Characterization and investigation of substrate specificity of the sugar aminotransferase WecE from E-coli K12
Characterization and investigation of substrate specificity of the sugar aminotransferase WecE from E-coli K12
复制标题
DOI：
10.1016/j.chembiol.2004.04.015
复制
发表时间：
2004-07-01
影响因子：
--
通讯作者：
Kim, BG
中科院分区：
文献类型：
--
作者：
Hwang, BY;Lee, HJ;Kim, BG
从大肠杆菌K12中克隆到编码糖转氨酶（SAT）的WecE基因，并在大肠杆菌BL21 （DE3）中表达。对该酶进行了纯化和表征。WecE分别以tdp -4-酮-6-脱氧-d -葡萄糖（TDP-D-Glc4O）和l -谷氨酸作为良好的氨基受体和给体，生成了tdp -4-氨基-4,6-二脱氧-d -半乳糖（TDP-Fuc4N），并通过NMR研究鉴定。WecE对tdp -4-酮-6-脱氧-d -甘露糖（tdp -d - man40o）也显示出类似的活性，但对gdp -4-酮-6-脱氧-d -甘露糖（gdp -d - man40o）没有活性，这表明核苷酸片段将成为决定SAT活性的胺受体底物特异性的关键因素。多重比对表明，SAT在活性位点周围有四个高度保守的基序，可分为三个亚群（VIalpha, VIbeta, VIbeta, VIalpha, VIalpha, VIalpha, VIalpha, VIalpha, VIbeta, VIalpha, VIalpha, VIalpha, VIalpha, VIalpha, VIalpha, VIalpha, VIalpha, VIalpha, VIalpha, VIbeta, VIalpha）。和VIgamma)可能与其底物特异性密切相关。
WecE gene, encoding a sugar aminotransferase (SAT), has been cloned from E. coli K12 and expressed in E. coli BL21 (DE3). The enzyme was purified and characterized. WecE used TDP-4-keto-6-deoxy-D-glucose (TDP-D-Glc4O) and L-glutamate as a good amino acceptor and donor, respectively, leading to the production of TDP-4-amino-4,6-dideoxy-D-galactose (TDP-Fuc4N), which was identified by NMR studies. WecE also showed a similar activity for TDP-4-keto 6-deoxy-D-mannose (TDP-D-Man4O), but no activity for GDP-4-keto-6-deoxy-D-mannose (GDP-D-Man4O), suggesting that the nucleotide moiety would become a key determinant to the substrate specificity of amine acceptor for the activity of the SAT. Multiple alignments showed that SATs have four highly conserved motifs located around the active site and could be divided into three subgroups (VIalpha, VIbeta, and VIgamma) that might be closely related with their substrate specificities.