Zhu P P, Reizer J, Peterkofsky A
Laboratory of Biochemical Genetics, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Protein Sci. 1994 Nov;3(11):2115-28. doi: 10.1002/pro.5560031125.
The region of the genome of Mycoplasma capricolum encompassing the genes for Enzymes I and IIAglc of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) was cloned and sequenced. Examination of the sequence revealed a unique arrangement of the pts operon. In all other bacterial species characterized thus far, the gene encoding Enzyme I (ptsI) in the pts operon is located immediately downstream of the gene (ptsH) encoding HPr, a general energy coupling protein of the PTS. In M. capricolum, ptsH and ptsI reside on 2 distinct operons at separate loci on the chromosome (Zhu PP, Reizer J, Reizer A, Peterkofsky A, 1993, J Biol Chem 268:26531-26540). In the present work, it is shown that the Mycoplasma Enzyme I gene is preceded by an open reading frame homologous to the product of the Escherichia coli kdtB gene and is followed by the gene (crr) encoding Enzyme IIAglc. Northern blot analysis indicated that ptsI and crr constitute a dicistronic operon that includes an independent promoter for the crr gene. Primer extension studies established the transcription start sites for the ptsI and crr genes. The products of the ptsI and crr genes are homologous to previously sequenced Enzymes I and IIAglc proteins but are more similar to the counterpart proteins from gram-positive than to those from gram-negative organisms. The deduced amino acid sequence of the Mycoplasma Enzyme I shows that it differs from other Enzymes I by having fewer acidic amino acids and more basic, amidated, and aromatic amino acids. The deduced amino acid sequence of the Mycoplasma Enzyme IIAglc indicates that it is the shortest (154 residues) of the proteins in this class and it is the only Enzyme IIAglc with a tryptophan and a cysteine residue. In vitro sugar phosphorylation studies with extracts from E. coli and Bacillus subtilis and purified proteins indicated that the Mycoplasma HPr is not a phosphoacceptor from the E. coli Enzyme I, whereas the Mycoplasma Enzyme IIAglc accepts and transfers phosphate from both E. coli and B. subtilis PTS components.
糖磷酸转移酶系统（PTS）的酶I和IIAglc基因的区域进行了克隆和测序。对该序列的检查揭示了pts操纵子的独特排列。在迄今为止已鉴定的所有其他细菌物种中，pts操纵子中编码酶I（ptsI）的基因位于编码HPr（PTS的一种通用能量偶联蛋白）的基因（ptsH）的紧邻下游。在山羊支原体中，ptsH和ptsI位于染色体上不同位点的两个不同操纵子上（朱PP、雷泽J、雷泽A、彼得科夫斯基A，1993年，《生物化学杂志》268:26531 - 26540）。在本研究中，发现山羊支原体酶I基因之前有一个与大肠杆菌kdtB基因产物同源的开放阅读框，之后是编码酶IIAglc的基因（crr）。Northern印迹分析表明，ptsI和crr构成一个双顺反子操纵子，其中包括crr基因的一个独立启动子。引物延伸研究确定了ptsI和crr基因的转录起始位点。ptsI和crr基因的产物与先前测序的酶I和IIAglc蛋白同源，但与革兰氏阳性菌的对应蛋白比与革兰氏阴性菌的对应蛋白更相似。山羊支原体酶I的推导氨基酸序列表明，它与其他酶I的不同之处在于酸性氨基酸较少，碱性、酰胺化和芳香族氨基酸较多。山羊支原体酶IIAglc的推导氨基酸序列表明，它是该类蛋白中最短的（154个残基），并且是唯一具有一个色氨酸残基和一个半胱氨酸残基的酶IIAglc。用大肠杆菌和枯草芽孢杆菌提取物及纯化蛋白进行的体外糖磷酸化研究表明，山羊支原体HPr不是大肠杆菌酶I的磷酸受体，而山羊支原体酶IIAglc接受并从大肠杆菌和枯草芽孢杆菌PTS组分转移磷酸。
