A Defined and Flexible Pocket Explains Aryl Substrate Promiscuity of the Cahuitamycin Starter Unit–Activating Enzyme CahJ

Tripathi, Ashootosh; Park, Sung Ryeol; Sikkema, Andrew P.; Cho, Hyo Je; Wu, Jianfeng; Lee, Brian; Xi, Chuanwu; Smith, Janet L.; Sherman, David H.

doi:10.1002/cbic.201800233

Cited by 18 publications

(22 citation statements)

References 20 publications

(24 reference statements)

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“…[26][27][28] Aryl acids frequently exhibit substrate promiscuity toward other structurally related aryl acids. [29] Recently, we described an engineered catalyst that is capable of activating non-native aryl acids with nitro, cyano, bromo, and iodo functionalities. [8] More recently, we have reported a detailed understanding of the NRPS code residues of aryl acid A-domains by grafting the NRPS codes of salicylic acid (Sal)-activating A-domains into the substrate binding pocket of EntE.…”

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confidence: 99%

Probing the Compatibility of an Enzyme‐Linked Immunosorbent Assay toward the Reprogramming of Nonribosomal Peptide Synthetase Adenylation Domains

et al. 2020

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An important challenge in natural product biosynthesis is the biosynthetic design and production of artificial peptides. One of the most promising strategies is reprogramming adenylation (A) domains to expand the substrate repertoire of nonribosomal peptide synthetases (NRPSs). Therefore, the precise detection of subtle structural changes in the substrate binding pockets of A domains might accelerate their reprogramming. Here we show that an enzyme-linked immunosorbent assay (ELISA) using a combination of small-molecule probes can detect the effects of substrate binding pocket residue substitutions in A-domains. When coupled with a set of aryl acid A-domain variants (total of nine variants), the ELISA can analyze the subtle differences in their active-site architectures. Furthermore, the ELISA-based screening was able to identify the variants with substrate binding pockets that accepted a non-cognate substrate from an original pool of 45. These studies demonstrate that ELISA is a reliable platform for providing insights into the active-site properties of A-domains and can be applied for the reprogramming of NRPS A-domains.

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confidence: 99%

Probing the Compatibility of an Enzyme‐Linked Immunosorbent Assay toward the Reprogramming of Nonribosomal Peptide Synthetase Adenylation Domains

et al. 2020

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“…The substrate specificity of an NRPS‐like protein that can activate 5‐methyl orsellinic acid has been engineered toward Ant with a 26‐fold improvement in enzyme specificity through bioinformatics analysis and mutagenesis . Aryl acid A‐domains frequently display the ability to catalyze adenylation toward a range of structurally related aryl acids . The substrate promiscuity would be afforded by a common benzoic acid (BA) recognition moiety.…”

Section: Methodsmentioning

confidence: 99%

An Engineered Aryl Acid Adenylation Domain with an Enlarged Substrate Binding Pocket

et al. 2019

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Adenylation (A) domains act as the gatekeepers of non‐ribosomal peptide synthetases (NRPSs), ensuring the activation and thioesterification of the correct amino acid/aryl acid building blocks. Aryl acid building blocks are most commonly observed in iron‐chelating siderophores, but are not limited to them. Very little is known about the reprogramming of aryl acid A‐domains. We show that a single asparagine‐to‐glycine mutation in an aryl acid A‐domain leads to an enzyme that tolerates a wide range of non‐native aryl acids. The engineered catalyst is capable of activating non‐native aryl acids functionalized with nitro, cyano, bromo, and iodo groups, even though no enzymatic activity of wild‐type enzyme was observed toward these substrates. Co‐crystal structures with non‐hydrolysable aryl‐AMP analogues revealed the origins of this expansion of substrate promiscuity, highlighting an enlargement of the substrate binding pocket of the enzyme. Our findings may be exploited to produce diversified aryl acid containing natural products and serve as a template for further directed evolution in combinatorial biosynthesis.

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“…Uncyclized shunt product cahuitamycin B (2) lacks the phenolate oxazoline and has no bioactivity. By recognizing that cahuitamycin C (3) is derived from an off-cluster starting material, the authors were able to engineer mutants deficient in starter-unit biosynthesis to generate mutasynthetic analogs via feeding experiments, resulting in cahuitamycins D (4), F 11 (6), and E (5), which lacks the piperazate moiety.…”

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Total Synthesis of the Reported Structure of Cahuitamycin A

Shapiro¹,

Post²,

Wuest

2020

Preprint

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In a 2016 screen of natural product extracts a new family of natural products, the cahuitamycins, was discovered and found to inhibit the formation of biofilms in the human pathogen <i>Acinetobacter baumannii</i>. The molecules contain an unusual piperazate residue that raises structure/function and biosynthesis questions and resemble iron-trafficking virulence factors from <i>A. baumannii</i>, suggesting a connection between metal homeostasis and biofilm-mediated pathogenicity. Here we disclose the first total synthesis of the reported structure of cahuitamycin A in a twelve-step longest linear sequence and 18% overall yield. Comparison of spectral data of the authentic natural product and synthetic target compound demonstrate that the reported structure is distinct from the isolated metabolite. Herein, we propose an alternative structure to reconcile our findings with the isolation report, setting the stage for future synthetic and biochemical investigations of an important class of natural products.

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A Defined and Flexible Pocket Explains Aryl Substrate Promiscuity of the Cahuitamycin Starter Unit–Activating Enzyme CahJ

Cited by 18 publications

References 20 publications

Probing the Compatibility of an Enzyme‐Linked Immunosorbent Assay toward the Reprogramming of Nonribosomal Peptide Synthetase Adenylation Domains

Probing the Compatibility of an Enzyme‐Linked Immunosorbent Assay toward the Reprogramming of Nonribosomal Peptide Synthetase Adenylation Domains

An Engineered Aryl Acid Adenylation Domain with an Enlarged Substrate Binding Pocket

Total Synthesis of the Reported Structure of Cahuitamycin A

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