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Cited by 35Microbial Natural Products and BiosynthesisChemical Synthesis and AnalysisBiochemical and Structural Characterization

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

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Heard, Stephanie C., Winter, Jaclyn M.·2024·Natural Product Reports
DOI 10.1039/d3np00064hPMC PMC11253843

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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.

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Figures

Fig. 1 Overview of gramicidin S biosynthesis. (A) Structure of gramicidin S. (B) NRPS-encoding genes from the grs cluster found in the genome of Brevibacillus brevis.177–179 (C) Domain organization of the core enzymes GrsA and GrsB, which use l-amino acids to build gramicidin S.179 A = Adenylation, T = thiolation, C = condensation, E = epimerization, TE = thioesterase. *The loading of l-Phe by module 1 is drawn, and it should be noted that the E domain catalyzes epimerization of this residue to d-Phe before it is transferred to l-Pro.
Fig. 1 Overview of gramicidin S biosynthesis. (A) Structure of gramicidin S. (B) NRPS-encoding genes from the grs cluster found in the genome of Brevibacillus brevis.177–179 (C) Domain organization of the core enzymes GrsA and GrsB, which use l-amino acids to build gramicidin S.179 A = Adenylation, T = thiolation, C = condensation, E = epimerization, TE = thioesterase. *The loading of l-Phe by module 1 is drawn, and it should be noted that the E domain catalyzes epimerization of this residue to d-Phe before it is transferred to l-Pro.CC BY · PubMed Central
Fig. 2 The catalytic cycle of an adenylation reaction. (A) An overview of the general two-step adenylation/thioesterification reaction. (B) The conformational changes that occur between the Acore and Asub regions of an adenylation domain upon interaction with its substrates and a neighboring T domain. Adapted from ref. 26 and 27. ATP = Adenosine triphosphate, AMP = adenosine monophosphate, PPi = inorganic phosphate, AA = acyl substrate, AA-AMP = acyl-adenosine monophosphate intermediate, Ppant = phosphopantetheine, O = open, A = adenylation, I = intermediate, and T = thiolation.
Fig. 2 The catalytic cycle of an adenylation reaction. (A) An overview of the general two-step adenylation/thioesterification reaction. (B) The conformational changes that occur between the Acore and Asub regions of an adenylation domain upon interaction with its substrates and a neighboring T domain. Adapted from ref. 26 and 27. ATP = Adenosine triphosphate, AMP = adenosine monophosphate, PPi = inorganic phosphate, AA = acyl substrate, AA-AMP = acyl-adenosine monophosphate intermediate, Ppant = phosphopantetheine, O = open, A = adenylation, I = intermediate, and T = thiolation.CC BY · PubMed Central
Fig. 3 Domain alternation of the Acore and Asub regions observed in AlphaFold2 models of GrsA_A. (A) The closed/adenylation conformation and (B) open/thiolation conformation. The Acore subdomain is colored light gray whereas the Asub subdomain is colored dark gray. Motifs A1–A7 are found in Acore, while Asub contains motifs A8–A10. The motifs are colored coded as A1: teal, A2: purple, A3: sky blue, A4: sand, A5: pale green, A6: dark blue, A7: dark brown, A8: yellow, A9: violet, and A10: aquamarine. Lysine residues 517 and 434 are shown in stick form and the hinge region is highlighted in red.
Fig. 3 Domain alternation of the Acore and Asub regions observed in AlphaFold2 models of GrsA_A. (A) The closed/adenylation conformation and (B) open/thiolation conformation. The Acore subdomain is colored light gray whereas the Asub subdomain is colored dark gray. Motifs A1–A7 are found in Acore, while Asub contains motifs A8–A10. The motifs are colored coded as A1: teal, A2: purple, A3: sky blue, A4: sand, A5: pale green, A6: dark blue, A7: dark brown, A8: yellow, A9: violet, and A10: aquamarine. Lysine residues 517 and 434 are shown in stick form and the hinge region is highlighted in red.CC BY · PubMed Central
Fig. 4 Structures of various proteinogenic and nonproteinogenic building blocks identified in the NRPS pathways whose structures are listed in Table 1. Boxed in red are l-α-amino acids, modified l-α-amino acids are boxed in pink, the d-amino acid is in cyan, β-amino acids are boxed in blue, aryl acids are shown in green, and the α-hydroxy acid and α-keto acid are boxed in orange and yellow, respectively. Structures not in boxes do not belong to any specific group.
Fig. 4 Structures of various proteinogenic and nonproteinogenic building blocks identified in the NRPS pathways whose structures are listed in Table 1. Boxed in red are l-α-amino acids, modified l-α-amino acids are boxed in pink, the d-amino acid is in cyan, β-amino acids are boxed in blue, aryl acids are shown in green, and the α-hydroxy acid and α-keto acid are boxed in orange and yellow, respectively. Structures not in boxes do not belong to any specific group.CC BY · PubMed Central
