4lem

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== Publication Abstract from PubMed ==
== Publication Abstract from PubMed ==
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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 Delta(1)-pyrroline-5-carboxylic acid (P5C) by the flavoenzyme proline dehydrogenase (PruB), and the second reaction involves converting the tautomeric form of P5C (glutamate-gamma-semialdehyde) into glutamate using the NAD(+)-dependent Delta(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 A 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.
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The use of small molecules as "silver bullets" that can bind to generate crosslinks between protein molecules has been advanced as a powerful means of enhancing success in protein crystallization (McPherson and Cudney, 2006). We have explored this approach in attempts to overcome an order-disorder phenomenon that complicated the structural analysis of the enzyme Delta1-pyrroline-5-carboxylate dehydrogenase from Mycobacterium tuberculosis (P5CDH, Mtb-PruA). Using the Silver Bullets Bio screen, we obtained new crystal packing using cobalamin as a co-crystallization agent. This crystal form did not display the order-disorder phenomenon previously encountered. Solution of the crystal structure showed that cobalamin molecules are present in the crystal contacts. Although the cobalamin binding probably does not have physiological relevance, it reflects similarities in the nucleotide-binding region of Mtb-PruA, with the nucleotide loop of cobalamin sharing the binding site for the adenine moiety of NAD+.
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Characterization of the proline-utilization pathway in Mycobacterium tuberculosis through structural and functional studies.,Lagautriere T, Bashiri G, Paterson NG, Berney M, Cook GM, Baker EN Acta Crystallogr D Biol Crystallogr. 2014 Apr 1;70(Pt 4):968-80. doi:, 10.1107/S1399004713034391. Epub 2014 Mar 19. PMID:24699642<ref>PMID:24699642</ref>
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Use of a "silver bullet" to resolve crystal lattice dislocation disorder: A cobalamin complex of Delta-pyrroline-5-carboxylate dehydrogenase from Mycobacterium tuberculosis.,Lagautriere T, Bashiri G, Baker EN J Struct Biol. 2014 Dec 31. pii: S1047-8477(14)00281-0. doi:, 10.1016/j.jsb.2014.12.007. PMID:25557497<ref>PMID:25557497</ref>
From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>
From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>

Revision as of 11:29, 4 March 2015

Crystal structure of the Delta-pyrroline-5-carboxylate dehydrogenase from Mycobacterium tuberculosis

4lem, resolution 2.27Å

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