Introduction of a Non‐Natural Amino Acid into a Nonribosomal Peptide Antibiotic by Modification of Adenylation Domain Specificity

Thirlway, Jenny; Lewis, Richard A.; Nunns, Laura; Nakeeb, Majid Al; Styles, Matthew Q.; Struck, Anna Winona; Smith, Colin P.; Micklefield, Jason

doi:10.1002/anie.201202043

Cited by 109 publications

(95 citation statements)

References 33 publications

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“…10 However, the former method characterizes only the first half-reaction catalyzed by an A domain (i.e., the acyl-adenylate formation reaction), but not the second half-reaction (i.e., the thioesterification reaction involving an ACP domain). Therefore, an apparent active substrate in this assay may form only an acyl adenylate intermediate that cannot be transferred to the ACP domain for subsequent reactions.…”

Section: Introductionmentioning

confidence: 99%

Characterization and Engineering of the Adenylation Domain of a NRPS-Like Protein: A Potential Biocatalyst for Aldehyde Generation

Wang

Zhao

2014

ACS Catal.

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The adenylation (A) domain acts as the first “gate-keeper” to ensure the activation and thioesterification of the correct monomer to nonribosomal peptide synthetases (NRPSs). Our understanding of the specificity-conferring code and our ability to engineer A domains are critical for increasing the chemical diversity of nonribosomal peptides (NRPs). We recently discovered a novel NRPS-like protein (ATEG_03630) that can activate 5-methyl orsellinic acid (5-MOA) and reduce it to 2,4-dihydroxy-5,6-dimethyl benzaldehyde. A NRPS-like protein is much smaller than multidomain NRPSs, but it still represents the thioesterification half-reaction, which is otherwise missed from a stand-alone A domain. Therefore, a NRPS-like protein may serve as a better model system for A domain engineering. Here, we characterize the substrate specificity of ATEG_03630 and conclude that the hydrogen-bond donor at the 4-position is crucial for substrate recognition. Next, we show that the substrate specificity of ATEG_03630 can be engineered toward our target substrate anthranilate via bioinformatics analysis and mutagenesis. The resultant mutant H358A increased its activity toward anthranilate by 10.9-fold, which led to a 26-fold improvement in specificity. Finally, we demonstrate one-pot chemoenzymatic synthesis of 4-hydroxybenzaldoxime from 4-hydroxybenzoic acid with high yield.

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Section: Introductionmentioning

confidence: 99%

Characterization and Engineering of the Adenylation Domain of a NRPS-Like Protein: A Potential Biocatalyst for Aldehyde Generation

Wang

Zhao

2014

ACS Catal.

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“…For instance, the specificity of the A domain of module 10 within the calcium-dependent antibiotic (CDA) NRPS was modified by the introduction of a single mutation (Lys278Gln), changing its specificity from (2 S ,3 R )-3-methyl Glu (mGlu)/Glu to (2 S ,3 R )-3-methyl Gln (mGln)/Gln to produce the Gln/mGln-containing CDA analogues ( 14 – 15 ) (Fig. 1) 24 . A similar point mutation in the Phe-specific A domain of the gramicidin synthetase GrsA (Trp239Ser) allowed for the introduction of an O -propargyl-Tyr residue 25 .…”

Section: Expanding Enzymatic Diversitymentioning

confidence: 99%

Reinvigorating natural product combinatorial biosynthesis with synthetic biology

2015

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Natural products continue to play a pivotal role in drug discovery efforts and in understanding human health. The ability to extend nature’s chemistry through combinatorial biosynthesis – altering functional groups, regiochemistry, and scaffold backbones through manipulation of biosynthetic enzymes – offers unique opportunities to create natural product analogues. Incorporating emerging synthetic biology techniques has the potential to further accelerate the refinement of combinatorial biosynthesis as a robust platform for the diversification of natural chemical drug leads. Two decades after the field originated, we discuss the current limitations, realities, and the state of the art of combinatorial biosynthesis, including the engineering of substrate specificity of biosynthetic enzymes and the development heterologous expression systems for biosynthetic pathways. We also propose a new perspective for the combinatorial biosynthesis of natural products that could reinvigorate drug discovery by using synthetic biology in combination with synthetic chemistry.

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“…NRPS enzymes seem to be more discerning of nonnative substrates than the ribosome for protein biosynthesis. While early work showed that downstream modules may block the elongation of peptide intermediates with the incorporation of nonnative substrates (Pavela-Vrancic et al, 1999; Uguru et al, 2004), more recent studies have successfully demonstrated the ability of reprogrammed A-domains to incorporate nonnative substrates into the final NRPs (Evans et al, 2011; Fischbach et al, 2007; Thirlway et al, 2012). …”

Section: Discussionmentioning

confidence: 99%

“…In an effort to alleviate these problems, Walsh and coworkers used directed evolution to improve the activity of chimeric assembly lines, which led to nearly 10-fold improvements in catalytic activity (Fischbach et al, 2007). A complimentary approach to reengineer A-domain specificity preserving the native protein-protein interactions within an NRPS module has been to use the nonribosomal peptide code and, more recently, computational structure-based redesign to rationally mutate residues important for substrate recognition (Chen et al, 2009; Eppelmann et al, 2002; Thirlway et al, 2012). In order to screen potential mutants more comprehensively, Kelleher and co-workers developed a novel workflow involving directed evolution to create a library of ~1,000 mutants based on the “nonribosomal peptide code” and mass spectrometry to measure the ability of the resulting mutants to support production of novel NRPs (Evans et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

Engineering the Substrate Specificity of the DhbE Adenylation Domain by Yeast Cell Surface Display

Zhang

Nelson

Bhuripanyo

et al. 2013

Chemistry & Biology

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SUMMARY The adenylation (A) domains of nonribosomal peptide synthetases (NRPSs) activate aryl acids or amino acids to launch their transfer through the NRPS assembly line for the biosynthesis of many medicinally important natural products. In order to expand the substrate pool of NRPSs, we developed a method based on yeast cell surface display to engineer the substrate specificities of the A-domains. We acquired A-domain mutants of DhbE that have 11- and 6-fold increases in kcat/Km with nonnative substrates 3-hydroxybenzoic acid and 2-aminobenzoic acid, respectively and corresponding 3- and 33-fold decreases in kcat/Km values with the native substrate 2,3-dihydroxybenzoic acid, resulting in a dramatic switch in substrate specificity of up to 200-fold. Our study demonstrates that yeast display can be used as a high throughput selection platform to reprogram the “nonribosomal code” of A-domains.

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Introduction of a Non‐Natural Amino Acid into a Nonribosomal Peptide Antibiotic by Modification of Adenylation Domain Specificity

Cited by 109 publications

References 33 publications

Characterization and Engineering of the Adenylation Domain of a NRPS-Like Protein: A Potential Biocatalyst for Aldehyde Generation

Characterization and Engineering of the Adenylation Domain of a NRPS-Like Protein: A Potential Biocatalyst for Aldehyde Generation

Reinvigorating natural product combinatorial biosynthesis with synthetic biology

Engineering the Substrate Specificity of the DhbE Adenylation Domain by Yeast Cell Surface Display

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