Characterization of the proline-utilization pathway in Mycobacterium tuberculosis through structural and functional studies.
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.
Structural Biology Laboratory, School of Biological Sciences and Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland 1010, New Zealand.
Thomas
T
Lagautriere
Structural Biology Laboratory, School of Biological Sciences and Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland 1010, New Zealand.
Ghader
G
Bashiri
Structural Biology Laboratory, School of Biological Sciences and Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland 1010, New Zealand.
Neil G
NG
Paterson
Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand.
Michael
M
Berney
Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand.
Gregory M
GM
Cook
Structural Biology Laboratory, School of Biological Sciences and Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland 1010, New Zealand.
Edward N
EN
Baker
D050842:1-Pyrroline-5-Carboxylate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D011392:Proline / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D040681:Structural Homology, Protein
D009243:NAD; D011392:Proline; D050842:1-Pyrroline-5-Carboxylate Dehydrogenase
Mycobacterium tuberculosis; PruA; crystal disorder; proline utilization