1. Characterization of Enzymes Catalyzing the Formation of the Nonproteinogenic Amino Acid <scp>l</scp>-Dap in Capreomycin Biosynthesis
- Author
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Sheng-Hsin Hsu, Shouqi Zhang, Tung-Kung Wu, Chin-Yuan Chang, Sheng Cih Huang, and Zhengren Xu
- Subjects
Models, Molecular ,chemistry.chemical_classification ,Binding Sites ,Stereochemistry ,Aminobutyrates ,Hydrolysis ,Glutamic acid ,Crystallography, X-Ray ,Condensation reaction ,Biochemistry ,Catalysis ,Streptomyces ,Substrate Specificity ,Amino acid ,chemistry.chemical_compound ,Enzyme ,Bacterial Proteins ,chemistry ,Biosynthesis ,Viomycin ,Nonribosomal peptide ,medicine ,Capreomycin ,NAD+ kinase ,medicine.drug - Abstract
Capreomycin (CMN) and viomycin (VIO) are nonribosomal peptide antituberculosis antibiotics, the structures of which contain four nonproteinogenic amino acids, including l-2,3-diaminopropionic acid (l-Dap), β-ureidodehydroalanine, l-capreomycidine, and β-lysine. Previous bioinformatics analysis suggested that CmnB/VioB and CmnK/VioK participate in the formation of l-Dap; however, the real substrates of these enzymes are yet to be confirmed. We herein show that starting from O-phospho-l-Ser (OPS) and l-Glu precursors, CmnB catalyzes the condensation reaction to generate a metabolite intermediate N-(1-amino-1-carboxyl-2-ethyl)glutamic acid (ACEGA), which undergoes NAD+-dependent oxidative hydrolysis by CmnK to generate l-Dap. Furthermore, the binding site of ACEGA and the catalytic mechanism of CmnK were elucidated with the assistance of three crystal structures, including those of apo-CmnK, the NAD+-CmnK complex, and CmnK in an alternative conformation. The CmnK-ACEGA docking model revealed that the glutamate α-hydrogen points toward the nicotinamide moiety. It provides evidence that the reaction is dependent on hydride transfer to form an imine intermediate, which is subsequently hydrolyzed by a water molecule to produce l-Dap. These findings modify the original proposed pathway and provide insights into l-Dap formation in the biosynthesis of other related natural products.
- Published
- 2020