Fig. 5 Chemical mimics of AMP used for structure determination of A domains. (A) The structure of AMP. (B) A generic 5′-O-sulfamoyl adenosine (AMS) probe. (C) A generic 5′-(vinylsulfonylamino) adenosine (AVS) probe.
Fig. 5 Chemical mimics of AMP used for structure determination of A domains. (A) The structure of AMP. (B) A generic 5′-O-sulfamoyl adenosine (AMS) probe. (C) A generic 5′-(vinylsulfonylamino) adenosine (AVS) probe.CC BY · PubMed Central
Fig. 6 Binding pockets of l-amino acid-activating adenylation domains. Residues involved in substrate interactions are shown for (A) l-α-Phe in GrsA_A (PDB 1AMU); (B) Gly-AMP in AlmE (PDB 4OXI); (C) Thr-AMP in Thr1 (PDB 5N9X); (D) α-me-l-Ser-AMP in FmoA3 (PDB 6LTB); (E) Cys-AMP in PchE (PDB 7EMY); and (F) Lys-AMP in Pls_A (PDB 7WEW). Active site residues are shown as sticks whereas bound substrates are highlighted in yellow. Polar contacts are shown as black dashes.
Fig. 6 Binding pockets of l-amino acid-activating adenylation domains. Residues involved in substrate interactions are shown for (A) l-α-Phe in GrsA_A (PDB 1AMU); (B) Gly-AMP in AlmE (PDB 4OXI); (C) Thr-AMP in Thr1 (PDB 5N9X); (D) α-me-l-Ser-AMP in FmoA3 (PDB 6LTB); (E) Cys-AMP in PchE (PDB 7EMY); and (F) Lys-AMP in Pls_A (PDB 7WEW). Active site residues are shown as sticks whereas bound substrates are highlighted in yellow. Polar contacts are shown as black dashes.CC BY · PubMed Central
Fig. 7 d-Amino acid-activating adenylation domains. (A) Residues interacting with d-Ala-AMP in DltA from Bacillus cereus (PDB 3DHV). (B) Residues interacting with ATP in DltA from Staphylococcus aureus (PDB 7VHV). Active site residues are shown as sticks whereas bound substrates are highlighted in yellow. Polar contacts are shown as black dashes.
Fig. 7 d-Amino acid-activating adenylation domains. (A) Residues interacting with d-Ala-AMP in DltA from Bacillus cereus (PDB 3DHV). (B) Residues interacting with ATP in DltA from Staphylococcus aureus (PDB 7VHV). Active site residues are shown as sticks whereas bound substrates are highlighted in yellow. Polar contacts are shown as black dashes.CC BY · PubMed Central
Fig. 8 β-Amino acid-activating adenylation domain. Residues interacting with β-me-l-Asp in the active site of VinN are highlighted (PDB 3WV5). Active site residues are shown as sticks whereas β-me-l-Asp is highlighted in yellow. Polar contacts are shown as black dashes.
Fig. 8 β-Amino acid-activating adenylation domain. Residues interacting with β-me-l-Asp in the active site of VinN are highlighted (PDB 3WV5). Active site residues are shown as sticks whereas β-me-l-Asp is highlighted in yellow. Polar contacts are shown as black dashes.CC BY · PubMed Central
Fig. 9 α-Hydroxy acid-activating adenylation domain. Residues interacting with AMP in the active site of the engineered TycA_A variant are highlighted (PDB 7YWK). Active site residues are shown as sticks whereas AMP is highlighted in yellow. Polar contacts are shown as black dashes.
Fig. 9 α-Hydroxy acid-activating adenylation domain. Residues interacting with AMP in the active site of the engineered TycA_A variant are highlighted (PDB 7YWK). Active site residues are shown as sticks whereas AMP is highlighted in yellow. Polar contacts are shown as black dashes.CC BY · PubMed Central
Fig. 10 α-Keto acid-activating adenylation domain. Residues interacting with α-ketoisocaproic acid-AMP (α-Kic-AMP) in the active site of StsA_A are highlighted (PDB 6ULX). Active site residues are shown as sticks whereas α-Kic-AMP is highlighted in yellow. Polar contacts are shown as black dashes.
Fig. 10 α-Keto acid-activating adenylation domain. Residues interacting with α-ketoisocaproic acid-AMP (α-Kic-AMP) in the active site of StsA_A are highlighted (PDB 6ULX). Active site residues are shown as sticks whereas α-Kic-AMP is highlighted in yellow. Polar contacts are shown as black dashes.CC BY · PubMed Central
Fig. 11 Structures of aryl acid-activating adenylation domains. Residues involved in substrate interactions are shown for (A) 2,3-dihydroxybenzoic acid and AMP in DhbE (PDB 1MD9); (B) salicylate and AMP in CahJ (PDB 5WM2); (C) anthraniloyl-AMP in AuaEII (PDB 4WV3) and (D) anthraniloyl-AMP in PqsA (PDB 5OE3). Active site residues are shown as sticks whereas substrates are highlighted in yellow. Polar contacts are shown as black dashes.
Fig. 11 Structures of aryl acid-activating adenylation domains. Residues involved in substrate interactions are shown for (A) 2,3-dihydroxybenzoic acid and AMP in DhbE (PDB 1MD9); (B) salicylate and AMP in CahJ (PDB 5WM2); (C) anthraniloyl-AMP in AuaEII (PDB 4WV3) and (D) anthraniloyl-AMP in PqsA (PDB 5OE3). Active site residues are shown as sticks whereas substrates are highlighted in yellow. Polar contacts are shown as black dashes.CC BY · PubMed Central
