TY - JOUR
T1 - NADP+ Expels both the Co-factor and a Substrate Analog from the Mycobacterium tuberculosis ThyX Active Site
T2 - Opportunities for Anti-bacterial Drug Design
AU - Sampathkumar, Parthasarathy
AU - Turley, Stewart
AU - Sibley, Carol Hopkins
AU - Hol, Wim G.J.
PY - 2006/6/30
Y1 - 2006/6/30
N2 - 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.
AB - 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.
KW - bivalent drugs
KW - flavin
KW - inhibitor design
KW - thymidylate synthase
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U2 - 10.1016/j.jmb.2006.04.061
DO - 10.1016/j.jmb.2006.04.061
M3 - Article
C2 - 16730023
AN - SCOPUS:33745270753
SN - 0022-2836
VL - 360
SP - 1
EP - 6
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 1
ER -