链霉素的生物合成。来自灰色链霉菌的dTDP-二氢链霉糖合酶以及来自灰色链霉菌和大肠杆菌Y10的dTDP-4-酮基-L-鼠李糖3,5-表异构酶。

Biosynthesis of streptomycin. dTDP-dihydrostreptose synthase from Streptomyces griseus and dTDP-4-keto-L-rhamnose 3,5-epimerase from S. griseus and Escherichia coli Y10.

作者信息

Wahl H P, Grisebach H

出版信息

Biochim Biophys Acta. 1979 May 10;568(1):243-52. doi: 10.1016/0005-2744(79)90291-2.

Abstract

dTDP-dihydrostreptose synthase from Streptomyces griseus was purfied about 50-fold by removal of protein with polyethyleneimine, (NH4)2SO4 fractionation and gel filtration on Ultrogel AcA44. The synthase preparation was free of dTDP-4-keto-L-rhamnose 3,5-epimerase (dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase, EC 5.1.3.13) activity. A new enzyme assay using Escherichia coli Y10 as source for the epimerase and dTDP-glucose 4,6-dehydratase (dTDP-glucose 4,6-hydro-lyase, EC 4.2.1.46) was developed. In the presence of excess epimerase the apparent Km for dTDP-4-keto-6-deoxy-D-glucose was determined to be 25 microM. The molecular weight of epimerase and synthase were determined by their elution volumes from a Sephadex G-100 column to be approx. 67,000 and 32,000, respectively. The pH optimum for the epimerase was between 7.5 and 8.5. The intermediate formation of dTDP-4-keto-L-rhamnose in the epimerase reaction could be shown by detection of 6-deoxy-[3H]talose after NaB3H4 reduction. Results which indicate the existence of dTDP-4-keto-6-rhamnose as a free intermediate in the epimerase reaction are reported.

摘要

通过用聚乙烯亚胺去除蛋白质、硫酸铵分级分离以及在Ultrogel AcA44上进行凝胶过滤，将灰色链霉菌的dTDP - 二氢链霉糖合酶纯化了约50倍。该合酶制剂不含dTDP - 4 - 酮 - L - 鼠李糖3,5 - 差向异构酶（dTDP - 4 - 酮 - 6 - 脱氧 - D - 葡萄糖3,5 - 差向异构酶，EC 5.1.3.13）活性。开发了一种新的酶活性测定方法，使用大肠杆菌Y10作为差向异构酶和dTDP - 葡萄糖4,6 - 脱水酶（dTDP - 葡萄糖4,6 - 氢裂解酶，EC 4.2.1.46）的来源。在存在过量差向异构酶的情况下，测定dTDP - 4 - 酮 - 6 - 脱氧 - D - 葡萄糖的表观Km为25 microM。通过它们从Sephadex G - 100柱上的洗脱体积确定差向异构酶和合酶的分子量分别约为67,000和32,000。差向异构酶的最适pH在7.5至8.5之间。通过硼氢化钠还原后检测6 - 脱氧 - [3H]塔罗糖，可以证明差向异构酶反应中dTDP - 4 - 酮 - L - 鼠李糖的中间产物形成。报道了表明dTDP - 4 - 酮 - 6 - 鼠李糖作为差向异构酶反应中游离中间产物存在的结果。