Zhang W, Reynolds K A
Institute for Structural Biology and Drug Discovery and Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, Virginia 23219, USA.
J Bacteriol. 2001 Mar;183(6):2071-80. doi: 10.1128/JB.183.6.2071-2080.2001.
The ratio of the major monensin analogs produced by Streptomyces cinnamonensis is dependent upon the relative levels of the biosynthetic precursors methylmalonyl-coenzyme A (CoA) (monensin A and monensin B) and ethylmalonyl-CoA (monensin A). The meaA gene of this organism was cloned and sequenced and was shown to encode a putative 74-kDa protein with significant amino acid sequence identity to methylmalonyl-CoA mutase (MCM) (40%) and isobutyryl-CoA mutase (ICM) large subunit (36%) and small subunit (52%) from the same organism. The predicted C terminus of MeaA contains structural features highly conserved in all coenzyme B12-dependent mutases. Plasmid-based expression of meaA from the ermE* promoter in the S. cinnamonensis C730.1 strain resulted in a decreased ratio of monensin A to monensin B, from 1:1 to 1:3. Conversely, this ratio increased to 4:1 in a meaA mutant, S. cinnamonensis WM2 (generated from the C730.1 strain by insertional inactivation of meaA by using the erythromycin resistance gene). In both of these experiments, the overall monensin titers were not significantly affected. Monensin titers, however, did decrease over 90% in an S. cinnamonensis WD2 strain (an icm meaA mutant). Monensin titers in the WD2 strain were restored to at least wild-type levels by plasmid-based expression of the meaA gene or the Amycolatopsis mediterranei mutAB genes (encoding MCM). In contrast, growth of the WD2 strain in the presence of 0.8 M valine led only to a partial restoration (<25%) of monensin titers. These results demonstrate that the meaA gene product is significantly involved in methylmalonyl-CoA production in S. cinnamonensis and that under the tested conditions the presence of both MeaA and ICM is crucial for monensin production in the WD2 strain. These results also indicate that valine degradation, implicated in providing methylmalonyl-CoA precursors for many polyketide biosynthetic processes, does not do so to a significant degree for monensin biosynthesis in the WD2 mutant.
肉桂链霉菌产生的主要莫能菌素类似物的比例取决于生物合成前体甲基丙二酰辅酶A（CoA）（莫能菌素A和莫能菌素B）和乙基丙二酰辅酶A（莫能菌素A）的相对水平。该生物体的meaA基因被克隆并测序，结果显示它编码一种推定的74 kDa蛋白质，与来自同一生物体的甲基丙二酰辅酶A变位酶（MCM）（40%）、异丁酰辅酶A变位酶（ICM）大亚基（36%）和小亚基（52%）具有显著的氨基酸序列同一性。预测的MeaA C末端含有在所有依赖辅酶B12的变位酶中高度保守的结构特征。在肉桂链霉菌C730.1菌株中，基于质粒从ermE*启动子表达meaA导致莫能菌素A与莫能菌素B的比例从1:1降至1:3。相反，在meaA突变体肉桂链霉菌WM2（通过使用红霉素抗性基因对C730.1菌株进行meaA插入失活产生）中，该比例增至4:1。在这两个实验中，总体莫能菌素效价均未受到显著影响。然而，在肉桂链霉菌WD2菌株（icm meaA突变体）中，莫能菌素效价确实下降了90%以上。通过基于质粒表达meaA基因或地中海拟无枝酸菌mutAB基因（编码MCM），WD2菌株中的莫能菌素效价恢复到至少野生型水平。相比之下，WD2菌株在0.8 M缬氨酸存在下生长仅导致莫能菌素效价部分恢复（<25%）。这些结果表明，meaA基因产物在肉桂链霉菌甲基丙二酰辅酶A的产生中起重要作用，并且在测试条件下，MeaA和ICM的存在对WD2菌株中莫能菌素的产生至关重要。这些结果还表明，缬氨酸降解虽然在许多聚酮生物合成过程中为提供甲基丙二酰辅酶A前体发挥作用，但在WD2突变体的莫能菌素生物合成中，其作用程度并不显著。
