Abstract
The pteridine glycosyltransferase (PGT) found in Chlorobium tepidum (CtPGT) catalyzes the conversion of L-threo-tetrahydrobiopterin to 1-O-(L-threo-biopterin-2'-yl)-β-N-acetylglucosamine using UDP-N-acetylglucosamine. The gene for CtPGT was cloned, and selenomethionine-derivatized protein was overexpressed and purified using various chromatographic techniques. The protein was crystallized by the hanging-drop vapour-diffusion method using 0.24 M triammonium citrate pH 7.0, 14%(w/v) PEG 3350 as a reservoir solution. Multiple-wavelength anomalous diffraction data were collected to 2.15 Å resolution from a single CtPGT crystal. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 189.61, b = 79.98, c=105.92 Å, β=120.5°.
Highlights
Tetrahydrobiopterin (BH4) is an essential cofactor for enzymes involved in neurotransmitter biosynthesis, hydroxylation of aromatic amino acids and nitric oxide (NO) synthesis (Werner et al, 2011) in mammals
There are a group of enzymes called pteridine glycosyltransferases (PGTs) which catalyse the transfer of sugar moieties from activated donor molecules such as UDP-glucose, UDP-xylose and UDPgalactose to specific pteridine acceptor molecules including BH4, biopterin and neopterin to produce various pteridine glycosides (Wachi et al, 1995)
The gene for CtPGT from C. tepidum was successfully cloned in the pProEX HTa expression vector
Summary
Tetrahydrobiopterin (BH4) is an essential cofactor for enzymes involved in neurotransmitter biosynthesis, hydroxylation of aromatic amino acids (phenylalanine hydroxylase and tryptophan hydroxylase) and nitric oxide (NO) synthesis (Werner et al, 2011) in mammals. (Chung et al, 2000) In these bacteria, there are a group of enzymes called pteridine glycosyltransferases (PGTs) which catalyse the transfer of sugar moieties from activated donor molecules such as UDP-glucose, UDP-xylose and UDPgalactose to specific pteridine acceptor molecules including BH4, biopterin and neopterin to produce various pteridine glycosides (Wachi et al, 1995). Chlorobium tepidum is a thermophilic, anaerobic phototrophic bacterium. It is one of the primitive model organisms used in the study of photosynthesis. A glycosidic l-threo-BH4, 1-O-(l-threo-biopterin-20-yl)- -N-acetyl glucosamine, exists (Cho et al, 1998, 1999). To understand the structure and mechanism of CtPGT, we isolated the corresponding gene from C. tepidum and cloned it for expression in E. coli. CtPGT was expressed, purified and crystallized for structural studies. A description of the expression, purification, crystallization and X-ray diffraction analysis of CtPGT is given below
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