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Cited by 15Microbial Natural Products and BiosynthesisAmino Acid Enzymes and MetabolismEnzyme Structure and Function

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Mechanistic and Structural Insights into a Divergent PLP-Dependent l -Enduracididine Cyclase from a Toxic Cyanobacterium

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Cordoza, Jennifer L., Chen, Percival Yang-Ting, Blaustein, Linnea R., Lima, Stella T., Fiore, Marli F., Chekan, Jonathan R., Moore, Bradley S., McKinnie, Shaun M. K.·2023·ACS Catalysis
DOI 10.1021/acscatal.3c01294PMC PMC10367076

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Abstract

ABSTRACT Cyclic arginine noncanonical amino acids (ncAAs) are found in several actinobacterial peptide natural products with therapeutically useful antibacterial properties. The preparation of ncAAs like enduracididine and capreomycidine currently takes multiple biosynthetic or chemosynthetic steps, thus limiting the commercial availability and applicability of these cyclic guanidine-containing amino acids. We recently discovered and characterized the biosynthetic pathway of guanitoxin, a potent freshwater cya-nobacterial neurotoxin, that contains an arginine-derived cyclic guanidine phosphate within its highly polar structure. The ncAA L-enduracididine is an early intermediate in guanitoxin biosynthesis and is produced by GntC, a unique pyridoxal-5’-phosphate (PLP)-dependent enzyme. GntC catalyzes a cyclodehydration from a stereoselectively γ-hydroxylated L-arginine precursor via a reaction that functionally and mechanistically diverges from previously established actinobacterial cyclic arginine ncAA pathways. Herein, we interrogate L-enduracididine biosynthesis from the cyanobacterium Sphaerospermopsis torques-reginae ITEP-024 using spectroscopic, stable isotope labeling techniques, and X-ray crystal structure-guided site-directed mutagenesis. GntC initially facilitates the reversible deprotonations of the α- and β-positions of its substrate prior to catalyzing an irreversible diastereoselective dehydration and subsequent intramolecular cyclization. The comparison of holo- and substrate bound GntC structures and activity assays on sitespecific mutants further identified amino acid residues that contribute to the overall catalytic mechanism. These interdisciplinary efforts at structurally and functionally characterizing GntC enables an improved understanding of how Nature divergently produces cyclic arginine ncAAs and generates additional tools for their biocatalytic production and downstream biological applications.

