Summers R G, Ali A, Shen B, Wessel W A, Hutchinson C R
Department of Bacteriology, University of Wisconsin, Madison 53706, USA.
Biochemistry. 1995 Jul 25;34(29):9389-402. doi: 10.1021/bi00029a015.
Streptomyces glaucescens, a Gram-positive soil bacterium, produces the polyketide antibiotic tetracenomycin (Tcm) C. To study possible biochemical connections between the biosynthesis of bacterial fatty acids and polyketides, the abundant acyl carrier protein (ACP) detected throughout the growth of the tetracenomycin (Tcm) C-producing S. glaucescens was purified to homogeneity and found to behave like many other ACPs from bacteria and plants (apparent M(r) of 20,000 on gel filtration chromatography, apparent M(r) of 3400-4800 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, and pI approximately 3.8). By using an oligodeoxynucleotide synthesized in accordance with the sequence of residues 25-36 of the ACP, the fabC gene encoding this protein was cloned, and expression of this gene in Escherichia coli yielded the ACP entirely as the active holoenzyme. Sequence analysis of 4.3 kilobases (kb) of DNA flanking fabC revealed the presence of three other genes oriented in the same transcriptional direction in the order fabD, fabH, fabC, and fabB. Each of the four genes is predicted to encode proteins with high sequence similarity to the following components of the E. coli fatty acid synthase (FAS): the FabD malonyl-coenzyme A:ACP acyltransferase (MAT), FabH 3-oxoacyl:ACP synthase III, AcpP ACP, and FabB 3-oxoacyl:ACP synthase I. Expression of the S. glaucescens fabD gene in E. coli produced active MAT able to catalyze in vitro the transfer of radioactive malonate from malonyl-coenzyme A to the E. coli AcpP and S. glaucescens FabC ACPs, as well as to the TcmM ACP component of the Tcm type II polyketide synthase [Shen, B., et al. (1992) J. Bacteriol 174, 3818-3821]. Expression of fabD also restored the high-temperature growth of the E. coli fabD89 mutant that bears a temperature-sensitive MAT. The latter finding and the close similarity between the organization of the S. glaucescens fabDHCB and E. coli FAS-encoding genes (fabH/fabD/fabG/acpP/fabF) suggest that the S. glaucescens genes encode FAS enzymes. Moreover, on the basis of its in vitro activity, it is possible that the S. glaucescens FabD MAT is responsible for charging the TcmM ACP with malonate in vivo, a key step in the synthesis of the deca(polyketide) precursor of Tcm C. This implies the existence of a functional connection between fatty acid and polyketide metabolism in this bacterium.
灰绿链霉菌是一种革兰氏阳性土壤细菌，能产生聚酮类抗生素四环素霉素（Tcm）C。为了研究细菌脂肪酸生物合成与聚酮类生物合成之间可能的生化联系，在产生四环素霉素（Tcm）C的灰绿链霉菌整个生长过程中检测到的丰富酰基载体蛋白（ACP）被纯化至同质，并发现其行为与许多其他来自细菌和植物的ACP相似（凝胶过滤色谱法上的表观分子量为20,000，还原条件下十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳上的表观分子量为3400 - 4800，pI约为3.8）。通过使用根据ACP第25 - 36位残基序列合成的寡脱氧核苷酸，克隆了编码该蛋白的fabC基因，该基因在大肠杆菌中的表达产生了完全作为活性全酶的ACP。对fabC侧翼4.3千碱基（kb）的DNA序列分析揭示了另外三个基因，它们以fabD、fabH、fabC和fabB的顺序在相同转录方向上排列。预测这四个基因中的每一个都编码与大肠杆菌脂肪酸合酶（FAS）的以下组分具有高度序列相似性的蛋白质：FabD丙二酰辅酶A：ACP酰基转移酶（MAT）、FabH 3 - 氧代酰基：ACP合酶III、AcpP ACP和FabB 3 - 氧代酰基：ACP合酶I。灰绿链霉菌fabD基因在大肠杆菌中的表达产生了活性MAT，能够在体外催化放射性丙二酸从丙二酰辅酶A转移到大肠杆菌AcpP和灰绿链霉菌FabC ACP上，以及转移到Tcm II型聚酮合酶的TcmM ACP组分上[沈波等人（199年）《细菌学杂志》174，3818 - 3821]。fabD的表达还恢复了携带温度敏感MAT的大肠杆菌fabD89突变体的高温生长。后一发现以及灰绿链霉菌fabDHCB与大肠杆菌FAS编码基因（fabH/fabD/fabG/acpP/fabF）的组织之间的密切相似性表明，灰绿链霉菌基因编码FAS酶。此外，基于其体外活性，灰绿链霉菌FabD MAT有可能在体内负责用丙二酸对TcmM ACP进行酰化，这是Tcm C的十（聚酮）前体合成中的关键步骤。这意味着该细菌中脂肪酸和聚酮代谢之间存在功能联系。
