Cloning, expression and enzymatic properties analysis of dihydrofolate reductase gene from the silkworm, Bombyx mori
Cloning, expression and enzymatic properties analysis of dihydrofolate reductase gene from the silkworm, Bombyx mori
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DOI：
10.1007/s11033-012-1905-4
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发表时间：
2012-10
影响因子：
2.8
通讯作者：
Wenjing Wang;Jun-shan Gao;Jing Wang;Chaoliang Liu;Y. Meng
中科院分区：
文献类型：
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作者：
Wenjing Wang;Jun-shan Gao;Jing Wang;Chaoliang Liu;Y. Meng
四氢生物蝶呤（BH 4）是芳香酸羟化酶的重要辅因子，该酶控制单胺神经递质的水平。BH 4缺乏与许多神经心理障碍有关。二氢叶酸还原酶（DHFR）在家蚕体壁中的主要色素成分Sepiapterin（SP）高浓度合成BH 4的补救途径中，可催化7，8-二氢生物蝶呤（DH 2）生成5，6，7，8-四氢生物蝶呤（BH 4）。本研究克隆了家蚕DHFR基因。BmDhfr位于支架Bm_199上，预测的基因模型为BGIBMGA 013340，其编码185个氨基酸的多肽，预测的分子量约为21 kDa。生化分析表明，重组BmDHFR蛋白具有较高的酶活性和适宜的底物参数。结合我们以前对B. mori（BmSPR）和家蚕突变体BmDHFR共表达，并将其作为底物，可能是一条工业化大规模提取家蚕SP，体外生产BH 4的有效途径。
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 silkwormBombyx morimutantlemon(lem) in high concentration. In this study, we report the cloning of DHFR gene from the silkwormB. mori(BmDhfr) and identification of enzymatic properties of BmDHFR.BmDhfris 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 ofB. mori(BmSPR) and thelemmutant, it may be an effective way to industrially extract SP from thelemsilkworms 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.