Characterization of the proline‐utilization pathway in Mycobacterium tuberculosis through structural and functional studies. (1st April 2014)

• Record Type: Journal Article
• Authors: Lagautriere, Thomas; Bashiri, Ghader; Paterson, Neil G.; Berney, Michael; Cook, Gregory M.; Baker, Edward N.
• Is Part Of: Acta crystallographica. Volume 70:Part 4(2014:Apr.)
• Page Start: 968
• Page End: 980
• Publication Date: 2014-04-01
• Subjects: Biomolecules -- Structure -- Periodicals; Physical biochemistry -- Periodicals; X-ray crystallography -- Periodicals; Crystallography -- Periodicals; 572
• DOI: 10.1107/S1399004713034391
• Languages: English
• ISSNs: 0907-4449

Abstract

<abstract abstract-type="main" xml:lang="en"> <title> <x xml:space="preserve">Abstract</x> </title> <p>The proline‐utilization pathway in <italic>Mycobacterium tuberculosis</italic> (<italic>Mtb</italic>) has recently been identified as an important factor in <italic>Mtb</italic> persistence <italic>in vivo</italic>, suggesting that this pathway could be a valuable therapeutic target against tuberculosis (TB). In <italic>Mtb</italic>, two distinct enzymes perform the conversion of proline into glutamate: the first step is the oxidation of proline into Δ<sup>1</sup>‐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<sup>+</sup>‐dependent Δ<sup>1</sup>‐pyrroline‐5‐carboxylic dehydrogenase (PruA). Here, the three‐dimensional structures of <italic>Mtb</italic>‐PruA, determined by X‐ray crystallography, in the apo state and in complex with NAD<sup>+</sup> are described at 2.5 and 2.1 Å resolution, respectively. The structure reveals a conserved NAD<sup>+</sup>‐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 <italic>Mtb</italic>‐PruA despite its reasonably high sequence identity to other PruA enzymes. Using recombinant PruA and PruB, the proline‐utilization pathway in <italic>Mtb</italic> has also been reconstituted <italic>in vitro</italic>. 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.</p> </abstract>