aminoacyl substrate with ATP to form an aminoacyl adenylate intermediate and then transfers it onto the cognate carrier protein (CP). The generated aminoacyl-CP is used for condensa-tion reactions by nonribosomal peptide synthetases or polyketide synthases (PKSs).…”

Section: Introductionmentioning

confidence: 99%

Engineering the Substrate Specificity of (S)‐β‐Phenylalanine Adenylation Enzyme HitB

Wang,

Miyanaga,

Chisuga

et al. 2024

ChemBioChem

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Adenylation enzymes catalyze the selective incorporation of aminoacyl building blocks in the biosynthesis of nonribosomal peptides and related natural products. Although β‐amino acid units are one of the important aminoacyl building blocks in natural product biosynthesis, very little is known about the engineering of β‐amino acid adenylation enzymes. In this study, we engineered the substrate specificity of the (S)‐β‐phenylalanine adenylation enzyme, HitB, involved in the biosynthesis of macrolactam polyketide hitachimycin. Based on the previously determined structure of HitB wild‐type, we mutated Phe328 and Ser293, which are located near the meta and ortho position of the (S)‐β‐phenylalanine moiety, respectively. As a result, the HitB F328V and F328L mutants efficiently activated meta‐substituted (S)‐β‐phenylalanine analogs, and the HitB T293G and T293S mutants efficiently activated ortho‐substituted (S)‐β‐phenylalanine analogs. Structural analysis of the HitB F328L and T293G mutants with the corresponding nonhydrolyzable intermediate analogs revealed an enlarged substrate binding pocket for (S)‐β‐phenylalanine analogs, providing detailed insights into the structural basis for creating enzyme substrate promiscuity. Our findings may be useful for production of various β‐amino acid‐containing natural product analogs.

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Section: Introductionmentioning

confidence: 99%

Engineering the Substrate Specificity of (S)‐β‐Phenylalanine Adenylation Enzyme HitB

Wang,

Miyanaga,

Chisuga

et al. 2024

ChemBioChem

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Altering glycopeptide antibiotic biosynthesis through mutasynthesis allows incorporation of fluorinated phenylglycine residues

Voitsekhovskaia,

Ho,

Klatt

et al. 2024

RSC Chem. Biol.

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Expanding the substrate selectivity of the fimsbactin biosynthetic adenylation domain, FbsH

Ahmed,

Balutowski,

Yang

et al. 2024

Preprint

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Nonribosomal peptide synthetases (NRPSs) produce diverse natural products including siderophores, chelating agents that many pathogenic bacteria produce to survive in low iron conditions. Engineering NRPSs to produce diverse siderophore analogs could lead to the generation of novel antibiotics and imaging agents that take advantage of this unique iron uptake system in bacteria. The highly pathogenic and antibiotic-resistant bacteria Acinetobacter baumannii produces fimsbactin, an unusual branched siderophore with iron-binding catechol groups bound to a serine or threonine side chain. To explore the substrate promiscuity of the assembly line enzymes, we report a structure-guided investigation of the stand-alone aryl adenylation enzyme FbsH. We report on structures bound to its native substrate 2,3-dihydroxybenzoic acid (DHB) as well as an inhibitor that mimics the adenylate intermediate. We produced enzyme variants with an expanded binding pocket that are more tolerant for analogs containing a DHB C4 modification. Wild-type and mutant enzymes were then used in an in vitro reconstitution analysis to assess the production of analogs of the final product as well as several early-stage intermediates. This analysis shows that some altered substrates progress down the fimsbactin assembly line to the downstream domains. However, analogs from alternate building blocks are produced at lower levels, indicating that selectivity exists in the downstream catalytic domains. These findings expand the substrate scope of producing condensation products between serine and aryl acids and identify the bottlenecks for chemoenzymatic production of fimsbactin analogs.

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Structural, biochemical and bioinformatic analyses of nonribosomal peptide synthetase adenylation domains

Abstract: This review highlights the utility of using adenylation domain structural data, biochemical assays, and computational predictions for prioritizing nonribosomal peptide pathways for natural product discovery.

Cited by 4 publications

References 196 publications

Engineering the Substrate Specificity of (S)‐β‐Phenylalanine Adenylation Enzyme HitB

Engineering the Substrate Specificity of (S)‐β‐Phenylalanine Adenylation Enzyme HitB

Altering glycopeptide antibiotic biosynthesis through mutasynthesis allows incorporation of fluorinated phenylglycine residues

Expanding the substrate selectivity of the fimsbactin biosynthetic adenylation domain, FbsH

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