ISOLATION AND CHARACTERIZATION OF AN ACYL-COENZYME-A CARBOXYLASE FROM AN ERYTHROMYCIN-PRODUCING STREPTOMYCES-ERYTHREUS
ISOLATION AND CHARACTERIZATION OF AN ACYL-COENZYME-A CARBOXYLASE FROM AN ERYTHROMYCIN-PRODUCING STREPTOMYCES-ERYTHREUS
复制标题
DOI：
10.1016/0003-9861(82)90222-3
复制
发表时间：
1982-01-01
影响因子：
3.9
通讯作者：
KOLATTUKUDY, PE
中科院分区：
文献类型：
--
作者：
HUNAITI, AR;KOLATTUKUDY, PE
S.红曲霉菌产生红霉素，可能是从甲基丙二酰辅酶A产生的，甲基丙二酰辅酶A可能是由丙酰辅酶A的羧化产生的。从S.用DEAE-cellulose亲和层析法、亲和素-琼脂糖凝胶和蓝色琼脂糖凝胶柱层析法分离纯化。该酶羧化丙酰辅酶A（100%），Km为0.09 mM，V为0.86 μ mol/mg/min，羧化乙酰辅酶A（16%），Km为0.17 mM，V为0.08 μ mol/mg/min，羧化丁酰辅酶A（7.7%），Km为0.67 mM，V为0.044 μ mol/mg/min。天然酶的MW为537，000，由MW分别为67，000和61，000的两种亚基组成，表明八聚体α 4 β 4类型的结构。生物素与大太阳黑子（α）有关。该酶的最适pH为7.5至7.8。它被K+、Rb+和Cs+刺激（3- 4倍），但不被Na+或Li+刺激，并被高浓度的NH 4+和Cl-抑制。柠檬酸盐和游离CoA都不能刺激酶。该酶是立体特异性的，并且仅从丙酰辅酶A的羧化产生S-甲基丙二酰辅酶A。目前的情况下，似乎是第一个酶可能参与红霉素生产被分离在均匀的形式。
S. erythraeus produces erythromycin presumably from methylmalonyl-CoA which might be generated by carboxylation of propionyl-CoA. A biotin-containing enzyme which carboxylates acetyl-CoA, propionyl-CoA and butyryl-CoA was purified to near homogeneity from S. erythraeus using DEAE-cellulose, affinity chromatography on monomeric avidin-Sepharose and blue Sepharose. The enzyme carboxylates propionyl-CoA (100%) with a Km of 0.09 mM and V of 0.86 .mu.mol/mg per min, acetyl-CoA (16%) with a Km of 0.17 mM and V of 0.08 .mu.mol/mg per min and butyryl-CoA (7.7%) with a Km of 0.67 mM and V of 0.044 .mu.mol/mg per min. The native enzyme has a MW of 537,000 and consists of 2 types of subunits with MW of 67,000 and 61,000, respectively, indicating an octameric .alpha.4.beta.4 type of structure. Biotin is associated with the large sunbunit (.alpha.). The enzyme has a pH optimum between 7.5 and 7.8. It is stimulated (3- to 4-fold) by K+, Rb+ and Cs+ but not by Na+ of Li+ and is inhibited by high concentrations of NH4+ and Cl-. Neither citrate nor free CoA stimulated the enzyme. The enzyme was stereospecific and generated only S-methylmalonyl-CoA from the carboxylation of propionyl-CoA. The present case appears to be the 1st enzyme possibly involved in erythromycin production to be isolated in homogeneous form.