Nonprocessive [2 + 2]e- off-loading reductase domains from mycobacterial nonribosomal peptide synthetases

Proc Natl Acad Sci U S A. 2012 Apr 10;109(15):5681-6. doi: 10.1073/pnas.1118680109. Epub 2012 Mar 26.


In mycobacteria, polyketide synthases and nonribosomal peptide synthetases (NRPSs) produce complex lipidic metabolites by using a thio-template mechanism of catalysis. In this study, we demonstrate that off-loading reductase (R) domain of mycobacterial NRPSs performs two consecutive [2 + 2]e(-) reductions to release thioester-bound lipopeptides as corresponding alcohols, using a nonprocessive mechanism of catalysis. The first crystal structure of an R domain from Mycobacterium tuberculosis NRPS provides strong support to this mechanistic model and suggests that the displacement of intermediate would be required for cofactor recycling. We show that 4e(-) reductases produce alcohols through a committed aldehyde intermediate, and the reduction of this intermediate is at least 10 times more efficient than the thioester-substrate. Structural and biochemical studies also provide evidence for the conformational changes associated with the reductive cycle. Further, we show that the large substrate-binding pocket with a hydrophobic platform accounts for the remarkable substrate promiscuity of these domains. Our studies present an elegant example of the recruitment of a canonical short-chain dehydrogenase/reductase family member as an off-loading domain in the context of assembly-line enzymology.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alcohols / metabolism
  • Binding Sites
  • Crystallography, X-Ray
  • Electrons*
  • Glycopeptides / chemistry
  • Glycopeptides / metabolism
  • Lipopeptides / chemistry
  • Lipopeptides / metabolism
  • Models, Molecular
  • Mycobacterium tuberculosis / enzymology*
  • NADP
  • Oxidation-Reduction
  • Oxidoreductases / chemistry
  • Oxidoreductases / metabolism
  • Peptide Synthases / chemistry*
  • Peptide Synthases / metabolism*
  • Protein Structure, Tertiary
  • Substrate Specificity


  • Alcohols
  • Glycopeptides
  • Lipopeptides
  • NADP
  • Oxidoreductases
  • Peptide Synthases
  • non-ribosomal peptide synthase

Associated data

  • PDB/4DQV