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Figures

Figure 1 (A) Bioactive antimicrobial nonribosomal peptides containing enduracididine (red) or capreomycidine (blue) cyclic arginine noncanonical amino acids (ncAAs). (B) Characterized biosynthetic strategies toward the construction of cyclic arginine enduracididine (left) and capreomycidine (right) ncAAs. Guanitoxin biosynthesis in cyanobacterium S. torques-reginae ITEP-024 proceeds through cyclic arginine intermediate l-enduracididine (2) via the cyclodehydration activity of PLP-dependent enzyme GntC. Pyridoxal-5′-phosphate (PLP) enzymology involved in cyclic arginine biosynthesis from actinobacterial mannopeptimycin (MppP, MppQ), viomycin (VioD), and streptolidine (OrfR) pathways.
Figure 1 (A) Bioactive antimicrobial nonribosomal peptides containing enduracididine (red) or capreomycidine (blue) cyclic arginine noncanonical amino acids (ncAAs). (B) Characterized biosynthetic strategies toward the construction of cyclic arginine enduracididine (left) and capreomycidine (right) ncAAs. Guanitoxin biosynthesis in cyanobacterium S. torques-reginae ITEP-024 proceeds through cyclic arginine intermediate l-enduracididine (2) via the cyclodehydration activity of PLP-dependent enzyme GntC. Pyridoxal-5′-phosphate (PLP) enzymology involved in cyclic arginine biosynthesis from actinobacterial mannopeptimycin (MppP, MppQ), viomycin (VioD), and streptolidine (OrfR) pathways.CC BY · PubMed Central
Figure 2 In vitro GntC assays with (A) native substrate 1 and (B) diastereomer 1′ show divergent UV–visible absorption spectra at 325 and 420 nm, corresponding to the ketimine and aldimine intermediates, respectively. R′ represents either a conserved lysine residue (internal aldimine) or an amine substrate (external aldimine).
Figure 2 In vitro GntC assays with (A) native substrate 1 and (B) diastereomer 1′ show divergent UV–visible absorption spectra at 325 and 420 nm, corresponding to the ketimine and aldimine intermediates, respectively. R′ represents either a conserved lysine residue (internal aldimine) or an amine substrate (external aldimine).CC BY · PubMed Central
Figure 3 Selective deuteration reveals that GntC does not deprotonate the γ-position of substrate 1. (A) Synthesis of selectively γ-deuterated substrate 1-dγ. (B) UPLC-MS spectra for l-FDAA-derivatized substrates (1 and 1-dγ; left) and products (2 and 2-dγ; right) in the absence and presence of in vitro GntC. The increase of +1 Da in product 2-dγ supports that the γ-deuterium atom is retained throughout the GntC mechanism.
Figure 3 Selective deuteration reveals that GntC does not deprotonate the γ-position of substrate 1. (A) Synthesis of selectively γ-deuterated substrate 1-dγ. (B) UPLC-MS spectra for l-FDAA-derivatized substrates (1 and 1-dγ; left) and products (2 and 2-dγ; right) in the absence and presence of in vitro GntC. The increase of +1 Da in product 2-dγ supports that the γ-deuterium atom is retained throughout the GntC mechanism.CC BY · PubMed Central
Figure 4 UPLC-MS analyses of l-FDAA-derivatized in vitro GntC D2O assays identify the incorporation of up to three deuterium atoms in γ-hydroxy-l-arginine substrates. (A) Increase of +3 Da is observed in linear substrate 1 and cyclic product 2 (1-d3 and 2-d3, respectively) in the presence of D2O and GntC. (B) Substrate diastereomer 1′ also shows an increase of +3 Da (1′-d3) in the presence of D2O-buffered GntC despite any cyclic product formation.
Figure 4 UPLC-MS analyses of l-FDAA-derivatized in vitro GntC D2O assays identify the incorporation of up to three deuterium atoms in γ-hydroxy-l-arginine substrates. (A) Increase of +3 Da is observed in linear substrate 1 and cyclic product 2 (1-d3 and 2-d3, respectively) in the presence of D2O and GntC. (B) Substrate diastereomer 1′ also shows an increase of +3 Da (1′-d3) in the presence of D2O-buffered GntC despite any cyclic product formation.CC BY · PubMed Central
Figure 5 1H NMR time-course experiments under D2O conditions identify that both the α- and β-hydrogen atoms of substrate 1 and product 2 are solvent exchanged during in vitro GntC assays. The predominant chemical species are depicted to the left of GntC reaction time points and compared to 1 (top, red trace) and 2 (bottom, blue trace) standards.
Figure 5 1H NMR time-course experiments under D2O conditions identify that both the α- and β-hydrogen atoms of substrate 1 and product 2 are solvent exchanged during in vitro GntC assays. The predominant chemical species are depicted to the left of GntC reaction time points and compared to 1 (top, red trace) and 2 (bottom, blue trace) standards.CC BY · PubMed Central
Figure 6 GntC X-ray crystal structure and site-directed mutagenesis identify important amino acid residues for catalysis. (A) GntC crystal structure with bound PLP-1 substrate (yellow) crystallized as a dimer of homodimers at 2.04 Å resolution (PDB ID: 8FFU, only one homodimer depicted for simplicity). The active site is at the interface of the homodimer between the green and teal monomers, consistent with other PLP-dependent enzyme structures. (B) GntC active site with Fo-Fc electron density map for bound PLP-1 (blue mesh, σ = 1.0) imposed on the ligand (left) and amino acid residues proposed to participate in substrate recognition and catalysis (right), including E9, S25, T84, and K219 (green monomer), and N52* and H250* (teal monomer). (C) Simplified active site comparison of GntC (green/teal) and capreomycidine cyclase OrfR residues (gray, PDB ID: 4M2M) with bound PLP-1 (yellow) and PLP-l-arg (gray) substrates, respectively. (D) In vitro enzyme assays for 2 production with soluble GntC mutants in comparison to wild-type (WT) GntC.
Figure 6 GntC X-ray crystal structure and site-directed mutagenesis identify important amino acid residues for catalysis. (A) GntC crystal structure with bound PLP-1 substrate (yellow) crystallized as a dimer of homodimers at 2.04 Å resolution (PDB ID: 8FFU, only one homodimer depicted for simplicity). The active site is at the interface of the homodimer between the green and teal monomers, consistent with other PLP-dependent enzyme structures. (B) GntC active site with Fo-Fc electron density map for bound PLP-1 (blue mesh, σ = 1.0) imposed on the ligand (left) and amino acid residues proposed to participate in substrate recognition and catalysis (right), including E9, S25, T84, and K219 (green monomer), and N52* and H250* (teal monomer). (C) Simplified active site comparison of GntC (green/teal) and capreomycidine cyclase OrfR residues (gray, PDB ID: 4M2M) with bound PLP-1 (yellow) and PLP-l-arg (gray) substrates, respectively. (D) In vitro enzyme assays for 2 production with soluble GntC mutants in comparison to wild-type (WT) GntC.CC BY · PubMed Central
Scheme 1 Proposed GntC Mechanism Based on Cumulative Spectroscopic, Heavy Isotope Labeling, NMR, and X-ray Crystallography and Site-Directed Mutagenesis Results
Scheme 1 Proposed GntC Mechanism Based on Cumulative Spectroscopic, Heavy Isotope Labeling, NMR, and X-ray Crystallography and Site-Directed Mutagenesis ResultsCC BY · PubMed Central

