Huang Lan, Li Dong-Yang, Wang Shao-Xiao, Zhang Shi-Ming, Chen Jun-Hui, Wu Xiang-Fu
State Key Laboratory of Pharmaceutical Biotechnology, Department of Biochemistry, Nanjing University, Nanjing 210093, China.
Acta Biochim Biophys Sin (Shanghai). 2005 Jun;37(6):371-8. doi: 10.1111/j.1745-7270.2005.00054.x.
Methionine synthase (MS) is grouped into two classes. Class One MS (MetH) and Class Two MS (MetE) share no homology and differ in their catalytic model. Based on the conserved sequences of metE genes from different organisms, a segment of the metE gene was first cloned from Pichia pastoris genomic DNA by PCR, and its 5' and 3' regions were further cloned by 5'- and 3'-rapid amplification of cDNA ends (RACE), respectively. The assembled sequence reveals an open reading frame encoding a polypeptide of 768 residues, and the deduced product shares 76% identity with MetE of Saccharomyces cerevisiae. P. pastoris methionine synthase (PpMetE) consists of two domains common to MetEs. The active site is located in the C-terminal domain, in which the residues involved in the interaction of zinc with substrates are conserved. Homologous expression of PpMetE in P. pastoris was achieved, and the heterologous expression of PpMetE in the S. cerevisiae strain XJB3-1D that is MetE-defective restored the growth of the mutant on methionine-free minimal media. The gene sequence has been submitted to GenBank/EMBL/DDBJ under accession No. AY601648.
甲硫氨酸合酶（MS）分为两类。一类甲硫氨酸合酶（MetH）和二类甲硫氨酸合酶（MetE）没有同源性，其催化模式也不同。基于不同生物体中metE基因的保守序列，首先通过PCR从巴斯德毕赤酵母基因组DNA中克隆了metE基因的一段序列，然后分别通过5'-和3'-cDNA末端快速扩增（RACE）进一步克隆其5'和3'区域。组装后的序列显示一个编码768个残基多肽的开放阅读框，推导产物与酿酒酵母的MetE有76%的同一性。巴斯德毕赤酵母甲硫氨酸合酶（PpMetE）由MetE共有的两个结构域组成。活性位点位于C末端结构域，其中锌与底物相互作用所涉及的残基是保守的。实现了PpMetE在巴斯德毕赤酵母中的同源表达，并且PpMetE在MetE缺陷的酿酒酵母菌株XJB3-1D中的异源表达恢复了突变体在无甲硫氨酸基本培养基上的生长。该基因序列已提交至GenBank/EMBL/DDBJ，登录号为AY601648。
