State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China.
TEDA School of Biological Sciences and Biotechnology, Nankai University, Tianjin Economic-Technological Development Area, Tianjin 300457, PR China.
Microbiology (Reading). 2011 Nov;157(Pt 11):3059-3070. doi: 10.1099/mic.0.051847-0. Epub 2011 Aug 18.
We characterized the de novo biosynthetic pathway of tetrahydrobiopterin (BH₄) in the lipid-producing fungus Mortierella alpina. The BH₄ cofactor is essential for various cell processes, and is probably present in every cell or tissue of higher organisms. Genes encoding two copies of GTP cyclohydrolase I (GTPCH-1 and GTPCH-2) for the conversion of GTP to dihydroneopterin triphosphate (H₂-NTP), 6-pyruvoyltetrahydropterin synthase (PTPS) for the conversion of H₂-NTP to 6-pyruvoyltetrahydropterin (PPH₄), and sepiapterin reductase (SR) for the conversion of PPH₄ to BH₄, were expressed heterologously in Escherichia coli. The recombinant enzymes were produced as His-tagged fusion proteins and were purified to homogeneity to investigate their enzymic activities. Enzyme products were analysed by HPLC and electrospray ionization-MS. Kinetic parameters and other properties of GTPCH, PTPS and SR were investigated. Physiological roles of BH₄ in M. alpina are discussed, and comparative analyses between GTPCH, PTPS and SR proteins and other homologous proteins were performed. The presence of two functional GTPCH enzymes has, as far as we are aware, not been reported previously, reflecting the unique ability of this fungus to synthesize both BH₄ and folate, using the GTPCH product as a common substrate. To our knowledge, this study is the first to report the comprehensive characterization of a BH₄ biosynthesis pathway in a fungus.
我们对产脂真菌枝顶孢霉（Mortierella alpina）中四氢生物蝶呤（BH₄）的从头生物合成途径进行了研究。BH₄ 辅因子对各种细胞过程至关重要，可能存在于高等生物的每个细胞或组织中。编码 GTP 环化水解酶 I（GTPCH-1 和 GTPCH-2）的基因，用于将 GTP 转化为二氢神经蝶呤三磷酸（H₂-NTP），6- 丙酮酰四氢蝶呤合酶（PTPS）用于将 H₂-NTP 转化为 6- 丙酮酰四氢蝶呤（PPH₄），以及蝶呤还原酶（SR）用于将 PPH₄ 转化为 BH₄，这些基因在大肠杆菌中被异源表达。重组酶被表达为 His 标记的融合蛋白，并被纯化至均一性，以研究它们的酶活性。通过 HPLC 和电喷雾电离-MS 分析酶产物。研究了 GTPCH、PTPS 和 SR 的酶动力学参数和其他特性。讨论了 BH₄ 在枝顶孢霉中的生理作用，并对 GTPCH、PTPS 和 SR 蛋白与其他同源蛋白进行了比较分析。据我们所知，这种真菌中存在两种功能齐全的 GTPCH 酶，这是以前未曾报道过的，反映了这种真菌具有独特的合成 BH₄ 和叶酸的能力，将 GTPCH 的产物作为共同的底物。据我们所知，这项研究首次报道了真菌中 BH₄ 生物合成途径的全面特征。
