Chen H, Tseng C C, Hubbard B K, Walsh C T
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Proc Natl Acad Sci U S A. 2001 Dec 18;98(26):14901-6. doi: 10.1073/pnas.221582098.
Four proteins, DpgA-D, required for the biosynthesis by actinomycetes of the nonproteinogenic amino acid monomer (S)-3,5-dihydroxyphenylglycine (Dpg), that is a crosslinking site in the maturation of vancomycin and teicoplanin antibiotic scaffolds, were expressed in Escherichia coli, purified in soluble form, and assayed for enzymatic activity. DpgA is a type III polyketide synthase, converting four molecules of malonyl-CoA to 3,5-dihydroxyphenylacetyl-CoA (DPA-CoA) and three free coenzyme A (CoASH) products. Almost no turnover was observed for DpgA until DpgB was added, producing a net k(cat) of 1-2 min(-1) at a 3:1 ratio of DpgB:DpgA. Addition of DpgD gave a further 2-fold rate increase. DpgC had the unusual catalytic capacity to convert DPA-CoA to 3,5-dihydroxyphenylglyoxylate, which is a transamination away from Dpg. DpgC performed a net CH(2) to C=O four-electron oxidation on the Calpha of DPA-CoA and hydrolyzed the thioester linkage with a k(cat) of 10 min(-1). Phenylacetyl-CoA was also processed, to phenylglyoxylate, but with about 500-fold lower k(cat)/K(M). DpgC showed no activity in anaerobic incubations, suggesting an oxygenase function, but had no detectable bound organic cofactors or metals. A weak enoyl-CoA hydratase activity was detected for both DpgB and DpgD.
放线菌生物合成非蛋白质ogenic氨基酸单体（S）-3,5-二羟基苯甘氨酸（Dpg）所需的四种蛋白质DpgA-D在大肠杆菌中表达，以可溶形式纯化，并测定其酶活性。Dpg是万古霉素和替考拉宁抗生素支架成熟过程中的交联位点。DpgA是一种III型聚酮合酶，将四分子丙二酰辅酶A转化为3,5-二羟基苯乙酰辅酶A（DPA-CoA）和三个游离辅酶A（CoASH）产物。在添加DpgB之前，几乎没有观察到DpgA的周转，以DpgB:DpgA为3:1的比例时，净催化常数k(cat)为1-2 min(-1)。添加DpgD使反应速率进一步提高2倍。DpgC具有将DPA-CoA转化为3,5-二羟基苯乙二醛的异常催化能力，这是一种与Dpg不同的转氨反应。DpgC对DPA-CoA的α-碳原子进行了净CH(2)到C=O的四电子氧化，并以10 min(-1)的催化常数水解硫酯键。苯乙酰辅酶A也被加工成苯乙二醛，但催化常数/米氏常数约低500倍。DpgC在厌氧培养中没有活性，表明其具有加氧酶功能，但没有可检测到的结合有机辅因子或金属。DpgB和DpgD都检测到微弱的烯酰辅酶A水合酶活性。
