title: NADP+ expels both the co-factor and a substrate analog from the Mycobacterium tuberculosis ThyX active site: opportunities for anti-bacterial drug design.
abstract: The novel flavin-dependent thymidylate synthase, ThyX, is absent in humans but several pathogenic bacteria depend exclusively on ThyX activity to synthesize thymidylate. Reduction of the enzyme-bound FAD by NADPH is suggested to be the critical first step in ThyX catalysis. We soaked Mycobacterium tuberculosis ThyX-FAD-BrdUMP ternary complex crystals in a solution containing NADP+ to gain structural insights into the reductive step of the catalytic cycle. Surprisingly, the NADP+ displaced both FAD and BrdUMP from the active site. In the resultant ThyX-NADP+ binary complex, the AMP moiety is bound in a deep pocket similar to that of the same moiety of FAD in the ternary complex, while the nicotinamide part of NADP+ is engaged in a limited number of contacts with ThyX. The additional 2'-phosphate group attached to the AMP ribose of NADP+ could be accommodated with minor rearrangement of water molecules. The newly introduced 2'-phosphate groups are engaged in water-mediated interactions across the non-crystallographic 2-fold axis of the ThyX tetramer, suggesting possibilities for design of high-affinity bivalent inhibitors of this intriguing enzyme.
authors.affiliation: Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, USA.
authors.forename: Parthasarathy
authors.initials: P
authors.lastname: Sampathkumar
authors.forename: Stewart
authors.initials: S
authors.lastname: Turley
authors.forename: Carol Hopkins
authors.initials: CH
authors.lastname: Sibley
authors.forename: Wim G J
authors.initials: WG
authors.lastname: Hol
mesh_terms: D000249:Adenosine Monophosphate / Q000737:chemistry; D000890:Anti-Infective Agents / Q000494:pharmacology*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D015195:Drug Design; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D005415:Flavins / Q000737:chemistry; D008958:Models, Molecular; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000378:metabolism*; D009249:NADP / Q000737:chemistry*; D012266:Ribose / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D000890:Anti-Infective Agents; D005415:Flavins; D005182:Flavin-Adenine Dinucleotide; D000249:Adenosine Monophosphate; D009249:NADP; D012266:Ribose