Bisubstrate analogue inhibitors of 6-hydroxymethyl-7,8- dihydropterin pyrophosphokinase: Synthesis and biochemical and crystallographic studies

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6- hydroxymethyl-7,8-dihydropterin (HP), leading to the biosynthesis of folate cofactors. Like other enzymes in the folate pathway, HPPK is an ideal target for the development of antimicrobial agents because the enzyme is essential for microorganisms but is absent from human and animals. Three bisubstrate analogues have been synthesized for HPPK and characterized by biochemical and X-ray crystallographic analyses. All three bisubstrate analogues consist of a pterin, an adenosine moiety, and a link composed of 2-4 phosphoryl groups. P-1-(6-Hydroxymethylpterin)-P-2-(5 ' - adenosyl)diphosphate (HP(2)A, 5) shows little affinity and inhibitory activity for E. coli HPPK, P-1-(6- Hydroxymethylpterin)-P-4-(5 ' -adenosyl)triphosphate (H(P)3A, 6) shows moderate affinity and inhibitory activity with K-d = 4.25 muM in the presence of Mg2+ and IC50 = 1.27 muM. P-1-(6- Hydroxymethylpterin)-P-4-(5 ' -adenosyl)tetraphosphate (HP(4)A, 7) shows the highest affinity and inhibitory activity with K-d = 0.47 muM in the presence of Mg2+ and IC50 = 0.44 muM The affinity of MgHP(4)A for HPPK is similar to 116 and 76 times higher than that of MgADP and 6-hydroxymethylpterin, respectively. The crystal structure of HPPK in complex with 7 (HPPK MgHP4A) has been determined at 1.85 Angstrom resolution with a crystallographic R factor of 0.185. The crystal structure shows that 7 occupies both HP- and ATP-binding sites and induces significant conformational changes in HPPK. The biochemical and structural studies of the bisubstrate analogues indicate that the bisubstrate analogue approach can produce more potent inhibitors for HPPK and the minimum length of the link for a bisubstrate analogue is similar to7 Angstrom.

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