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

Thomas Lagautriere, Ghader Bashiri, Neil G. Paterson, Michael Berney, Gregory M. Cook, Edward N. Baker

Research output: Contribution to journalArticle

10 Scopus citations

Abstract

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 Δ1-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 Δ1-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.

Original languageEnglish (US)
Pages (from-to)968-980
Number of pages13
JournalActa Crystallographica Section D: Biological Crystallography
Volume70
Issue number4
DOIs
StatePublished - Apr 1 2014
Externally publishedYes

Keywords

  • Mycobacterium tuberculosis
  • PruA
  • crystal disorder
  • proline utilization

ASJC Scopus subject areas

  • Structural Biology

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