NADP super(+) Expels both the Co-factor and a Substrate Analog from the Mycobacterium tuberculosis ThyX Active Site: Opportunities for Anti-bacterial Drug Design

• Published in: Journal of molecular biology Vol. 360; no. 1; pp. 1 - 6
• Main Authors: Sampathkumar, P, Turley, S, Sibley, CH, Hol, WGJ
• Format: Journal Article
• Language: English
• Published: 30-06-2006
• Subjects: Mycobacterium tuberculosis
• ISSN: 0022-2836
• DOI: 10.1016/j.jmb.2006.04.061

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 super(+) to gain structural insights into the reductive step of the catalytic cycle. Surprisingly, the NADP super(+) displaced both FAD and BrdUMP from the active site. In the resultant ThyX-NADP super(+) 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 super(+) is engaged in a limited number of contacts with ThyX. The additional 2'-phosphate group attached to the AMP ribose of NADP super(+) 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.