TY - JOUR
T1 - Characterization of the proline-utilization pathway in Mycobacterium tuberculosis through structural and functional studies
AU - Lagautriere, Thomas
AU - Bashiri, Ghader
AU - Paterson, Neil G.
AU - Berney, Michael
AU - Cook, Gregory M.
AU - Baker, Edward N.
PY - 2014/4
Y1 - 2014/4
N2 - 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.
AB - 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.
KW - Mycobacterium tuberculosis
KW - PruA
KW - crystal disorder
KW - proline utilization
UR - http://www.scopus.com/inward/record.url?scp=84898727266&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84898727266&partnerID=8YFLogxK
U2 - 10.1107/S1399004713034391
DO - 10.1107/S1399004713034391
M3 - Article
C2 - 24699642
AN - SCOPUS:84898727266
SN - 0907-4449
VL - 70
SP - 968
EP - 980
JO - Acta Crystallographica Section D: Biological Crystallography
JF - Acta Crystallographica Section D: Biological Crystallography
IS - 4
ER -