Han L, Reynolds K A
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland at Baltimore, 21201, USA.
J Bacteriol. 1997 Aug;179(16):5157-64. doi: 10.1128/jb.179.16.5157-5164.1997.
ccr encoding crotonyl coenzyme A (CoA) reductase (CCR), which catalyzes the conversion of crotonyl-CoA to butyryl-CoA in the presence of NADPH, was previously cloned from Streptomyces collinus. We now report that a complete open reading frame, designated meaA, is located downstream from ccr. The predicted gene product showed 35% identity with methylmalonyl-CoA mutases from various sources. In addition, the predicted amino acid sequences of S. collinus ccr and meaA exhibit strong similarity to that of adhA (43% identity), a putative alcohol dehydrogenase gene, and meaA (62% identity) of Methylobacterium extorquens, respectively. Both adhA and meaA are involved in the assimilation of C1 and C2 compounds in an unknown pathway in the isocitrate lyase (ICL)-negative Methylobacterium. We have demonstrated that S. collinus can grow with acetate as its sole carbon source even though there is no detectable ICL, suggesting that in this organism ccr and meaA may also be involved in a pathway for the assimilation of C2 compounds. Previous studies with streptomycetes provided a precedent for a pathway that initiates with the condensation of two acetyl-CoA molecules to form butyryl-CoA, which is then transformed to succinyl-CoA with two separate CoB12-mediated rearrangements and a series of oxidations. The biological functions of ccr and meaA in this process were investigated by gene disruption. A ccr-blocked mutant showed no detectable crotonyl-CoA reductase activity and, compared to the wild-type strain, exhibited dramatically reduced growth when acetate was the sole carbon source. An meaA-blocked mutant also exhibited reduced growth on acetate. However, both methylmalonyl-CoA mutase and isobutyryl-CoA mutase, which catalyze the two CoB12-dependent rearrangements in this proposed pathway, were shown to be present in the meaA-blocked mutant. These results suggested that both ccr and meaA are involved in a novel pathway for the growth of S. collinus when acetate is its sole carbon source.
ccr编码巴豆酰辅酶A（CoA）还原酶（CCR），该酶在NADPH存在的情况下催化巴豆酰辅酶A转化为丁酰辅酶A，此前已从链霉菌中克隆得到。我们现在报告，一个完整的开放阅读框，命名为meaA，位于ccr的下游。预测的基因产物与来自各种来源的甲基丙二酰辅酶A变位酶具有35%的同一性。此外，链霉菌ccr和meaA的预测氨基酸序列分别与推定的醇脱氢酶基因adhA（同一性为43%）和扭脱甲基杆菌的meaA（同一性为62%）表现出高度相似性。adhA和meaA都参与了异柠檬酸裂合酶（ICL）阴性甲基杆菌中一条未知途径中C1和C2化合物的同化作用。我们已经证明，即使没有可检测到的ICL，链霉菌也能以乙酸盐作为唯一碳源生长，这表明在这种生物体中，ccr和meaA也可能参与了C2化合物的同化途径。先前对链霉菌的研究为一条途径提供了先例，该途径始于两个乙酰辅酶A分子的缩合形成丁酰辅酶A，然后通过两个独立的钴胺素B12介导的重排和一系列氧化反应转化为琥珀酰辅酶A。通过基因破坏研究了ccr和meaA在此过程中的生物学功能。一个ccr阻断突变体没有可检测到的巴豆酰辅酶A还原酶活性，与野生型菌株相比，当乙酸盐作为唯一碳源时，其生长显著降低。一个meaA阻断突变体在乙酸盐上的生长也降低。然而，在meaA阻断突变体中显示存在催化该拟议途径中两个钴胺素B12依赖性重排的甲基丙二酰辅酶A变位酶和异丁酰辅酶A变位酶。这些结果表明，当乙酸盐是其唯一碳源时，ccr和meaA都参与了链霉菌生长的一条新途径。
