Cloning, expression and enzymatic properties analysis of dihydrofolate reductase gene from the silkworm, Bombyx mori.
Tetrahydrobiopterin (BH4) is an essential cofactor for aromatic acid hydroxylases, which control the levels of monoamine neurotransmitters. BH4 deficiency has been associated with many neuropsychological disorders. Dihydrofolate reductase (DHFR) can catalyze 7,8-dihydrobiopterin to 5,6,7,8-tetrahydrobiopterin (BH4) in the salvage pathway of BH4 synthesis from sepiapterin (SP), a major pigment component contained in the integument of silkworm Bombyx mori mutant lemon (lem) in high concentration. In this study, we report the cloning of DHFR gene from the silkworm B. mori (BmDhfr) and identification of enzymatic properties of BmDHFR. BmDhfr is located on scaffold Bm_199 with a predicted gene model BGIBMGA013340, which encodes a 185-aa polypeptide with a predicted molecular mass of about 21 kDa. Biochemical analyses showed that the recombinant BmDHFR protein exhibited high enzymatic activity and suitable parameters to substrate. Together with our previous studies on SP reductase of B. mori (BmSPR) and the lem mutant, it may be an effective way to industrially extract SP from the lem silkworms in large scale to produce BH4 in vitro by co-expressing BmSPR and BmDHFR and using the extracted SP as a substrate in the future.
School of Life Sciences, Anhui Agricultural University, 130 West Changjiang Road, Hefei 230036, China. jingbaobao8758@163.com
Wenjing
W
Wang
Junshan
J
Gao
Jing
J
Wang
Chaoliang
C
Liu
Yan
Y
Meng
D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives / Q000096:biosynthesis; D012831:Bombyx / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli; D005492:Folic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D007814:Larva / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D019476:Insect Proteins; D011994:Recombinant Proteins; D001708:Biopterins; C010920:dihydrofolate; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase; C003402:sapropterin