title: Biochemical characterization of the tetrahydrobiopterin synthesis pathway in the oleaginous fungus Mortierella alpina.
abstract: 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.
authors.affiliation: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China.
authors.forename: Hongchao
authors.initials: H
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China.
authors.forename: Bo
authors.initials: B
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China.
authors.forename: Guangfei
authors.initials: G
authors.lastname: Hao
authors.affiliation: TEDA School of Biological Sciences and Biotechnology, Nankai University, Tianjin Economic-Technological Development Area, Tianjin 300457, PR China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Feng
authors.affiliation: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China.
authors.forename: Haiqin
authors.initials: H
authors.lastname: Chen
authors.affiliation: TEDA School of Biological Sciences and Biotechnology, Nankai University, Tianjin Economic-Technological Development Area, Tianjin 300457, PR China.
authors.forename: Lu
authors.initials: L
authors.lastname: Feng
authors.affiliation: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China.
authors.forename: Jianxin
authors.initials: J
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China.
authors.forename: Hao
authors.initials: H
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China.
authors.forename: Yong Q
authors.initials: YQ
authors.lastname: Chen
authors.affiliation: TEDA School of Biological Sciences and Biotechnology, Nankai University, Tianjin Economic-Technological Development Area, Tianjin 300457, PR China.
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, PR China.
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D001708:Biopterins / Q000031:analogs & derivatives* / Q000096:biosynthesis; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D006136:GTP Cyclohydrolase / Q000235:genetics / Q000378:metabolism*; D015394:Molecular Structure; D020094:Mortierella / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D019761:Phosphorus-Oxygen Lyases / Q000235:genetics / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D001708:Biopterins; D000429:Alcohol Oxidoreductases; C022630:sepiapterin reductase; D006136:GTP Cyclohydrolase; D019761:Phosphorus-Oxygen Lyases; C043497:6-pyruvoyltetrahydropterin synthase; C003402:sapropterin