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Metadata

Authors
Cordoza, J. L., Chen, P. Y.-T., Blaustein, L. R., Lima, S. T., Fiore, M. F., Chekan, J. R., Moore, B. S., & McKinnie, S. M. K.
Affiliations
  • University of California, Santa Cruz (US)
  • Scripps Institution of Oceanography (US)
  • Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California, La Jolla, California 92093, United States
  • Universidade de São Paulo (BR)
  • University of North Carolina at Greensboro (US)
Corresponding author
McKinnie, Shaun M. K.
ORCID
Venue
ACS Catalysis
Publisher
American Chemical Society (ACS)
Published
2023-07-11
Volume
13
Issue
14
Pages
9817-9828
ISSN
2155-5435 (ISSN-L)
DOI
10.1021/acscatal.3c01294
PMC
PMC10367076
OpenAlex
W4383875873
Cited by
15
References
1
Citations by year
2026: 5 · 2025: 4 · 2024: 6
FWCI
2.34
Type
journal article
Language
en
Open access
Licence
Locations
  • ACS Catalysis (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • eScholarship (California Digital Library) (submittedVersion, cc-by)
  • PubMed Central (submittedVersion, cc-by-nc-nd)
Funders
  • National Institute of Environmental Health Sciences (R21-ES032056)
  • Funda??o de Amparo ? Pesquisa do Estado de S?o Paulo (2017/06869-0)
  • University of California, Santa Cruz
  • National Institute of General Medical Sciences (R25-GM051765-18)
  • Simons Foundation
Topics
  • Life Sciences › Immunology and Microbiology › Applied Microbiology and Biotechnology › Microbial Natural Products and Biosynthesis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Amino Acid Enzymes and Metabolism
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Enzyme Structure and Function
Keywords
pyridoxal phosphate-dependent enzymes, cyanobacterial neurotoxins, intramolecular cyclization, catalytic mechanism, site-directed mutagenesis, crystal structure, stable isotope labeling, biocatalytic production

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