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

Sheng-Hsin Hsu, Shouqi Zhang, Sheng Cih Huang, Tung-Kung Wu, Zhengren Xu, Chin-Yuan Chang*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

Capreomycin (CMN) and viomycin (V10) are non-ribosomal peptide antituberculosis antibiotics, the structures of which contain four nonproteinogenic amino acids, including L-2,3-diaminopropionic acid (L-Dap), beta-ureidodehydroalanine, L-capreomycidine, and beta-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 alpha-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.

Original languageEnglish
Pages (from-to)77-84
Number of pages8
JournalBiochemistry
Volume60
Issue number1
DOIs
StatePublished - 12 Jan 2021

Keywords

  • VIOMYCIN BIOSYNTHESIS
  • CRYSTAL-STRUCTURE
  • STRUCTURAL BASIS
  • GENE-CLUSTER
  • L-SERINE
  • VIOC
  • ZWITTERMICIN
  • RESISTANCE
  • OXYGENASE
  • FEATURES

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