Purification and Characterization of the Folate Catabolic Enzyme p-Aminobenzoyl-Glutamate Hydrolase from Escherichia coli
Purification and Characterization of the Folate Catabolic Enzyme p-Aminobenzoyl-Glutamate Hydrolase from Escherichia coli
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DOI：
10.1128/jb.01362-09
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发表时间：
2010-05-01
影响因子：
3.2
通讯作者：
Carter, Eric L.
中科院分区：
文献类型：
--
作者：
Green, Jacalyn M.;Hollandsworth, Ryan;Carter, Eric L.
大肠杆菌染色体的abg基因座包括编码蛋白质（AbgA、AbgB和AbgT）的三个基因，所述蛋白质使得能够摄取和利用叶酸分解产物对氨基苯甲酰谷氨酸盐（PABA-GLU）。我们报告的纯化和表征的对氨基苯甲酰谷氨酸水解酶（PGH）全酶编码的abgA和abgB。使用携带在AbgB的羧基末端上具有六组氨酸标签的abgAB的质粒和随后的金属亲和层析（MAC）完成一步纯化。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析显示，预期质量的AbgB和AbgA有两个亚基（类似于53-kDa和类似于47-kDa蛋白质）; N-末端测序证实了亚基鉴定，氨基酸分析得出亚基比例为1：1。尺寸排阻色谱法结合光散射分析纯化的PGH显示了206 kDa的主要分子量和400至500 kDa的次要组分。两个峰均含有PGH活性，SDS-PAGE显示含有活性的级分由AbgA和AbgB两者组成。MAC纯化的PGH被氯化锰高度刺激。MAC纯化的PGH的动力学分析显示PABA-GLU的K（m）值为60 +/- 0.08 μ M，比活性为63，300 +/- 600 nmol min（-1）mg（-1）。叶酸和多种二肽是PGH的不良底物。该位点为E.大肠杆菌染色体可以编码叶酸催化途径的一部分。
The abg locus of the Escherichia coli chromosome includes three genes encoding proteins (AbgA, AbgB, and AbgT) that enable uptake and utilization of the folate breakdown product, p-aminobenzoyl-glutamate (PABA-GLU). We report on the purification and characterization of the p-aminobenzoyl-glutamate hydrolase (PGH) holoenzyme encoded by abgA and abgB. One-step purification was accomplished using a plasmid carrying abgAB with a hexahistidine tag on the carboxyl terminus of AbgB and subsequent metal affinity chromatography (MAC). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed two subunits (similar to 53-kDa and similar to 47-kDa proteins) of the expected masses of AbgB and AbgA; N-terminal sequencing confirmed the subunit identification, and amino acid analysis yielded a 1: 1 ratio of the subunits. Size exclusion chromatography coupled with light-scattering analysis of purified PGH revealed a predominant molecular mass of 206 kDa and a minor component of 400 to 500 kDa. Both peaks contained PGH activity, and SDS-PAGE revealed that fractions containing activity were composed of both AbgA and AbgB. MAC-purified PGH was highly stimulated by manganese chloride. Kinetic analysis of MAC-purified PGH revealed a K(m) value for PABA-GLU of 60 +/- 0.08 mu M and a specific activity of 63,300 +/- 600 nmol min(-1) mg(-1). Folic acid and a variety of dipeptides served as poor substrates of PGH. This locus of the E. coli chromosome may encode a portion of a folate catabolism pathway.