Hahn F M, Hurlburt A P, Poulter C D
Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.
J Bacteriol. 1999 Aug;181(15):4499-504. doi: 10.1128/JB.181.15.4499-4504.1999.
Isopentenyl diphosphate isomerase catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In eukaryotes, archaebacteria, and some bacteria, IPP is synthesized from acetyl coenzyme A by the mevalonate pathway. The subsequent isomerization of IPP to DMAPP activates the five-carbon isoprene unit for subsequent prenyl transfer reactions. In Escherichia coli, the isoprene unit is synthesized from pyruvate and glyceraldehyde-3-phosphate by the recently discovered nonmevalonate pathway. An open reading frame (ORF696) encoding a putative IPP isomerase was identified in the E. coli chromosome at 65.3 min. ORF696 was cloned into an expression vector; the 20.5 kDa recombinant protein was purified in three steps, and its identity as an IPP isomerase was established biochemically. The gene for IPP isomerase, idi, is not clustered with other known genes for enzymes in the isoprenoid pathway. E. coli FH12 was constructed by disruption of the chromosomal idi gene with the aminoglycoside 3'-phosphotransferase gene and complemented by the wild-type idi gene on plasmid pFMH33 with a temperature-sensitive origin of replication. FH12/pFMH33 was able to grow at the restrictive temperature of 44 degrees C and FH12 lacking the plasmid grew on minimal medium, thereby establishing that idi is a nonessential gene. Although the V(max) of the bacterial protein was 20-fold lower than that of its yeast counterpart, the catalytic efficiencies of the two enzymes were similar through a counterbalance in K(m)s. The E. coli protein requires Mg(2+) or Mn(2+) for activity. The enzyme contains conserved cysteine and glutamate active-site residues found in other IPP isomerases.
异戊烯基二磷酸异构酶催化异戊烯基二磷酸（IPP）和二甲基烯丙基二磷酸（DMAPP）的相互转化。在真核生物、古细菌和一些细菌中，IPP通过甲羟戊酸途径由乙酰辅酶A合成。随后IPP异构化为DMAPP激活了五碳异戊二烯单元，用于后续的异戊烯基转移反应。在大肠杆菌中，异戊二烯单元通过最近发现的非甲羟戊酸途径由丙酮酸和3-磷酸甘油醛合成。在大肠杆菌染色体65.3分钟处鉴定出一个编码假定IPP异构酶的开放阅读框（ORF696）。将ORF696克隆到表达载体中；通过三步纯化出20.5 kDa的重组蛋白，并通过生化方法确定其为IPP异构酶。IPP异构酶基因idi不与类异戊二烯途径中其他已知的酶基因聚集在一起。通过用氨基糖苷3'-磷酸转移酶基因破坏染色体idi基因构建了大肠杆菌FH12，并由具有温度敏感复制起点的质粒pFMH33上的野生型idi基因进行互补。FH12/pFMH33能够在44℃的限制温度下生长，而缺乏质粒的FH12能在基本培养基上生长，从而证明idi是一个非必需基因。尽管细菌蛋白的V(max)比酵母对应物低20倍，但通过K(m)的平衡，两种酶的催化效率相似。大肠杆菌蛋白的活性需要Mg(2+)或Mn(2+)。该酶含有在其他IPP异构酶中发现的保守的半胱氨酸和谷氨酸活性位点残基。
