Biosynthesis of tetrahydrofolate. Stereochemistry of dihydroneopterin aldolase

7,8-Dihydroneopterin aldolase catalyzes the formation of the tetrahydrofolate precursor, 6-hydroxymethyl-7,8-dihydropterin, and is a potential target for antimicrobial and anti-parasite chemotherapy. The last step of the enzyme-catalyzed reaction is believed to involve the protonation of an enol type intermediate. In order to study the stereochemical course of that reaction step, 1',2',3',6,7-13C5]dihydroneopterin was treated with aldolase in deuterated buffer. The resulting, partially deuterated 6alpha,6,7-13C3]6-hydroxymethyl-7,8-dihydropterin was converted to partially deuterated 6-(R)-6,7,9,11-13C4]5,10-methylenetetrahydropteroate by a sequence of three enzyme-catalyzed reactions followed by treatment with 13C]formaldehyde. The product was analyzed by multinuclear NMR spectroscopy. The data show that the carbinol group of enzymatically formed 6-hydroxymethyl-dihydropterin contained 2H predominantly in the pro-S position.