AnPRT (anthranilate phosphoribosyltransferase), required for the biosynthesis of tryptophan, is essential for the virulence of Mycobacterium tuberculosis (Mtb). AnPRT catalyses the Mg2+-dependent transfer of a phosphoribosyl group from PRPP (5′-phosphoribosyl-1′-pyrophosphate) to anthranilate to form PRA (5′-phosphoribosyl anthranilate). Mtb-AnPRT was shown to catalyse a sequential reaction and significant substrate inhibition by anthranilate was observed. Antimycobacterial fluoroanthranilates and methyl-substituted analogues were shown to act as alternative substrates for Mtb-AnPRT, producing the corresponding substituted PRA products. Structures of the enzyme complexed with anthranilate analogues reveal two distinct binding sites for anthranilate. One site is located over 8 Å (1 Å=0.1 nm) from PRPP at the entrance to a tunnel leading to the active site, whereas in the second, inner, site anthranilate is adjacent to PRPP, in a catalytically relevant position. Soaking the analogues for variable periods of time provides evidence for anthranilate located at transient positions during transfer from the outer site to the inner catalytic site. PRPP and Mg2+ binding have been shown to be associated with the rearrangement of two flexible loops, which is required to complete the inner anthranilate-binding site. It is proposed that anthranilate first binds to the outer site, providing an unusual mechanism for substrate capture and efficient transfer to the catalytic site following the binding of PRPP.
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Research Article|
June 13 2014
Alternative substrates reveal catalytic cycle and key binding events in the reaction catalysed by anthranilate phosphoribosyltransferase from Mycobacterium tuberculosis
Tammie V. M. Cookson;
Tammie V. M. Cookson
1
*Maurice Wilkins Centre for Molecular Biodiscovery, Biomolecular Interaction Centre and Department of Chemistry, University of Canterbury, 20 Kirkwood Avenue, Christchurch 8140, New Zealand
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Alina Castell;
†Maurice Wilkins Centre for Molecular Biodiscovery and School of Biological Sciences, University of Auckland, 3 Symonds Street, Auckland 1142, New Zealand
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Esther M. M. Bulloch;
Esther M. M. Bulloch
†Maurice Wilkins Centre for Molecular Biodiscovery and School of Biological Sciences, University of Auckland, 3 Symonds Street, Auckland 1142, New Zealand
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Genevieve L. Evans;
Genevieve L. Evans
†Maurice Wilkins Centre for Molecular Biodiscovery and School of Biological Sciences, University of Auckland, 3 Symonds Street, Auckland 1142, New Zealand
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Francesca L. Short;
Francesca L. Short
3
†Maurice Wilkins Centre for Molecular Biodiscovery and School of Biological Sciences, University of Auckland, 3 Symonds Street, Auckland 1142, New Zealand
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Edward N. Baker;
Edward N. Baker
†Maurice Wilkins Centre for Molecular Biodiscovery and School of Biological Sciences, University of Auckland, 3 Symonds Street, Auckland 1142, New Zealand
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J. Shaun Lott;
J. Shaun Lott
4
†Maurice Wilkins Centre for Molecular Biodiscovery and School of Biological Sciences, University of Auckland, 3 Symonds Street, Auckland 1142, New Zealand
4Correspondence may be addressed to either of these authors (email s.lott@auckland.ac.nz or emily.parker@canterbury.ac.nz).
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Emily J. Parker
Emily J. Parker
4
*Maurice Wilkins Centre for Molecular Biodiscovery, Biomolecular Interaction Centre and Department of Chemistry, University of Canterbury, 20 Kirkwood Avenue, Christchurch 8140, New Zealand
4Correspondence may be addressed to either of these authors (email s.lott@auckland.ac.nz or emily.parker@canterbury.ac.nz).
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Publisher: Portland Press Ltd
Received:
February 13 2014
Revision Received:
April 07 2014
Accepted:
April 09 2014
Accepted Manuscript online:
April 09 2014
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2014 Biochemical Society
2014
Biochem J (2014) 461 (1): 87–98.
Article history
Received:
February 13 2014
Revision Received:
April 07 2014
Accepted:
April 09 2014
Accepted Manuscript online:
April 09 2014
Citation
Tammie V. M. Cookson, Alina Castell, Esther M. M. Bulloch, Genevieve L. Evans, Francesca L. Short, Edward N. Baker, J. Shaun Lott, Emily J. Parker; Alternative substrates reveal catalytic cycle and key binding events in the reaction catalysed by anthranilate phosphoribosyltransferase from Mycobacterium tuberculosis. Biochem J 1 July 2014; 461 (1): 87–98. doi: https://doi.org/10.1042/BJ20140209
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