Hsieh Y J, Kolattukudy P E
Ohio State University Biotechnology Center, Columbus 43210.
J Bacteriol. 1994 Feb;176(3):714-24. doi: 10.1128/jb.176.3.714-724.1994.
Malonyl-coenzyme A (malonyl-CoA) decarboxylase is widely distributed in prokaryotes and eukaryotes. However, the biological function of this enzyme has not been established in any organism. To elucidate the structure and function of this enzyme, the malonyl-CoA decarboxylase gene from Saccharopolyspora erythraea (formerly Streptomyces erythreaus) was cloned and sequenced. This gene would encode a polypeptide of 417 amino acids. The deduced amino acid sequence matched the experimentally determined amino acid sequences of 25 N-terminal residues each of the enzyme and of an internal peptide obtained by proteolysis of the purified enzyme. This decarboxylase showed homology with aminoglycoside N6'-acetyltransferases of Pseudomonas aeruginosa, Serratia marcescens, and Klebsiella pneumoniae. Northern (RNA) blot analysis revealed a single transcript. The transcription initiation site was 220 bp upstream of the start codon. When expressed in Escherichia coli, the S. erythraea malonyl-CoA decarboxylase gene yielded a protein that cross-reacted with antiserum prepared against S. erythraea malonyl-CoA decarboxylase and catalyzed decarboxylation of [3-14C]malonyl-CoA to acetyl-CoA and 14CO2. The S. erythraea malonyl-CoA decarboxylase gene was disrupted by homologous recombination using an integrating vector pWHM3. The gene-disrupted transformant did not produce immunologically cross-reacting 45-kDa decarboxylase, lacked malonyl-CoA decarboxylase activity, and could not produce erythromycin. Exogenous propionate restored the ability to produce erythromycin. These results strongly suggest that the decarboxylase provides propionyl-CoA for erythromycin synthesis probably via decarboxylation of methylmalonyl-CoA derived from succinyl-CoA, and therefore the malonyl-CoA decarboxylase gene is designated eryM. The gene disrupted mutants also did not produce pigments.
丙二酰辅酶A（丙二酰 - CoA）脱羧酶广泛分布于原核生物和真核生物中。然而，该酶在任何生物体中的生物学功能尚未明确。为阐明该酶的结构和功能，克隆并测序了来自糖多孢红霉菌（原红色链霉菌）的丙二酰辅酶A脱羧酶基因。该基因编码一个由417个氨基酸组成的多肽。推导的氨基酸序列与该酶25个N端残基以及通过对纯化酶进行蛋白水解获得的一个内部肽段的实验测定氨基酸序列相匹配。这种脱羧酶与铜绿假单胞菌、粘质沙雷氏菌和肺炎克雷伯菌的氨基糖苷N6'-乙酰转移酶具有同源性。Northern（RNA）印迹分析显示有一个单一转录本。转录起始位点在起始密码子上游220 bp处。当在大肠杆菌中表达时，糖多孢红霉菌丙二酰辅酶A脱羧酶基因产生一种蛋白质，该蛋白质与针对糖多孢红霉菌丙二酰辅酶A脱羧酶制备的抗血清发生交叉反应，并催化[3-14C]丙二酰辅酶A脱羧生成乙酰辅酶A和14CO2。使用整合载体pWHM3通过同源重组破坏了糖多孢红霉菌丙二酰辅酶A脱羧酶基因。基因破坏的转化体不产生免疫交叉反应的45 kDa脱羧酶，缺乏丙二酰辅酶A脱羧酶活性，且不能产生红霉素。外源性丙酸恢复了产生红霉素的能力。这些结果强烈表明，该脱羧酶可能通过琥珀酰辅酶A衍生的甲基丙二酰辅酶A的脱羧作用为红霉素合成提供丙酰辅酶A，因此将丙二酰辅酶A脱羧酶基因命名为eryM。基因破坏的突变体也不产生色素。
