Characterization of the proline-utilization pathway in Mycobacterium tuberculosis through structural and functional studies

Acta Crystallogr D Biol Crystallogr. 2014 Apr;70(Pt 4):968-80. doi: 10.1107/S1399004713034391. Epub 2014 Mar 19.


The proline-utilization pathway in Mycobacterium tuberculosis (Mtb) has recently been identified as an important factor in Mtb persistence in vivo, suggesting that this pathway could be a valuable therapeutic target against tuberculosis (TB). In Mtb, two distinct enzymes perform the conversion of proline into glutamate: the first step is the oxidation of proline into Δ(1)-pyrroline-5-carboxylic acid (P5C) by the flavoenzyme proline dehydrogenase (PruB), and the second reaction involves converting the tautomeric form of P5C (glutamate-γ-semialdehyde) into glutamate using the NAD(+)-dependent Δ(1)-pyrroline-5-carboxylic dehydrogenase (PruA). Here, the three-dimensional structures of Mtb-PruA, determined by X-ray crystallography, in the apo state and in complex with NAD(+) are described at 2.5 and 2.1 Å resolution, respectively. The structure reveals a conserved NAD(+)-binding mode, common to other related enzymes. Species-specific conformational differences in the active site, however, linked to changes in the dimer interface, suggest possibilities for selective inhibition of Mtb-PruA despite its reasonably high sequence identity to other PruA enzymes. Using recombinant PruA and PruB, the proline-utilization pathway in Mtb has also been reconstituted in vitro. Functional validation using a novel NMR approach has demonstrated that the PruA and PruB enzymes are together sufficient to convert proline to glutamate, the first such demonstration for monofunctional proline-utilization enzymes.

Keywords: Mycobacterium tuberculosis; PruA; crystal disorder; proline utilization.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 1-Pyrroline-5-Carboxylate Dehydrogenase / chemistry*
  • 1-Pyrroline-5-Carboxylate Dehydrogenase / metabolism
  • Crystallography, X-Ray
  • Models, Molecular
  • Mycobacterium tuberculosis / enzymology*
  • NAD / chemistry
  • NAD / metabolism
  • Nuclear Magnetic Resonance, Biomolecular
  • Proline / metabolism
  • Protein Structure, Quaternary
  • Protein Structure, Tertiary
  • Structural Homology, Protein


  • NAD
  • Proline
  • 1-Pyrroline-5-Carboxylate Dehydrogenase

Associated data

  • PDB/4IDM
  • PDB/4IDS
  • PDB/4IHI
  • PDB/4JDC