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(3)-(5'-adenosyl)triphosphate (HP(3)A, 6) shows moderate affinity and inhibitory activity with K(d) = 4.25 microM in the presence of Mg(2+) and IC(50) = 1.27 microM. 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 microM in the presence of Mg(2+) and IC(50) = 0.44 microM. The affinity of MgHP(4)A for HPPK is approximately 116 and 76 times higher than that of MgADP and 6-hydroxymethylpterin, respectively. The crystal structure of HPPK in complex with 7 (HPPK.MgHP(4)A) has been determined at 1.85 A 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 approximately 7 A.
Shi
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Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824, USA.
Blaszczyk
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J
Ji
X
X
Yan
H
H
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000138:chemical synthesis* / Q000737:chemistry; D000900:Anti-Bacterial Agents / Q000138:chemical synthesis* / Q000737:chemistry; D018360:Crystallography, X-Ray; D017854:Diphosphotransferases / Q000037:antagonists & inhibitors*; D004791:Enzyme Inhibitors / Q000138:chemical synthesis* / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry; D005470:Fluorometry; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D015394:Molecular Structure; D011485:Protein Binding; D011622:Pterins / Q000138:chemical synthesis* / Q000737:chemistry; D013329:Structure-Activity Relationship
D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; C426026:P(1)-(6-hydroxymethylpterin)-P(4)-(5'-adenosyl)tetraphosphate; D011622:Pterins; D000255:Adenosine Triphosphate; D017854:Diphosphotransferases; C019684:2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase