Characterization of Enzymes Catalyzing the Formation of the Nonproteinogenic Amino Acid l-Dap in Capreomycin Biosynthesis

Biochemistry. 2021 Jan 12;60(1):77-84. doi: 10.1021/acs.biochem.0c00808. Epub 2020 Dec 23.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aminobutyrates / metabolism*
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / metabolism*
  • Binding Sites
  • Capreomycin / biosynthesis*
  • Catalysis
  • Crystallography, X-Ray
  • Hydrolysis
  • Models, Molecular
  • Streptomyces / enzymology*
  • Substrate Specificity

Substances

  • Aminobutyrates
  • Bacterial Proteins
  • Capreomycin
  • 2,4-diaminobutyric acid