Fig. 12 Adenylation domains that activate unique building blocks. (A) Residues lining the binding pocket in SidN (PDB 3ITE). (B) Residues interacting with (2S,3R)-β-hydroxy-p-nitro-homophenylalanine (hnh) in ObiF1 (PDB 6N8E). Active site residues are shown as sticks whereas hnh is highlighted in yellow. Polar contacts are shown as black dashes.
Fig. 12 Adenylation domains that activate unique building blocks. (A) Residues lining the binding pocket in SidN (PDB 3ITE). (B) Residues interacting with (2S,3R)-β-hydroxy-p-nitro-homophenylalanine (hnh) in ObiF1 (PDB 6N8E). Active site residues are shown as sticks whereas hnh is highlighted in yellow. Polar contacts are shown as black dashes.CC BY · PubMed Central
Fig. 13 Structures of teicoplanin and teicoplanin-like adenylation domains. Residues involved in substrate interactions are shown for (A) l-Hpg in Tcp9_A1core-tei (PDB 8GIC); and (B) l-leucine in the mutated Tcp9_A1 variant Tcp9A1core-ANC3 (PDB 8GKM). Active site residues are shown as sticks whereas substrates are highlighted in yellow. Polar contacts are shown as black dashes and the water molecule coordinating residue H237 and E201 in panel A is designated as a red sphere.
Fig. 13 Structures of teicoplanin and teicoplanin-like adenylation domains. Residues involved in substrate interactions are shown for (A) l-Hpg in Tcp9_A1core-tei (PDB 8GIC); and (B) l-leucine in the mutated Tcp9_A1 variant Tcp9A1core-ANC3 (PDB 8GKM). Active site residues are shown as sticks whereas substrates are highlighted in yellow. Polar contacts are shown as black dashes and the water molecule coordinating residue H237 and E201 in panel A is designated as a red sphere.CC BY · PubMed Central
Fig. 14 In vitro methods for interrogating adenylation domain substrate specificity. Solid arrows represent the adenylation reaction, and dashed arrows represent the various detection methods branching from it. Blue text indicates methods that require instrumentational analysis, while black text indicates biochemical methods. Colorimetric and fluorometric assays are color-coded based on the wavelength of light they measure. ATP = Adenosine triphosphate, ADP = adenosine diphosphate, AMP = adenosine monophosphate, PPi = inorganic pyrophosphate, Pi = inorganic phosphate, CAS = Chrome Azurol S, NADH = nicotinamide adenine dinucleotide reduced, NAD+ = nicotinamide adenine dinucleotide, l-Gln = l-glutamine, F6P = fructose-6-phosphate, PPi-PFK = PPi-dependent phosphofructokinase, F1,6P = fructose-1,6-bisphosphate, GAP = glyceraldehyde-3-phosphate, DHAP = dihydroxyacetone phosphate, TPI = triose phosphate isomerase, GDH = glycerol-3-phosphate dehydrogenase, UDP = uridine diphosphate, UT = glucose-1-phosphate uridylyltransferase, UTP = uridine triphosphate, G1P = glucose-1-phosphate, PGM = phosphoglucomutase, G6P = glucose-6-phosphate, G6PD = glucose-6-phosphate dehydrogenase, 6PGL = 6-phosphono-d-glucono-1,5-lactone, ADK = adenosine kinase, PEP = phosphoenolpyruvate, PK = pyruvate kinase, LDH = lactate dehydrogenase, HPLC = high performance liquid chromatography, LC-MSx = liquid chromatography tandem mass spectrometry.
Fig. 14 In vitro methods for interrogating adenylation domain substrate specificity. Solid arrows represent the adenylation reaction, and dashed arrows represent the various detection methods branching from it. Blue text indicates methods that require instrumentational analysis, while black text indicates biochemical methods. Colorimetric and fluorometric assays are color-coded based on the wavelength of light they measure. ATP = Adenosine triphosphate, ADP = adenosine diphosphate, AMP = adenosine monophosphate, PPi = inorganic pyrophosphate, Pi = inorganic phosphate, CAS = Chrome Azurol S, NADH = nicotinamide adenine dinucleotide reduced, NAD+ = nicotinamide adenine dinucleotide, l-Gln = l-glutamine, F6P = fructose-6-phosphate, PPi-PFK = PPi-dependent phosphofructokinase, F1,6P = fructose-1,6-bisphosphate, GAP = glyceraldehyde-3-phosphate, DHAP = dihydroxyacetone phosphate, TPI = triose phosphate isomerase, GDH = glycerol-3-phosphate dehydrogenase, UDP = uridine diphosphate, UT = glucose-1-phosphate uridylyltransferase, UTP = uridine triphosphate, G1P = glucose-1-phosphate, PGM = phosphoglucomutase, G6P = glucose-6-phosphate, G6PD = glucose-6-phosphate dehydrogenase, 6PGL = 6-phosphono-d-glucono-1,5-lactone, ADK = adenosine kinase, PEP = phosphoenolpyruvate, PK = pyruvate kinase, LDH = lactate dehydrogenase, HPLC = high performance liquid chromatography, LC-MSx = liquid chromatography tandem mass spectrometry.CC BY · PubMed Central
Stephanie C. Heard
Stephanie C. HeardCC BY · PubMed Central
Jaclyn M. Winter
Jaclyn M. WinterCC BY · PubMed Central

