title: Structure and inhibition of subunit I of the anthranilate synthase complex of Mycobacterium tuberculosis and expression of the active complex.
abstract: The tryptophan-biosynthesis pathway is essential for Mycobacterium tuberculosis (Mtb) to cause disease, but not all of the enzymes that catalyse this pathway in this organism have been identified. The structure and function of the enzyme complex that catalyses the first committed step in the pathway, the anthranilate synthase (AS) complex, have been analysed. It is shown that the open reading frames Rv1609 (trpE) and Rv0013 (trpG) encode the chorismate-utilizing (AS-I) and glutamine amidotransferase (AS-II) subunits of the AS complex, respectively. Biochemical assays show that when these subunits are co-expressed a bifunctional AS complex is obtained. Crystallization trials on Mtb-AS unexpectedly gave crystals containing only AS-I, presumably owing to its selective crystallization from solutions containing a mixture of the AS complex and free AS-I. The three-dimensional structure reveals that Mtb-AS-I dimerizes via an interface that has not previously been seen in AS complexes. As is the case in other bacteria, it is demonstrated that Mtb-AS shows cooperative allosteric inhibition by tryptophan, which can be rationalized based on interactions at this interface. Comparative inhibition studies on Mtb-AS-I and related enzymes highlight the potential for single inhibitory compounds to target multiple chorismate-utilizing enzymes for TB drug discovery.
authors.affiliation: School of Biological Sciences, University of Auckland, 3a Symonds Steet, Private Bag 90210, Auckland 1142, New Zealand.
authors.forename: Ghader
authors.initials: G
authors.lastname: Bashiri
authors.affiliation: School of Biological Sciences, University of Auckland, 3a Symonds Steet, Private Bag 90210, Auckland 1142, New Zealand.
authors.forename: Jodie M
authors.initials: JM
authors.lastname: Johnston
authors.affiliation: School of Biological Sciences, University of Auckland, 3a Symonds Steet, Private Bag 90210, Auckland 1142, New Zealand.
authors.forename: Genevieve L
authors.initials: GL
authors.lastname: Evans
authors.affiliation: School of Biological Sciences, University of Auckland, 3a Symonds Steet, Private Bag 90210, Auckland 1142, New Zealand.
authors.forename: Esther M M
authors.initials: EM
authors.lastname: Bulloch
authors.affiliation: School of Biological Sciences, University of Auckland, 3a Symonds Steet, Private Bag 90210, Auckland 1142, New Zealand.
authors.forename: David C
authors.initials: DC
authors.lastname: Goldstone
authors.affiliation: School of Biological Sciences, University of Auckland, 3a Symonds Steet, Private Bag 90210, Auckland 1142, New Zealand.
authors.forename: Ehab N M
authors.initials: EN
authors.lastname: Jirgis
authors.affiliation: School of Biological Sciences, University of Auckland, 3a Symonds Steet, Private Bag 90210, Auckland 1142, New Zealand.
authors.forename: Silke
authors.initials: S
authors.lastname: Kleinboelting
authors.affiliation: School of Biological Sciences, University of Auckland, 3a Symonds Steet, Private Bag 90210, Auckland 1142, New Zealand.
authors.forename: Alina
authors.initials: A
authors.lastname: Castell
authors.affiliation: School of Biological Sciences, University of Auckland, 3a Symonds Steet, Private Bag 90210, Auckland 1142, New Zealand.
authors.forename: Rochelle J
authors.initials: RJ
authors.lastname: Ramsay
authors.affiliation: School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Manos-Turvey
authors.affiliation: School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Payne
authors.affiliation: School of Biological Sciences, University of Auckland, 3a Symonds Steet, Private Bag 90210, Auckland 1142, New Zealand.
authors.forename: J Shaun
authors.initials: JS
authors.lastname: Lott
authors.affiliation: School of Biological Sciences, University of Auckland, 3a Symonds Steet, Private Bag 90210, Auckland 1142, New Zealand.
authors.forename: Edward N
authors.initials: EN
authors.lastname: Baker
mesh_terms: D000878:Anthranilate Synthase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D053898:Biosynthetic Pathways; D018360:Crystallography, X-Ray; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008958:Models, Molecular; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000378:metabolism; D011487:Protein Conformation; D055503:Protein Multimerization; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D014364:Tryptophan / Q000378:metabolism*; D014376:Tuberculosis / Q000382:microbiology*
chemical_list: D004791:Enzyme Inhibitors; D021122:Protein Subunits; D014364:Tryptophan; D000878:Anthranilate Synthase
keywords: Mycobacterium tuberculosis; anthranilate synthase complex; inhibitor design; tryptophan biosynthesis