Biosynthetic enzymes of tetrahydrolimipterin from green sulfur bacterium Chlorobium limicola

Based on the structure of limipterin (Cha, Pfleiderer, and Yim, Helv. Chim. Acta, 78: 600-614, 1995), the biosynthetic pathway for the newly identified pterin glycoside was investigated. It was demonstrated that tetrahydrolimipterin (H₄-limipterin) can be synthesized from GTP by the enzymes GTP cyclohydrolase I, 6-pyruvoyltetrahydropterin (PTP) synthase, sepiapterin reductase and limipterin synthase, present in the extract of Chlorobium limicola. Limipterin synthase (UDP-N-acetylglucosamine:5,6,7,8- tetrahydro-L-biopterin 2'-O-β-N-acetylglucosaminyl transferase) catalyzed the condensation of tetrahydrobiopterin (H₄-biopterin) with UDP-N-a- cetylglucosamine in the presence of dithiothreitol and MnCl₂. It could also produce D-tepidopterin, [1-O-(D-threo-biopterin-2'-yl)-β-N- acetylglucosamine] when 5,6,7,8-tetrahydro-D-threobiopterin and UDP-N- acetylglucosamine were used as substrates. Substrate analogues such as UTP, UDP and UDP-N-acetylgalactosamine inhibit the enzyme activity. The K(m) values for tetrahydrobiopterin and UDP-N-a-cetylglucosamine were 42.2 μM and 124.3 μM, respectively. Optimum pH and temperature were pH 8.0 and 37°C. The molecular weight of the enzyme was calculated to be 46,300 dalton from a calibrated Superdex 75 and the subunit molecular weight was estimated at 46,000 dalton by SDS-PAGE. These results suggest that limipterin synthase exists as a monomer. Biosynthetic intermediates of H₄-limipterin such as H₂NTP, 6-PTP, and H₄-biopterin were identified in vitro using purified GTP cyclohydrolase I, PTP synthase, sepiapterin reductase, and limipterin synthase. From the HPLC and TLC analyses of the enzymic intermediater, it could be concluded that H₄-limipterin comes from GTP by way of H₄-biopterin in Chlorobium limicola.