Characterization and Structural Determination of CmnG‐A, the Adenylation Domain That Activates the Nonproteinogenic Amino Acid Capreomycidine in Capreomycin Biosynthesis

Chen, I-Hsuan; Cheng, Tao; Wang, Yung-Lin; Huang, Szu-Jo; Hsiao, Yu-Hsuan; Lai, Yi-Ting; Toh, Shu-Ing; Chu, John; Rudolf, Jeffrey D.; Chang, Chin-Yuan

doi:10.1002/cbic.202200563

Cited by 7 publications

(2 citation statements)

References 42 publications

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“…Two isomeric forms currently exist wherein the guanidine moiety is cyclized at either the γ- or β-position of the arginine backbone, creating either the 5-membered enduracididine or 6-membered capreomycidine scaffolds, respectively. Once constructed, enduracididine and capreomycidine ncAAs can be directly incorporated into nonribosomal peptide synthetase (NRPS) products via specific adenylation domain activation, 7 or further transformed by oxidative enzymes 8,9 into other cyclic guanidine-containing natural products. Recent advances have shown that capreomycidines can be divergently biosynthesized on NRPSs via dehydrogenation and thioester-mediated Michael addition reactions in the faulknamycin and muraymycin biosynthetic pathways.…”

Section: Introductionmentioning

confidence: 99%

Mechanistic and structural insights into a divergent PLP-dependent L-enduracididine cyclase from a toxic cyanobacterium

Cordoza¹,

Chen

Blaustein³

et al. 2023

Preprint

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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 cyanobacterial 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 site-specific 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.

show abstract

Section: Introductionmentioning

confidence: 99%

Mechanistic and structural insights into a divergent PLP-dependent L-enduracididine cyclase from a toxic cyanobacterium

Cordoza¹,

Chen

Blaustein³

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…Two isomeric forms currently exist wherein the guanidine moiety is cyclized at either the γ- or β-position of the arginine backbone, creating either the five-membered enduracididine or six-membered capreomycidine scaffolds, respectively. Once constructed, enduracididine and capreomycidine ncAAs can be directly incorporated into nonribosomal peptide synthetase (NRPS) products via specific adenylation domain activation or further transformed by oxidative enzymes , into other cyclic guanidine-containing natural products. Recent advances have shown that capreomycidines can be divergently biosynthesized on NRPSs via dehydrogenation and thioester-mediated Michael addition reactions in the faulknamycin and muraymycin biosynthetic pathways. , …”

Section: Introductionmentioning

confidence: 99%

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

et al. 2023

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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 cyanobacterial 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 spectroscopy, stable isotope labeling techniques, and X-ray crystallography structure-guided site-directed mutagenesis. GntC initially facilitates the reversible deprotonations of the α- and β-positions of its substrate before catalyzing an irreversible diastereoselective dehydration and subsequent intramolecular cyclization. The comparison of holo- and substrate-bound GntC structures and activity assays on site-specific mutants further identified amino acid residues that contribute to the overall catalytic mechanism. These interdisciplinary efforts at structurally and functionally characterizing GntC enable an improved understanding of how nature divergently produces cyclic arginine ncAAs and generate additional tools for their biocatalytic production and downstream biological applications.

show abstract

Biosynthesis and recruitment of reactive amino acids in nonribosomal peptide assembly lines

Ehinger,

Hertweck

2024

Current Opinion in Chemical Biology

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Characterization and Structural Determination of CmnG‐A, the Adenylation Domain That Activates the Nonproteinogenic Amino Acid Capreomycidine in Capreomycin Biosynthesis

Cited by 7 publications

References 42 publications

Mechanistic and structural insights into a divergent PLP-dependent L-enduracididine cyclase from a toxic cyanobacterium

Mechanistic and structural insights into a divergent PLP-dependent L-enduracididine cyclase from a toxic cyanobacterium

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

Biosynthesis and recruitment of reactive amino acids in nonribosomal peptide assembly lines

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