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Cites

Show all 199 references

Research works printed but not linked: 5

  1. Ishikawa, 2019, Curr. Top. Microbiol. Immunol., 420, 321
  2. N.Kadi and G. L.Challis , Methods in Enzymology , 2009 , vol. 458 , pp. 431–457
  3. D. V.Santi , R. W.Webster and W. W.Cleland , Methods in Enzymology , 1974 , vol. 29 , pp. 620–627
  4. Pfleiderer, 1960, Biochem. Z., 333, 302
  5. Kautsar, 2020, Nucleic Acids Res., 48, D454

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Metadata

Authors
Heard, S. C., & Winter, J. M.
Affiliations
University of Utah (US)
Corresponding author
  • Heard, Stephanie C.
  • Winter, Jaclyn M.
ORCID
Venue
Natural Product Reports
Publisher
Royal Society of Chemistry (RSC)
Published
2024
Volume
41
Issue
7
Pages
1180-1205
ISSN
  • 0265-0568 (ISSN-L)
  • 1460-4752
DOI
10.1039/d3np00064h
PMC
PMC11253843
OpenAlex
W4392872704
Cited by
35
References
199
Citations by year
2026: 15 · 2025: 15 · 2024: 5
FWCI
2.18
Type
journal article
Language
en
Open access
Licence
Locations
  • Natural Product Reports (publishedVersion, cc-by)
  • ChemRxiv (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
National Institutes of Health (1R01AI155694)
Topics
  • Life Sciences › Immunology and Microbiology › Applied Microbiology and Biotechnology › Microbial Natural Products and Biosynthesis
  • Physical Sciences › Chemistry › Organic Chemistry › Chemical Synthesis and Analysis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Structural Characterization
Keywords
nonribosomal peptide synthetase, adenylation domain, natural product discovery, protein structure, biochemical tests, in silico prediction, biosynthetic gene clusters
MeSH terms
Biological Products, Peptide Synthases, Molecular Structure, Computational Biology

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Heard, S. C., & Winter, J. M. (2024). Structural, biochemical and bioinformatic analyses of nonribosomal peptide synthetase adenylation domains. Natural Product Reports. https://doi.org/10.1039/d3np00064h

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